U.S. patent number RE34,606 [Application Number 07/556,918] was granted by the patent office on 1994-05-10 for modified enzymes and methods for making same.
This patent grant is currently assigned to Genencor, Inc.. Invention is credited to Richard R. Bott, David A. Estell, James A. Wells.
United States Patent |
RE34,606 |
Estell , et al. |
May 10, 1994 |
Modified enzymes and methods for making same
Abstract
A cloned subtilsin gene has been modified at specific sites to
cause amino acid substitutions at certain spots in the enzyme. The
modified enzyme, preferably produced by Bacillus, is useful in
combination with detergents.
Inventors: |
Estell; David A. (San Mateo,
CA), Wells; James A. (Burlingame, CA), Bott; Richard
R. (Burlingame, CA) |
Assignee: |
Genencor, Inc. (So. San
Francisco, CA)
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Family
ID: |
24462011 |
Appl.
No.: |
07/556,918 |
Filed: |
July 20, 1990 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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Reissue of: |
614612 |
May 29, 1984 |
04760025 |
Jul 26, 1988 |
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Current U.S.
Class: |
510/392; 930/240;
435/221; 930/200; 510/530; 435/222 |
Current CPC
Class: |
C12N
15/102 (20130101); C11D 3/386 (20130101); C12N
9/54 (20130101); C12N 1/205 (20210501); C12R
2001/07 (20210501); C12N 15/75 (20130101) |
Current International
Class: |
C11D
3/386 (20060101); C12N 9/52 (20060101); C12N
9/54 (20060101); C11D 3/38 (20060101); C12N
009/56 (); C12N 009/54 (); C12N 015/03 (); C12P
019/34 () |
Field of
Search: |
;435/91,172.1,172.3,221,222,188,252.5 ;252/547,174.12
;935/10,11,14,29,74 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0328229 |
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Aug 1989 |
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EP |
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88/01038 |
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Oct 1988 |
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WO |
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Other References
Svendsen, I. 1976. Carlsberg Res. Commun. 41, 237-291. .
Robertus, et al. 1972. Biochemistry 11, 2439-2448. .
Wright et al. 1969. Nature. 221, 235-241. .
Rastetter, W. H. 1983. Trends, Biotechnol. 1, 80-84. .
Wells et al. 1983. Nuc. Acids Res. 11, 7911-7923. .
Ferrari et al. 1983. J. Bacteriol. 154, 1513-1515. .
Hutchinson et al. 1978. J. Biol. Chem. 253, 6551-6550. .
Wallace et al. 1981. Nucleic Acids Research 9, 3647-3656. .
Stahl. et al. 1984. J. Bacteriol. 158, 411-418. .
Patterson et al. 1979. J. Gen. Microbiol. 144, 75-85. .
Nedkov et al. 1983. Hoppe-Seyler's Z. Physiol. Chem. 346 (11),
1537-1540 C.A. 100:81934r. .
Kerjan et al. 1979. Eur. J. Biochem 98, 353-362. .
Uehara et al. 1979. J. Bacteriol. 139(2), 583-590. .
Polgar et al. 1981. Biochim. Biophys. Acta. 667, 351-354. .
Stauffer and Etson, J. Biol. Chem. (1969) 244:5333-5338. .
Brot and Weissbach, All Types of Biochemistry and Biophysics (1983)
223:271-281. .
Zoller and Smith, Methods In Enzymology (1983) 100:468-500. .
Polgar and Bender, Advances In Enzymology (1970) 33:381-400. .
Winter, et al., Nature (1982) 299:756-758. .
Wilkinson, et al., Nature (1984) 307:187-188. .
Estell, et al., J. Biol. Chem. (1985) 260:6518-6521..
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Primary Examiner: Low; Christopher S. F.
Attorney, Agent or Firm: Horn; Margaret A.
Claims
We claim: .[.1. A subtilisin enzyme having a different amino acid
at a site +32, +155, +104, +222, +166, +64, +33, +169, +189, +217,
or +157 than subtilisin naturally produced by Bacillus
amyloliquefaciens..]. .[.2. A composition comprising an enzyme
according to claim 1 in combination with a detergent..]. .[.3. A
composition according to claim 2 wherein the detergent is a linear
alkyl benzene sulfonate, alkyl benzene sulfate,
sulfated linear alcohol, or ethoxylated linear alcohol..]. .Iadd.4.
A substantially pure modified subtilisin substituted at the residue
position equivalent to asp+32 of the Bacillus amyloliquefaciens
subtilisin shown in FIG. 1B with one of the other nineteen
naturally occurring amino acids shown in FIG. 17. .Iaddend.
.Iadd.5. A substantially pure modified subtilisin substituted at
the residue position equivalent to asn+155 of the Bacillus
amyloliquefaciens subtilisin shown in FIG. 1B, with one of the
other nineteen naturally occurring amino acids shown in FIG. 17.
.Iaddend. .Iadd.6. A substantially pure modified subtilisin
substituted at the residue position equivalent to tyr+104 of the
Bacillus amyloliquefaciens subtilisin shown in FIG. 1B with one of
the other nineteen amino acids shown in FIG. 17. .Iaddend. .Iadd.7.
A substantially pure modified subtilisin substituted at the residue
position equivalent to met+222 of the Bacillus amyloliquefaciens
subtilisin shown in FIG. 1B with one of the other nineteen amino
acids as shown in FIG. 17. .Iaddend. .Iadd.8. A substantially pure
modified subtilisin substituted at the residue position equivalent
to gly+166 of the Bacillus amyloliquefaciens subtilisin shown in
FIG. 1B with one of the other nineteen naturally occurring amino
acids shown in FIG. 17. .Iaddend. .Iadd.9. A substantially pure
modified subtilisin substituted at the residue position equivalent
to his+64 of the Bacillus amyloliquefaciens subtilisin shown in
FIG. 1B with one of the other nineteen naturally occurring amino
acids shown in FIG.
17. .Iaddend. .Iadd.10. A substantially pure modified subtilisin
substituted at the residue position equivalent to ser+33 of the
Bacillus amyloliquefaciens subtilisin shown in FIG. 1B with one of
the other nineteen naturally occurring amino acids as shown in FIG.
17. .Iaddend. .Iadd.11. A substantially pure modified subtilisin
substituted at the residue position equivalent to gly+169 of the
Bacillus amyloliquefaciens subtilisin shown in FIG. 1B with one of
the other nineteen naturally occurring amino acids shown in FIG.
17. .Iaddend. .Iadd.12. A substantially pure modified subtilisin
substituted at the residue position equivalent to phe+189 of the
Bacillus amyloliquefaciens subtilisin shown in FIG. 1B with one of
the other nineteen naturally occurring amino acids shown in FIG.
17. .Iaddend. .Iadd.13. A substantially pure modified subtilisin
substituted at the residue position equivalent to tyr+217 of the
Bacillus amyloliquefaciens subtilisin shown in FIG. 1B with one of
the other nineteen naturally occurring amino acids shown in FIG.
17. .Iaddend. .Iadd.14. A substantially pure modified subtilisin
substituted at the residue position equivalent to glu+156 of the
Bacillus amyloliquefaciens subtilisin shown in FIG. 1B with one of
the other nineteen naturally occurring amino acids shown in FIG.
17. .Iaddend. .Iadd.15. A substantially pure modified subtilisin
substituted at the position equivalent to ala+152 of the Bacillus
amyloliquefaciens subtilisin shown in FIG. 1B with one of the other
nineteen naturally occurring amino acids shown in FIG. 17.
.Iaddend. .Iadd.16. A modified subtilisin according to claim 7
wherein said subtilisin has an improved pH activity profile when
compared to said subtilisin having the amino acid naturally
occurring in said subtilisin at the residue position equivalent to
met+222. .Iaddend. .Iadd.17. A modified subtilisin according to
claim 8 wherein said subtilisin has improved substrate specificity
when compared to said subtilisin having the amino acid naturally
occurring in said subtilisin at the residue position equivalent to
gly+166. .Iaddend. .Iadd.18. A modified subtilisin according to
claim 7 wherein said subtilisin has improved oxidative stability
when compared to said subtilisin having the amino acid naturally
occurring in said subtilisin at the residue position equivalent to
met+222. .Iaddend. .Iadd.19. A modified subtilisin according to
claim 18 wherein the amino acid substituted at the residue position
equivalent to met+222 is selected from the group consisting of ala,
ser or cys. .Iaddend. .Iadd.20. A substantially pure modified
subtilisin having improved oxidative stability wherein the
stability is effected by deleting one or more methionine,
tryptophan, cysteine or lysine residue in said subtilisin and
substituting another amino acid other than one of methionine,
tryptophan, cysteine or lysine, for said deleted amino acid
residue. .Iaddend. .Iadd.21. A substantially pure modified
subtilisin resulting from the expression of DNA which DNA encodes
for the amino acid sequence of subtilisin substituted at the
residue position equivalent to asp+32, asn+155, tyr+104, met+222,
gly+166, his+64, ser+33, gly+169, phe+189, tyr+217, glu+156, or
ala+152 of the Bacillus amyloliquefaciens subtilisin shown in FIG.
1B, wherein the substituted amino acid is one of the other nineteen
naturally occurring amino acids shown in FIG. 17, and wherein said
DNA is obtained by direct recombinant mutagenesis to a precursor
DNA. .Iaddend. .Iadd.22. A composition comprising an enzyme
according to any one of the claims 4-15 in combination with a
detergent. .Iaddend. .Iadd.23. A composition according to claim 22
wherein the detergent is a linear alkyl benzene sulfonate, kyl
benzene sulfate, sulfated linear alcohol, or ethoxylated linear
alcohol. .Iaddend.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
Cross-reference is made to .[.application Ser. No. 509,419 filed
June 24, 1983, and its continuation-in-part Ser. No. 614,616. Cross
reference is made to application Ser. No. 614,617, application Ser.
No. 614,615, and application Ser. No. 614,491, all of which are
filed concurrently herewith..]. .Iadd.the following related
applications, all of which were filed on May 29, 1984 concurrently
with U.S. Application Ser. No. 06/614,612 now issued as U.S. Pat.
No. 4,760,025, and subject to reissue application Ser. No. 556,918
filed Jul. 20, 1990: (a) U.S. application Ser. No. 06/614,615
(abandoned), in favor of U.S. application Ser. No. 07/488,433
(pending) filed Feb. 29, 1990; U.S. application Ser. No. 07/511,972
(abn) filed Apr. 17, 1990; U.S. application Ser. No. 07/334,081
(abn) filed Apr. 4, 1989; U.S. application Ser. No. 07/041,885
(abn) filed Apr. 23, 1987; (b) U.S. application Ser. No. 06/614,616
(abandoned), in favor of U.S. application Ser. No. 352,326 (abn)
filed May 15, 1989; and (c) U.S. application Ser. No. 06/614,491
(abandoned), in favor of U.S. application Ser. No. 07/539,283 (abn)
filed Jun. 14, 1990, and the continuation thereof U.S. application
Ser. No. 07/760,833 filed Sep. 16, 1991 (pending). .Iaddend.
BACKGROUND
This invention relates to the production and manipulation of
proteins using recombinant techniques in suitable hosts. More
specifically, the invention relates to the production of
procaryotic proteases such as subtilsin and neutral protease using
recombinant microbial host cells, to the synthesis of heterologous
proteins by microbial hosts, and to the directed mutagenesis of
enzymes in order to modify the characteristics thereof.
Various bacteria are known to secrete proteases at some stage in
their life cycles. Bacillus species produce two major extracellular
proteases, a neural protease (a metalloprotease inhibited by EDTA)
and an alkaline protease (or subtilsin, a serine endoprotease).
Both generally are produced in greatest quantity after the
exponential growth phase, when the culture enters stationary phase
and begins the process of sporulation. The physiological role of
these two proteases is not clear. They have been postulated to play
a role in sporulation (J. Hoch, 1976, Adv. Genet." 18:69-98; P.
Piggot et al., 1976, "Bact. Rev." 40:908-962; and F. Priest, 1977,
"Bact. Rev." 41:711-753), to be involved in the regulation of cell
wall turnover (L. Jolliffe et al., 1980, "J. Bact." 141:1199-1208),
and to be scavenger enzymes (Priest, Id.). The regulation of
expression of the protease genes is complex. They appear to be
coordinately regulated in concert with sporulation, since mutants
blocked in the early stages of sporulation exhibit reduced levels
of both the alkaline and neutral protease. Additionally, a number
of pleiotropic mutations exist which affect the level of expression
of proteases and other secreted gene products, such as amylase and
levansucrase (Priest, Id.).
Subtilisin has found considerable utility in industrial and
commercial applications (see U.S. Pat. No. 3,623,957 and J. Millet,
1970, "J. Appl. Bact." 33:207). For example, subtilisins and other
proteases are commonly used in detergents to enable removal of
protein-based stains. They also are used in food processing to
accommodate the proteinaceous substances present in the food
preparations to their desired impact on the composition.
Classical mutagenesis of bacteria with agents such as radiation or
chemicals has produced a plethora of mutant strains exhibiting
different properties with respect to the growth phase at which
protease excretion occurs as well as the timing and activity levels
of excreted protease. These strains, however, do not approach the
ultimate potential of the organisms because the mutagenic process
is essentially random, with tedious selection and screening
required to identify organisms which even approach the desired
characteristics. Further, these mutants are capable of reversion to
the parent or wild-type strain. In such event the desirable
property is lost. The probability of reversion is unknown when
dealing with random mutagenesis since the type and site of mutation
is unknown or poorly characterized. This introduces considerable
uncertainty into the industrial process which is based on the
enzyme-synthesizing bacterium. Finally, classical mutagenesis
frequently couples a desirable phenotype, e.g. low protease levels,
with an undesirable character such as excessive premature cell
lysis.
Special problems exist with respect to the proteases which are
excreted by Bacillus. For one thing, since at least two such
proteases exist, screening for the loss of only one is difficult.
Additionally, the large number of pleiotropic mutations affecting
both sporulation and protease production make the isolation of true
protease mutations difficult.
Temperature sensitive mutants of the neutral protease gene have
been obtained by conventional mutagenic techniques, and were used
to map the position of the regulatory and structural gene in the
Bacillus subtilis chromosome (H. Uehara et al., 1979, "J. Bact."
139:583-590). Additionally, a presumed nonsense mutation of the
alkaline protease gene has been reported (C. Roitsch et al., 1983,
"J. Bact." 155:145-152).
Bacillus temperature sensitive mutants have been isolated that
produce inactive serine protease or greatly reduced levels of
serine protease. These mutants, however, are asporogenous and show
a reversion frequency to the wild-type of about from 10.sup.-7 to
10.sup.-8 (F. Priest, Id. p. 719). These mutants are unsatisfactory
for the recombinant production of heterologous proteins because
asporogenous mutants tend to lyse during earlier stages of their
growth cycle in minimal medium than do sporogenic mutants, thereby
prematurely releasing cellular contents (including intracellular
proteases) into the culture supernatant. The possibility of
reversion also is undesirable since wild-type revertants will
contaminate the culture supernatant with excreted proteases.
Bacillus sp. have been proposed for the expression of heterologous
proteins, but the presence of excreted proteases and the potential
resulting hydrolysis of the desired product has retarded the
commercial acceptance of Bacillus as a host for the expression of
heterologous proteins. Bacillus megaterium mutants have been
disclosed that are capable of sporulation and which do not express
a sporulation-associated protease during growth phases. However,
the assay employed did not exclude the presence of other proteases,
and the protease in question is expressed during the sporulation
phase (C. Loshon et al., 1982, "J. Bact." 150:303--311). This, of
course, is the point at which heterologous protein would have
accumulated in the culture and be vulnerable. It is an objective
herein to construct a Bacillus strain that is substantially free of
extracellular neutral and alkaline protease during all phases of
its growth cycle and which exhibits substantially normal
sporulation characteristics. A need exists for non-revertible,
otherwise normal protease deficient organisms that can then be
transformed with high copy number plasmids for the expression of
heterologous or homologous proteins.
Enzymes having characteristics which vary from available stock are
required. In particular, enzymes having enhanced oxidation
stability will be useful in extending the shelf life and bleach
compatibility of proteases used in laundry products. Similarly,
reduced oxidation stability would be useful in industrial processes
that require the rapid and efficient quenching of enzymatic
activity.
Modifying the pH-activity profiles of an enzyme would be useful in
making the enzymes more efficient in a wide variety of processes,
e.g. broadening the pH-activity profile of a protease would produce
an enzyme more suitable for both alkaline and neutral laundry
products. Narrowing the profile, particularly when combined with
tailored substrate specificity, would make enzymes in a mixture
more compatible, as will be further described herein.
Mutations of procaryotic carbonyl hydrolases (principally proteases
but including lipases) will facilitate preparation of a variety of
different hydrolases, particularly those having other modified
properties such as Km, Kcat, Km/Kcat ratio and substrate
specificity. These enzymes can then be tailored for the particular
substrate which is anticipated to be present, for example in the
preparation of peptides or for hydrolytic processes such as laundry
uses.
Chemical modification of enzymes is known. For example, see I.
Svendsen, 1976, "Carlsberg Res. Commun." 41 (5):237-291. These
methods, however, suffer from the disadvantages of being dependent
upon the presence of convenient amino acid residues, are frequently
nonspecific in that they modify all accessible residues with common
side chains, and are not capable of reaching inaccessible amino
acid residues without further processing, e.g. denaturation, that
is generally not completely reversible in reinstituting activity.
To the extent that such methods have the objective of replacing one
amino acid residue side chain for another side chain or equivalent
functionality, then mutagenesis promises to supplant such
methods.
Predetermined, site-directed mutagenesis of tRNA synthetase in
which a cys residue is converted to serine has been reported (G.
Winter et al., 1982, "Nature" 299:758-758; A. Wilkinson et al.,
1984, "Nature" 307:187-188). This method is not practical for large
scale mutagenesis. It is an object herein to provide a convenient
and rapid method for mutating DNA by saturation mutagenesis.
SUMMARY
A method for producing procaryotic carbonyl hydrolase such as
subtilisin and neutral protease in recombinant host cells is
described in which expression vectors containing sequences which
encode desired subtilisin or neutral protease, including the pro,
pre, or prepro forms of these enzymes, are used to transform hosts,
the host cultured and desired enzymes recovered. The coding
sequence may correspond exactly to one found in nature, or may
contain modifications which confer desirable properties on the
protein that is produced, as is further described below.
The novel strains then are transformed with at least one DNA moiety
encoding a polypeptide not otherwise expressed in the host strain,
the transformed strains cultured and the polypeptide recovered from
the culture. Ordinarily, the DNA moiety is a directed mutant of a
host Bacillus gene, although it may be DNA encoding a eucaryotic
(yeast or mammalian) protein. The novel strains also serve as hosts
for protein expressed from a bacterial gene derived from sources
other than the host genome, or for vectors expressing these
heterologous genes, or homologous genes from the host genome. In
the latter event enzymes such as amylase are obtained free of
neutral protease or subtilisin. In addition, it is now possible to
obtain neutral protease in culture which is free of enzymatically
active subtilisin, and vice-versa.
One may, by splicing the cloned genes for procaryotic carbonyl
hydrolase into a high copy number plasmid, synthesize the enzymes
in enhanced yield compared to the parental organisms. Also
disclosed are modified forms of such hydrolases, including the pro
and prepro zymogen forms of the enzymes, the pre forms, and
directed mutations thereof.
A convenient method is provided for saturation mutagenesis, thereby
enabling the rapid and efficient generation of a plurality of
mutations at any one site within the coding region of a protein,
comprising:
(a) obtaining a DNA moiety encoding at least a portion of said
precursor protein;
(b) identifying a region within the moiety;
(c) substituting nucleotides for those already existing within the
region in order to create at least one restriction enzyme site
unique to the moiety, whereby unique restriction sites 5' and 3' to
the identified region are made available such that neither alters
the amino acids coded for by the region as expressed;
(d) synthesizing a plurality of oligonucleotides, the 5' and 3'
ends of which each contain sequences capable of annealing to the
restriction enzyme sites introduced in step (c) and which, when
ligated to the moiety, are expressed as substitutions, deletions
and/or insertions of at least one amino acid in or into said
precursor protein;
(e) digesting the moiety of step (c) with restriction enzymes
capable of cleaving the unique sites; and
(f) ligating each of the oligonucleotides of step (d) into the
digested moiety of step (e) whereby a plurality of mutant DNA
moieties are obtained.
By the foregoing method or others known in the art, a mutation is
introduced into isolated DNA encoding a procaryotic carbonyl
hydrolase which, upon expression of the DNA, results in the
substitution, deletion or insertion of at least one amino acid at a
predetermined site in the hydrolase. This method is useful in
creating mutants of wild type proteins (where the "precursor"
protein is the wild type) or reverting mutants to the wild type
(where the "precursor" is the mutant.
Mutant enzymes are recovered which exhibit oxidative stability
and/or pH-activity profiles which differ from the precursor
enzymes. Procaryotic carbonyl hydrolases having varied Km, Kcat,
Kcat/Km ratio and substrate specificity also are provided
herein.
The mutant enzymes obtained by the methods herein are combined in
known fashion with surfactants or detergents to produce novel
compositions useful in the laundry or other cleaning arts.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the sequence of a functional B. amyloliquefaciens
subtilisin gene.
In FIG. 1A, the entire functional sequence for B.
amyloliquefaciens, including the promoter and ribosome binding
site, are present on a 1.5 kb fragment of the B. amyloliquefaciens
genome.
FIG. 1B shows the nucleotide sequence of the coding strand,
correlated with the amino acid sequence of the protein. Promoter
(p) ribosome binding site (rbs) and termination (term) regions of
the DNA sequence are also shown.
FIG. 2 shows the results of replica nitrocellulose filters of
purified positive clones probed with Pool 1 (Panel A) and Pool 2
(Panel B) respectively.
FIG. 3 shows the restriction analysis of the subtilisin expression
plasmid (pS4). pBS42 vector sequences (4.5 kb) are shown in solid
while the insert sequence (4.4 kb) is shown dashed.
FIG. 4 shows the results of SDS-PAGE preformed on supernatants from
cultures transformed with pBS42 and pS4.
FIG. 5 shows the construction of the shuttle vector pBS42.
FIG. 6 shows a restriction map for a sequence including the B.
subtilis subtilisin gene.
FIG. 7 is the sequence of a functional B. subtilis subtilisin
gene.
FIG. 8 demonstrates a construction method for obtaining a deletion
mutant of a B. subtilis subtilisin gene.
FIG. 9 discloses the restriction map for a B. subtilis neutral
protease gene.
FIG. 10 is a nucleotide sequence for a B. subtilis neutral protease
gene.
FIG. 11 demonstrates the construction of a vector containing a B.
subtilis neutral protease gene.
FIGS. 12, 13 and 16 disclose embodiments of the mutagenesis
technique provided herein.
FIG. 14 shows the enhanced oxidation stability of a subtilisin
mutant.
FIG. 15 demonstrates a change in the pH-activity profile of a
subtilisin mutant when compared to the wild type enzyme.
.Iadd.FIG. 17 is a reproduction in pertinent-part of Table C.
.Iaddend.
DETAILED DESCRIPTION
Procaryotic carbonyl hydrolases are enzymes which hydrolyze
compounds containing ##STR1## bonds in which X is oxygen or
nitrogen. They principally include hydrolases, e.g. lipases and
peptide hydrolases, e.g. subtilisins or metalloproteases. Peptide
hydrolases include .alpha.-aminoacylpeptide hydrolase,
peptidylamino-acid hydrolase, acylamino hydrolase, serine
carboxypeptidase, metallocarboxypeptidase, thiol proteinase,
carboxlproteinase and metalloprotinease. Serine, metallo, thiol and
acid proteases are included, as well as endo and exo-proteases.
Subtilisins are serine proteinases which generally act to cleave
internal peptide bonds of protein or peptides. Metalloproteases are
exo- or endoproteases which require a metal ion cofactor for
activity.
A number of naturally occurring mutants of subtilisin or neutral
protease exist, and all may be employed with equal effect herein as
sources for starting genetic material.
These enzymes and their genes may be obtained from many procaryotic
organisms. Suitable examples include gram negative organisms such
as E. coli or pseudomonas and gram positive bacteria such as
micrococcus or bacillus.
The genes encoding the carbonyl hydrolase may be obtained in accord
with the general method herein. As will be seen from the examples,
this comprises synthesizing labelled probes having putative
sequences encoding regions of the hydrolase of interest, preparing
genomic libraries from organisims expressing the hydrolase, and
screening the libraries for the gene of hybridization to the
probes. Positively hybridizing clones ar mapped and sequenced. The
cloned genes are ligated into an expression vector (which also may
be the cloning vector) with requisite regions for replication in
the host, the plasmid transfected into a host for enzyme synthesis
and the recombina host cells cultured under conditions favoring
enzyme synthesis, usually selection pressure such as is supplied by
the presence of an antibiotic, the resistance to which is encoded
by the vector. Culture under these conditions results in enzyme
yields multifold greater than the wild type enzyme synthesis of the
parent organism, even if it is the parent organism that is
transformed.
"Expression vector" refers to a DNA construct containing a DNA
sequence which is operably linked to a suitable control sequence
capable of effecting the expression of said DNA in a suitable host.
Such control sequences include a promoter to effect transcription,
an optional operator sequence to control such transcription, a
sequence encoding suitable mRNA ribosome binding sites, and
sequences which control termination of transcription and
translation. The vector may be a plasmid, a phage particle, or
simply a potential genomic insert. Once transformed into a suitable
host, the vector may replicate and function independently of the
host genome, or may, in some instances, integrate into the genome
itself. In the present specification, "plasmid" and "vector" are
sometimes used interchangeably as the plasmid is the most commonly
used form of vector at present. However, the invention is intended
to include such other forms of expression vectors which serve
equivalent functions and which are, or become, known in the
art.
"Recombinant host cells" refers to cells which have been
transformed or transfected with vectors constructed using
recombinant DNA techniques. As relevant to the present invention,
recombinant host cells are those which produce procaryotic carbonyl
nydrolases in its various forms by virtue of having been
transformed with expression vectors encoding these proteins. The
recombinant host cells may or may not have produced a form of
carbonyl hydrolase prior to transformation.
"Operably linked" when describing the relationship between two DNA
regions simply means that they are functionally related to each
other. For example, a presequence is operably linked to a peptide
if it functions as a signal sequence, participating in the
secretion of the mature form of the protein most probably involving
cleavage of the signal sequence. A promoter is operably linked to a
coding sequence if it controls the transcription of the sequence; a
ribosome binding site is operably linked to a coding sequence if it
is positioned so as to permit translation.
"Prohydrolase" refers to a hydrolase which contains additional
N-terminal amino acid resides which render the enzyme inactive but,
when removed, yield an enzyme. Many proteolytic enzymes are found
in nature as translational proenzyme products and, in the absence
of post-translational products, are expressed in this fashion.
"Presequence" refers to a signal sequence of amino acids bound to
the N-terminal portion of the hydrolase which may participate in
the secretion of the hydrolase. Presequences also may be modified
in the same fashion as is described here, including the
introduction of predetermined mutations. When bound to a nydrolase,
the subject protein becomes a "prehydrolase". Accordingly, relevant
prehydrolase for the purposes herein are presubtilisin and
preprosubtilisin. Prehydrolases are produced by deleting the "pro"
sequence (or at least that portion of the pro sequence that
maintains the enzyme in its inactive state) from a prepro coding
region, and then expressing the prehydrolase. In this way the
organism excretes the active rather than proenzyme.
The cloned carbonyl hydrolase is used to transform a host cell in
order to express the hydrolase. This will be of interest where the
hydrolase has commercial use in its unmodified form, as for example
subtilisin in laundry products as noted above. In the preferred
embodiment the hydrolase gene is ligated into a high copy. number
plasmid. This plasmid replicates in hosts in the sense that it
contains the well-known elements necessary for plasmid replication:
a promoter operably linked to the gene in question (which may be
supplied as the gene's own homologous promotor if it is recognized,
i.e., transcribed, by the host), a transcription termination and
polyadenylation region (necessary for stability of the mRNA
transcribed by the host from the hydrolase gene) which is exogenous
or is supplied by the endogenous terminator region of the hydrolase
gene and, desirably, a selection gene such as an antibiotic
resistance gene that enables continuous cultural maintenance of
plasmid-infected host cells by growth in antibiotic-containing
media. High copy number plasmids also contain an origin of
replication for the host, thereby enabling large numbers of
plasmids to be generated in the cytoplasm without chromosonal
limitations. However, it is within the scope herein to integrate
multiple copies of the hydrolase gene into host genome. This is
facilitated by bacterial strains which are particularly susceptible
to homologous recombination. The resulting host cells are termed
recombinant host cells.
Once the carbonyl hydrolase gene has been cloned, a number of
modifications are undertaken to enhance the use of the gene beyond
synthesis of the wild type or precursor enzyme. A precursor enzyme
is the enzyme prior to its modification as described in this
application. Usually the precursor is the enzyme as expressed by
the organism which donated the DNA modified in accord herewith. The
term "precursor" is to be understood as not implying that the
product enzyme was the result of manipulation of the precursor
enzyme per se.
In the first of these modifications, the gene may be detected from
a recombination positive (rec.sup.+) organism containing a
homologous gene. This is accomplished by recombination of an in
vitro deletion mutation of the cloned gene with the genome of the
organism. Many strains of organisms such as E.coli and Bacillus are
known to be capable of recombination. All that is needed is for
regions of the residual DNA from the deletion mutant to recombine
with homologous regions of the candidate host. The deletion may be
within the coding region (leaving enzymatically inactive
polypeptides) or include the entire coding region as long as
homologous flanking regions (such as promoters or termination
regions) exist in the host. Acceptability of the host for
recombination deletion mutants is simply determined by screening
for the deletion of the transformed phenotype. This is most readily
accomplished in the case of carbonyl hydrolase by assaying host
cultures for loss of the ability to cleave a chromogenic substrate
otherwise hydrolyzed by the hydrolase.
Transformed hosts contained the protease deletion mutants are
useful for synthesis of products which are incompatible with
proteolytic enzymes. These hosts by definition are incapable of
excreting the deleted proteases described herein, yet are
substantially normally sporulating. Also the other growth
characteristics of the transformants are substantially like the
parental organism. Such organisms are useful in that it is expected
they will exhibit comparatively less inactivation of heterologous
proteins than the parents, and these hosts do have growth
characteristics superior to known protease-deficient organisms.
However, the deletion of neutral protease and subtilisin as
described in this application does not remove all of the
proteolytic activity of Bacillus. It is believed that intracellular
proteases which are not ordinarily excreted extracellularly "leak"
or diffuse from the cells during late phases of the culture. These
intracellular proteases may or may not be subtilisin or neutral
protease as those enzymes are defined herein. Accordingly, the
novel Bacillus strains herein are incapable of excreting the
subtilisin and/or neutral protease enzymes which ordinarfly are
excreted extracellularly in the parent strains. "Incapable" means
not revertible to the wild type. Reversion is a finite probability
that exists with the heretofore known protease-deficient, naturally
occurring strains since there is no assurance that the phenotype of
such strains is not a function of a readily revertible mutation,
e.g. a point mutation. This is to be contrasted with the extremely
large deletions provided herein.
The deletion mutant-transformed host cells herein are free of genes
encoding enzymatically active neutral protease or subtilisin, which
genes are defined by those being substantially homologous with the
genes set forth in FIGS. 1, 7 or 10. "Homologous" genes contain
coding regions capable of hybridizing under high stringency
conditions with the genes shown in FIGS. 1, 7 or 10.
The microbial strains containing carbonyl hydrolase deletion
mutants are useful in two principal processes. In one embodiment
they are advantageous in the fermentative production of products
ordinarily expressed by a host that are desirably uncontaminated
with the protein encoded by the deletion gene. An example is
fermentative synthesis of amylase, where contaminant proteases
interfere in many industrial uses for amylase. The novel strains
herein relieve the art from part of the burden of purifying such
products free of contaminating carbonyl hydrolases.
In a second principal embodiment, subtilisin and neutral protease
deletion-mutant strains are useful in the synthesis of protein
which is not otherwise encoded by the strain. These proteins will
fall within one of two classes. The first class consists of
proteins encoded by genes exhibiting no substantial
pretransformation homology with those of the host. These may be
proteins from other procaryotes but ordinarily are eucaryotic
proteins from yeast or higher eucaryotic organisms, particularly
mammals. The novel strains herein serve as useful hosts for
expressible vectors containing genes encoding such proteins because
the probability for proteolytic degradation of the expressed,
non-homologous proteins is reduced.
The second group consists of mutant host genes exhibiting
substantial pretransformation homology with those of the host.
These include mutations of procaryotic carbonyl hydrolases such as
subtilisin and neutral protease, as well as microbial (rennin, for
example rennin from the genus Mucor). These mutants are selected in
order to improve the characteristics of the precursor enzyme for
industrial uses.
A novel method is provided to facilitate the construction and
identification of such mutants. First, the gene encoding the
hydrolase is obtained and sequenced in whole or in part. Then the
sequence is scanned for a point at which it is desired to make a
mutation (deletion, insertion or substitution) of one or more amino
acids in the expressed enzyme. The sequences flanking this point
are evaluated for the presence of restriction sites for replacing a
short segment of the gene with an oligonucleotide pool which when
expressed will encode various mutants. Since unique restriction
sites are generally not present at locations within a convenient
distance from the selected point (from 10 to 15 nucleotides), such
sites are generated by substituting nucleotides in the gene in such
a fashion that neither the reading frame nor the amino acids
encoded are changed in the final construction. The task of locating
suitable flanking regions and evaluating the needed changes to
arrive at two unique restriction site sequences is made routine by
the redundancy of the genetic code, a restriction enzyme map of the
gene and the large number of different restriction enzymes. Note
that if a fortuitous flanking unique restriction site is available,
the above method need be used only in connection with the flanking
region which does not contain a site.
Mutation of the gene in order to change its sequence to conform to
the desired sequence is accomplished by M13 primer extension in
accord with generally known methods. Once the gene is cloned, it is
digested with the unique restriction enzymes and a plurality. of
end termini-complementary oligonucleotide cassettes are ligated
into the unique sites. The mutagenesis is enormously simplified by
this method because all of the oligonucleotides can be synthesized
so as to have the same restriction sites, and no synthetic linkers
are necessary to create the restriction sites.
The number of commercially available restriction enzymes having
sites not present in the gene of interest is generally large. A
suitable DNA sequence computer search program simplifies the task
of finding potential 5' and 3' unique flanking sites. A primary.
constraint is that any mutation introduced in creation of the
restriction site must be silent to the final constructed amino acid
coding sequence. For a candidate restriction site 5' to the target
codon a sequence must exist in the gene which contains at least all
the nucleotides but for one in the recognition sequence 5' to the
cut of the candidate enzyme. For example, the blunt cutting enzyme
SmaI (CCC/GGG) would be a 5' candidate if a nearby 5' sequence
contained NCC, CNC, or CCN. Furthermore, if N needed to be altered
to C this alteration must leave the amino acid coding sequence
intact. In cases where a permanent silent mutation is necessary to
introduce a restriction site one may want to avoid the introduction
of a rarely used codon. A similar situation for SmaI would apply
for 3' flanking sites except the sequence NGG, GNG, or GGN must
exist. The criteria for locating candidate enzymes is most relaxed
for blunt cutting enzymes and most stringent for 4 base overhang
enzymes. In general many candidate sites are available. For the
codon-222 target described herein a BalI site (TGG/CCA) could have
been engineered in one base pair 5' from the KpnI site. A 3'EcoRV
site (GAT/ATC) could have been employed 11 base pairs 5' to the
PstI site. A cassette having termini ranging from a blunt end up to
a four base-overhang will function without difficulty. In
retrospect, this hypothetical EcoRV site would have significantly
shortened the oligonucleotide cassette employed (9 and 13 base
pairs) thus allowing greater purity and lower pool bias problems.
Flanking sites should obviously be chosen which cannot themselves
ligate so that ligation of the oligonucleotide cassette can be
assured in a single orientation.
The mutation per se need not be predetermined. For example, an
oligonucleotide cassette or fragment is randomly mutagenized with
nitrosoguanidine or other mutagen and then in turn ligated into the
hydrolase gene at a predetermined location.
The mutant carbonyl hydrolases expressed upon transformation of the
suitable hosts are screened for enzymes exhibiting desired
characteristics, e.g. substrate specificity, oxidation stability,
pH-activity profiles and the like.
A change in substrate specificity is defined as a difference
between the Kcat/Km ratio of the precursor enzyme and that of the
mutant. The Kcat/Km ratio is a measure of catalytic efficiency.
Procaryotic carbonyl hydrolases with increased or diminished
Kcat/Km ratios are described in the examples. Generally, the
objective will be to secure a mutant having a greater (numerically
larger) Kcat/Km ratio for a given substrate, thereby enabling the
use of the enzyme to more efficiently act on a target substrate. An
increase in Kcat/Kcm ratio for one substrate may be is accompanied
by a reduction in Kcat/Km ratio for another substrate. This is a
shift in substrate specificity, and mutants exhibiting such shifts
have utility where the precursors are undesirable, e.g. to prevent
undesired hydrolysis of a particular substrate in an admixture of
substrates.
Kcat and Km are measured in accord with known procedures, or as
described in Example 18.
Oxidation stability is a further objective which is accomplished by
mutants described in the examples. The stability may be enhanced or
diminished as is desired for various uses. Enhanced stability is
effected by deleting one or more methionine, tryptophan, cysteine
or lysine residues and, optionally, substituting another amino acid
residue not one of methionine, tryptophan, cysteine or lysine. The
opposite substitutions result in diminished oxidation stability.
The substituted residue is preferably alanyl, but neutral residues
also are suitable.
Mutants are provided which exhibit modified pH-activity profiles. A
pH-activity profile is a plot of pH against enzyme activity and may
be constructed as illustrated in Example 19 or by methods known in
the art. It may be desired to obtain mutants with broader profiles,
i.e., those having greater activity at certain pH than the
precursor, but no significantly greater activity at any pH, or
mutants with sharper profiles, i.e. those having enhanced activity
when compared to the precursor at a given pH, and lesser activity
elsewhere.
The foregoing mutants preferably are made within the active site of
the enzymes as these mutations are most likely to influence
activity. However, mutants at other sites important for enzyme
stability or conformation are useful. In the case of Bacillus
subtilisin or its pre, prepro and pro forms, mutations at
tyrosine-1, aspartate+32, asparagine+155, tyrosine+104,
methionine+222, glycine+166, histidine+64, glycine+169,
phenylalanine+189, serine+33, serine+221, tyrosine+217,
glutamate+156 and/or alanine+152 produce mutants having changes in
the characteristics described above or in the processing of the
enzyme. Note that these amino acid position numbers are those
assigned to B. amyloliquefaciens subtilisin as seen from FIG. 7. It
should be understood that a deletion or insertion in the N-terminal
direction from a given position will shift the relative amino acid
positions so that a residue will not occupy its original or wild
type numerical position. Also, allelic differences and the
variation among various procaryotic species will result in
positions shifts, so that position 169 in such subtilisins will not
be occupied by glycine. In such cases the new positions for glycine
will be considered equivalent to and embraced within the
designation glycine+169. The new position for glycine+169 is
readily identified by scanning the subtilisin in question for a
region homologous to glycine+169 in FIG. 7.
One or more, ordinarily up to about 10, amino acid residues may be
mutated. However, there is no limit to the number of mutations that
are to be made aside from commercial practicality.
The enzymes herein may be obtained as salts. It is clear that the
ionization state of a protein will be dependent on the pH of the
surrounding medium, if it is in solution, or of the solution from
which it is prepared, if it is in solid form. Acidic proteins are
commonly prepared as, for example, the ammonium, sodium, or
potassium salts; basic proteins as the chlorides, sulfates, or
phosphates. Accordingly, the present application includes both
electrically neutral and salt forms of the designated carbonyl
hydrolases, and the term carbonyl hydrolase refers to the organic
structural backbone regardless of ionization state.
The mutants are particularly useful in the food processing and
cleaning arts. The carbonyl hydrolases, including mutants, are
produced by fermentation as described herein and recovered by
suitable techniques. See for example K. Anstrup, 1974, Industrial
Aspects of Biochemistry, ed. B. Spencer pp. 23-46. They are
formulated with detergents or other surfactants in accord with
methods known per se for use in industrial processes, especially
laundry. In the latter case the enzymes are combined with
detergents, builders, bleach and/or fluorescent whitening agents as
is known in the art for proteolytic enzymes. Suitable detergents
include linear alkyl benzene sulfonates, alkyl ethoxylated sulfate,
sulfated linear alcohol or ethoxylated linear alcohol. The
compositions may be formulated in granular or liquid form. See for
example U.S. Pat. Nos. 3,623,957; 4,404,128; 4,381,247; 4,404,115;
4,318,818; 4,261,868; 4,242,219; 4,142,999; 4,111,855; 4,011,169;
4,090,973; 3,985,686; 3,790,482; 3,749,671; 3,560,392; 3,558,498;
and 3,557,002.
The following disclosure is intended to serve as a representation
of embodiments herein, and should not be construed as limiting the
scope of this application.
Glossary of Experimental Manipulations
In order to simplify the Examples certain frequently occurring
methods will be referenced by shorthand phrases.
Plasmids are designated by a small p preceeded and/or followed by
capital letters and/or numbers. The starting plasmids herein are
commercially available, are available on an unrestricted basis, or
can be constructed from such available plasmids in accord with
published procedures.
"Klenow treatment" refers to the process of filling a recessed 3'
end of double stranded DNA with deoxyribonucleotides complementary
to the nucleotides making up the protruding 5' end of the DNA
strand. This process is usually used to fill in a recessed end
resulting from a restriction enzyme cleavage of DNA. This creates a
blunt or flush end, as may. be required for further ligations.
Treatment with Klenow is accomplished by reacting (generally for 15
minutes at 15.degree. C.) the appropriate complementary
deoxyribonucleotides with the DNA to be filled in under the
catalytic activity (usually 10 units) of the Klenow fragment of E.
coli DNA polymerase I ("Klenow"). Klenow and the other reagent
needed are commercially available. The procedure has been published
extensively. See for example T. Maniatis et al., 1982, Molecular
Cloning, pp. 107-108.
"Digestion" of DNA refers to catalytic cleavage of the DNA with an
enzyme that acts only at certain locations in the DNA. Such enzymes
are called restriction enzymes, and the sites for which each is
specific is called a restriction site. "Partial" digestion refers
to incomplete digestion by a restriction enzyme, i.e., conditions
are chosen that result in cleavage of some but not all of the sites
for a given restriction endonuclease in a DNA substrate. The
various restriction enzymes. used herein are commercially available
and their reaction conditions, cofactors and other requirements as
established by the enzyme suppliers were used. Restriction enzymes
commonly are designated by. abbreviations composed of a capital
letter followed by other letters and then, generally, a number
representing the microorganism from which each restriction enzyme
originally was obtained. In general, about 1 .mu.g of plasmid or
DNA fragment is used with about 1 unit of enzyme in about 20 .mu.l
of buffer solution. Appropriate buffers and substrate amounts for
particular restriction enzymes are specified by the manufacturer.
Incubation times of about 1 hour at 37.degree. C. are ordinarily
used, but may vary in accordance with the supplier's instructions.
After incubation, protein is removed by extraction with phenol and
chloroform, and the digested nucleic acid is recovered from the
aqueous fraction by precipitation with ethanol. Digestion with a
restriction enzyme infrequently is followed with bacterial alkaline
phosphatase hydrolysis of the terminal 5' phosphates to prevent the
two restriction cleaved ends of a DNA fragment from "circularizing"
or forming a closed loop that would impede insertion of another DNA
fragment at the restriction site. Unless otherwise stated,
digestion of plasmids is not followed by 5' terminal
dephosphorylation. Procedures and reagents for dephosphorylation
are conventional (T. Maniatis et al., Id., pp. 133-134).
"Recovery" or "isolation" of a given fragment of DNA from a
restriction digest means separation of the digest on 6 percent
polyacrylamide gel electrophoresis, identification of the fragment
of interest by molecular weight (using DNA fragments of known
molecular weight as markers), removal of the gel section containing
the desired fragment, and separation of the gel from DNA. This
procedure is known generally. For example, see R. Lawn et al.,
1981, "Nucleic Acids Res." 9:6103-6114, and D. Goeddel et al.,
(1980) "Nucleic Acids Res." 8:4057.
"Southern Analysis" is a method by which the presence of DNA
sequences in a digest or DNA-containing composition is confirmed by
hybridization to a known, labelled oligonucleotide or DNA fragment.
For the purposes herein, Southern analysis shall means separation
of digests on 1 percent agarose and depurination as described by G.
Wahl et al., 1979, "Proc. Nat. Acad. Sci. U.S.A." 76:3683-3687,
transfer to nitrocellulose by the method of E. Southern, 1975, "J.
Mol. Biol." 98:503-517, and hybridization as described by T.
Maniatis et al., 1978, "Cell" 15:687-701.
"Transformation" means introducing DNA into an organism so that the
DNA is replicable, either as an extrachromosomal element or
chromosomal integrant. Unless otherwise stated, the method used
herein for transformation of E. coli is the CaCl.sub.2 method of
Mandel et al., 1970, "J. Mol. Biol." 53:154, and for Bacillus, the
method of Anagnostopoulos et al., 1961, "J. Bact." 81:791-746.
"Ligation" refers to the process of forming phosphodiester bonds
between two double stranded nucleic acid fragments (T. Maniatis et
al., Id., p. 146). Unless otherwise stated, ligation was
accomplished using known buffers and conditions with 10 units of T4
DNA ligase ("ligase") per 0.5 .mu.g of approximately equimolar
amounts of the DNA fragments to be ligated. Plasmids from the
transformants were prepared, analyzed by restriction mapping and/or
sequenced by the method of Messing, et al., 1981, "Nucleic Acids
Res.", 9:309.
"Preparation" of DNA from transformants means isolating plasmid DNA
from microbial culture. Unless otherwise stated, the alkaline/SDS
method of Maniatis et al., Id. p. 90, was used.
"Oligonucleotides" are short length single or double stranded
polydeoxynucleotides which were chemically synthesized by the
method of Crea et al., 1980, "Nucleic Acids Res." 8:2331-2348
(except that mesitylene nitrotriazole was used as a condensing
agent) and then purified on polyacrylamide gels.
All literature citations are expressly incorporated by
reference.
EXAMPLE 1
Preparation of a Genomic DNA Library from B. amyloliquifaciens and
Isolation of its Subtilisin Gene
The known amino acid sequence of the extracellular B.
amyloliquefaciens permits the construction of a suitable probe
mixture. The sequence of the mature subtilisin is included (along
with the additional information contributed by the present work) in
FIG. 1. All codon ambiguity for the sequence of amino acids at
position 117 through 121 is covered by a pool of eight
oligonucleotides of the sequence ##STR2##
Chromosomal DNA isolated from B. amyloliquefaciens (ATCC No. 23844)
as described by J. Marmur, "J. Mol, Biol.", 3:208, was partially
digested by Sau 3A, and the fragments size selected and ligated
into the BamH1 site of dephosphorylated pBS42. (pBS42 is shuttle
vector containing origins of replication effective both in E. coli
and Bacillus. It is prepared as described in Example 4). The Sau3A
fragment containing vectors were transformed into E. coli K12
strain 294 (ATCC No. 31446) according to the method of M. Mandel,
et al., 1970, "J. Mol. Bio." 53:154 using 80-400 nanograms of
library DNA per 250 .mu.L of competent cells.
Cells from the transformation mixture were plated at a density of
1-5.times.10.sup.3 transformants per 15 Omm plate containing LB
medium+12.5 .mu.g/ml chloramphenicol, and grown overnight at
37.degree. C. until visible colonies appeared. The plates were then
replica plated onto BA85 nitrocellulose filters overlayed on
LB/chloramphenicol plates. The replica plates were grown 10-12
hours at 37.degree. C. and the filters transferred to fresh plates
containing LB and 150 .mu.g/ml spectinomycin to amplify the plasmid
pool.
After overnight incubation at 37.degree. C., filters were processed
essentially as described by Grunstein and Hogness, 1975, "Proc.
Natl. Acad. Sci. (USA)" 72: 3961. Out of approximately 20,000
successful transformants, 25 positive colonies were found. Eight of
these positives were streaked to purify individual clones. 24
clones from each streak were grown in microtiter wells, stamped on
to two replica filters, and probed as described above with either
##STR3## which differ by only one nucleotide. As shown in FIG. 2,
pool 1 hybridized to a much greater extent to all positive clones
than did pool 2, suggesting specific hybridization.
Four out of five miniplasmid preparations (Maniatis et al., Id.)
from positive clones gave identical restriction digest patterns
when digested with Sau3A or HincII. The plasmid isolated from one
of these four identical colonies by the method of Maniatis et al.,
Id. had the entire correct gene sequence and was designated pS4.
The characteristics of this plasmid as determined by restriction
analysis are shown in FIG. 3.
EXAMPLE 2
Expression of the Subtilisin Gene
Bacillus subtilis I-168 (Catalog No. 1-A1, Bacillus Genetic Stock
Center) was transformed with pS4 and and a single chloramphenicol
resistant transformant then grown in minimal medium. After 24
hours, the culture was centrifuged and both the supernatant (10-200
.mu.l) and pellet assayed for proteolytic activity by measuring the
change in absorbance per minute at 412 nm using 1 ml of the
chromogenic substrate succinyl-L-ala-ala-pro-phe-p-nitroanilide
(0.2 .mu.M) in 0.1M sodium phosphate (pH 8.0) at 25.degree. C. A B.
subtilis I-168 culture transformed with pBS42 used as a control
showed less than 1/200 of the activity shown by the pS4 transformed
culture. Greater than 95 percent of the protease activity of the
pS4 culture was present in the supernatant, and was completely
inhibited by treatment with phenylmethylsulfonyl fluoride (PMSF)
but not by EDTA.
Aliquots of the supernatants were treated with PMSF and EDTA to
inhibit all protease activity and analyzed by 12 percent SDS-PAGE
according to the method of Laemmli, U.K., 1970 "Nature", 227: 680.
To prepare the supernatants, 16 .mu.L of supernatant was treated
with 1mM PMSF, 10 mM EDTA for 10 minutes, and boiled with 4 .mu.L
of 5x concentrated SDS sample buffer minus .beta.-mercaptoethanol.
The results of Coomassie stain on runs using supernatants of cells
transformed with pS4, pBS42, and untransformed B. amyloliquefaciens
are shown in FIG. 4. Lane 3 shows authentic subtilisin from B.
amyloliquefaciens. Lane 2 which is the supernatant from pBS42
transformed B. subtilis, does not give the 31,000 MW band
associated with subtilisin which is exhibited by Lane 1 from pS4
transformed hosts. The approximately 31,000 MW band result from
subtilisin is characteristic of the slower mobility shown by the
known M.W. 27,500 subtilisin preparations in general.
EXAMPLE 3
Sequencing of the B. amyloliquefaciens Subtilisin Gene
The entire sequence of an EcoRI-BamHI fragment (wherein the EcoRI
site was constructed by conversion of the HincII site) of pS4 was
determined by the method of F. Sanger, 1977, "Proc. Natl. Acad. Sci
(USA)", 74:5463. Referring to the restriction map shown in FIG. 3,
the BamHI-PvuII fragment was found to hybridize with pool 1
oligonucleotides by Southern analysis. Data obtained from
sequencing of this fragment directed the sequencing of the
remaining fragments (e.g. PvuII-HincII and AvaI-AvaI). The results
are shown in FIG. 1.
Examination of the sequence confirms the presence of codons for the
mature subtilisin corresponding to that secreted by the B.
amyloliquefaciens. Immediately upstream from this sequence is a
series of 107 codons beginning with the GTG start codon at -107.
Codon -107 to approximately codon -75 encodes an amino acid
sequence whose characteristics correspond to that of known signal
sequences. (Most such signal sequences are 18-30 amino acids in
length, have hydrophobic cores, and terminate in a small
hydrophobic amino acid.) Accordingly, examination of the sequence
data would indicate that codons -107 to approximately -75 encode
the signal sequence; the remaining intervening codons between -75
and -1 presumably encode a prosequence.
EXAMPLE 4
Construction of pBS42
pBS42 is formed by three-way ligation of fragments derived from
pUB11, pC194, and pBR322 (see FIG. 5). The fragment from pUB110 is
the approximately 2600 base pair fragment between the HpaII site at
1900 and the BamH1 site at 4500 and contains an origin of
replication operable in Bacillus: T. Grycztan, et al., 1978 "J.
Bacteriol.", 134: 318 (1978); A. Jalanko, et al., 1981 "Gene", 14:
325. The BamHI site was tested with Klenow. The pBR322 portion is
the 1100 base pair fragment between the PvuII site at 2067 and the
Sau3A site at 3223 which contains the E. coli origin of
replication: F. Bolivar, et al., 1977 "Gene", 2: 95;-J Sutcliffe,
1978, Cold Spring Harbor Symposium 43: I, 77. The pC194 fragment is
the 1200 base pair fragment between the HpaII site at 973 and the
Sau3A site at 2006 which contains the gene for chloramphenicol
resistance expressible in both E. coli and B subtilis; S. Ehrlich,
"Proc. Natl. Acad. Sci. (USA)", 74:1680; S. Horynuchi et al., 1982,
"J. Bacteriol." 150: 815.
pBS42 thus contains origins of replication operable both in E. coli
and in Bacillus and an expressible gene for chloramphenicol
resistance.
EXAMPLE 5
Isolation and Sequencing of the B. subtilis Subtilisin Gene
B. subtilis 1168 chromosomal DNA was digested with EcoRI and the
fragments resolved on gel electrophoresis. A single 6 kb fragment
hybridized to a [.alpha.-.sup.32 P] CTP nick translation - labeled
fragment obtained from the C-terminus of the subtilisin structural
gene in pS4, described above. The 6 kb fragment was electroluted
and ligated into pBS42 which had been digested and EcoRI and
treated with bacterial alkaline phosphatase. E. coli ATCC 31446 was
transformed with the ligation mixture and transformants selected by
growth on LB agar containing 12.5 .mu.g chloramphenicol/ml. Plasmid
DNA was prepared from a pooled suspension of 5,000 transformed
colonies. This DNA was transformed into B. subtilis BG84, a
protease deficient strain, the preparation of which is described in
Example 8 below. Colonies which produced protease were screened by.
plating on LB agar plus 1.5 percent w/w Carnation powdered nonfat
skim milk and 5 .mu.g chloramphenicol/ml (hereafter termed skim
milk selection plates) and observing for zones of clearance
evidencing proteolytic activity.
Plasmid DNA was prepared from protease producing colonies, digested
with EcoRI, and examined by Southern analysis for the presence of
the 6 kb EcoRI insert by hybridization to the .sup.32 P-labelled
C-terminus fragment of the subtilisin structural gene from B.
amyloliquefaciens. A positive clone was identified and the plasmid
was designated pS168.1. B. subtilis BG84 transformed with pS168.1
excreted serine protease at a level 5-fold over that produced in B.
subtilis 1168. Addition of EDTA to the supernatants did not affect
the assay results, but the addition of PMSF (phenylmethylsulfonyl
fluoride) to the supernatants reduced protease activity to levels
undetectable in the assay described in Example 8 for strain
BG84.
A restriction map of the 6.5 kb EcoRI insert is shown in FIG. 6.
The subtilisin gene was localized to within the 2.5 kb KpnI-EcoRI
fragment by subcloning various restriction enzyme digests and
testing for expression of subtilisin in B. subtilis BG84. Southern
analysis with the labelled fragment from the C-terminus of the B.
amyloliquefaciens subtilisin gene as a probe localized the
C-terminus of the B. subtilis gene to within or part of the 631 by
HincII fragment B in the center of this subclone (see FIG. 6). The
tandem HincII fragments B, C, and D and HincII-EcoRI fragment E
(FIG. 6) were ligated into the M13 vectors mp8 or mp9 and sequenced
in known fashion (J. Messing et al., 1982, "Gene" 19:209-276) using
dideoxy chain termination (F. Sanger et al., 1977, "Proc. Nat.
Acad. Sci. U.S.A." 74:5463-5467). The sequence of this region is
shown in FIG. 7. The first 23 amino acids are believed to be a
signal peptide. The remaining 83 amino acids between the signal
sequence and the mature coding sequence constitute the putative
"pro" sequence. The overlined nucleotides at the 3' end of the gene
are believed to be transcription terminator regions. Two possible
Shine-Dalgarno sequences are underlined upstream from the mature
start codon.
EXAMPLE 6
Manufacture of an Inactivating Mutation of the B. subtilis
Subtilisin Gene
A two step ligation, shown in FIG. 8, was required to construct a
plasmid carrying a defective gene which would integrate into the
Bacillus chromosome. In the first step, pS168.1, which contained
the 6.5 kb insert originally recovered from the B. subtilis genomic
library as described in Example 5 above, was digested with EcoRI,
the reaction products treated with Klenow, the DNA digested with
HincII, and the 800 bp EcoRI-HincII fragment E (see FIG. 6) that
contains, in part, the 5' end of the B. subtilis subtilisin gene,
was recovered. This fragment was ligated into pJH101(pJH101is
available from J. Hoch (Scripps) and is described by F. A. Ferrari
et al., 1983, "J. Bact." 134:318-329) that had been digested with
HincII and treated with bacterial alkaline phosphotase. The
resultant plasmid, pIDV1, contained fragment E in the orientation
shown in FIG. 8. In the second step, pS168.1 was digested with
HincII and the 700 bp HincII fragment B, which contains the 3' end
of the subtilisin gene, was recovered. pIDV1 was digested at its
unique HincII site and fragment B ligated to the linearized
plasmid, transformed in E. coli ATCC 31,446, and selected on LB
plates containing 12.5 .mu.g chloramphenicol/ml or 20 .mu.g
ampicillin/ml. One resulting plasmid, designated pIDV1.4, contained
fragment B in the correct orientation with respect to fragment E.
This plasmid pIDV1.4, shown in FIG. 8, is a deletion derivative of
the subtilisin gene containing portions of the 5' and 3' flanking
sequences as well.
B. subtilis BG77, a partial protease-deficient mutant (Prt.sup.+/-)
prepared in Example 8 below was transformed with pIDV1.4. Two
classes of chloramphenicol resistant (Cm.sup.r) transformants were
obtained. Seventy-five percent showed the same level of proteases
as BG77 (Prt.sup.+/-) and 25 percent were almost completely
protease deficient (Prt.sup.-) as observed by relative zones of
clearing on plates containing LB agar plus skim milk. The Cm.sup.r
Prt.sup.- transformants could not be due to a single crossover
integration of the plasmid at the homologous regions for fragment E
or B because, in such a case, the gene would be uninterrupted and
the phenotype would be Prt.sup.+/-. In fact, when either of
fragments E or B were ligated independently into pJH101and
subsequently transformed into B. subtilis BG77, the protease
deficient phenotype was not observed. The Cm.sup.r phenotype of
Cm.sup.r Prt.sup.- pIDV1.4 transformants was unstable in that
Cm.sup.s Prt- derivatives could be isolated from Cm.sup.r Prt.sup.
- cultures at a frequency of about 0.1 percent after 10 generations
of growth in minimal medium in the absence of antibiotic selection.
One such derivative was obtained and designated BG2018. The
deletion was transferred into IA84 (a BGSC strain carrying two
auxotrophic mutations flanking the subtilisin gene) by PSB1
transduction. The derivative organism was designated BG2019.
EXAMPLE 7
Preparation of a Genomic DNA Library from B. subtilis and Isolation
of its Neural Protease Gene
The partial amino acid sequence of a neutral protease of B.
subtilis is disclosed by P. Levy et al. 1975, "Proc. Nat. Acad.
Sci. USA" 72:4341-4345. A region of the enzyme (Asp Gln Met Ile Tyr
Gly) was selected from this published sequence in which the least
redundancy existed in the potential codons for the amino acids in
the region. 24 combinations were necessary to cover all the
potential coding sequences, as described below. ##STR4##
Four pools, each containing six alternatives, were prepared as
described above in Example 1. The pools were labelled by
phosphorylization with [.gamma.-.sup.32 p] ATP.
The labelled pool containing sequences conforming closest to a
unique sequence in a B. subtilis genome was selected by digesting
B. subtilis (1A72, Bacillus Genetic Stock Center) DNA with various
restriction enzymes, separating the digests on an electrophoresis
gel, and hybridizing each of the four probe pools to each of the
blotted digests under increasingly stringent conditions until a
single band was seen to nybridize. Increasingly stringent
conditions are those which tend to disfavor hybridization, e.g.,
increases in formamide concentration, decreases in salt
concentration and increases in temperature. At 37.degree. C. in a
solution of 5.times. Denhardt's, 5.times. SSC, 50 mM NaPO.sub.4 pH
6.8 and 20 percent formamide, only pool 4 would hybridize to a
blotted digest. These were selected as the proper hybridization
conditions to be used for the neutral protease gene and pool 4 was
used as the probe.
A lambda library of B. subtilis strain BGSC 1-A72 was prepared in
conventional fashion by partial digestion of the Bacillus genomic
DNA by Sau3A, separated of the partial digest by molecular weight
on an electrophoresis gel, elution of 15-20 kb fragments (R. Lawn
et al., 1981, "Nucleic Acids Res." 9:6103-6114), and ligation of
the fragments to BamHI digested charon 30 phage using a Packagene
kit from Promega Biotec.
E. coli DP50supF was used as the host for the phage library,
although any known host for Charon lambda phage is satisfactory.
The E. coli was plated with the library phage and cultured, after
which plaques were assayed for the presence of the neutral protease
gene by transfer to nitrocellulose and screening with probe pool 4
(Benton and Davis, 1977, "Science" 196:180-182). Positive plaques
were purified through two rounds of single plaque purification, and
two plaques were chosen for further study, designated .gamma.NPRG1
and .gamma.NPRG2. DNA was prepared from each phage by restriction
enzyme hydrolysis and separation on electrophoresis gels. The
separated fragments were blotted and hybridized to labelled pool 4
oligonucleotides. This disclosed that .gamma.NPRG1 contained a 2400
bp HindIII hybridizing fragment, but no 4300 EcoRI fragment, while
.gamma.NPRG2 contained a 4300 bp EcoRI fragment, but no 2400 by
HindIII fragment.
The 2400 bp .gamma.NPRG1 fragment was subcloned into the HindIII
site of pJH101by the following method. .gamma.NPRG1 was digested by
HindIII, the digest fractionated by electrophoresis and the 2400 by
fragment recovered from the gel. The fragment was ligated to
alkaline phosphatase-treated HindIII digested pJH101and the
ligation mixture used to transform E. coli ATCC 31446 by the
calcium chloride shock method of V. Herschfield et al., 1974,
"Proc. Nat. Acad. Sci. (U.S.A.)" 79:3455-3459). Transformants were
identified by selecting colonies capable of growth on plates
containing LB medium plus 12.5 .mu.g chloramphenicol/ml.
Transformants colonies yielded several plasmids. The orientation of
the 2400 bp fragment in each plasmid was determined by conventional
restriction analysis (orientation is the sense reading or
transcriptional direction of the gene fragment in relation to the
reading direction of the expression vector into which it is
ligated.) Two plasmids with opposite orientations were obtained and
designated pNPRsubH6 and pNPRsubH1.
The 4300 bp EcoRI fragment of .gamma.NPRG2 was subcloned into
pBR325 by the method described above for the 2400 bp fragment
except that .gamma.NPRG2 was digested with EcoRI and the plasmid
was alkaline phosphatase-treated, EcoRI-digested pBR325. pBR325 is
described by. F. Bolivar, 1978, "Gene" 4:121-136. Two plasmids were
identified in which the 4300 bp insert was present in different
orientations. These two plasmids were designated pNPRsubRI and
pNPRsubRIb.
EXAMPLE 8
Characterization of B. subtilis Neutral Protease Gene
The pNPRsubH1 insert was sequentially digested with different
restriction endonucleases and blot hybridized with labelled pool 4
in order to prepare a restriction map of the insert (for general
procedures of restriction mapping see T. Maniatis et al., Id., p.
377). A 430 bp RsaI fragment was the smallest fragment that
hybridized to probe pool 4. The RsaI fragment was ligated into the
SmaI site of M13 mp8 (J. Messing et al., 1982, "Gene" 19:269-276
and J. Messing in Methods in Enzymology, 1983, R. Wu et al., Eds.,
101:20-78) and the sequence determined by the chain-terminating
dideoxy method (F. Sanger et al., 1977, "Proc. Nat. Acad. Sci.
U.S.A." 74:5463-5467). Other restriction fragments from the
pNPRsubH1 insert were ligated into appropriate sites in M13 mp8 or
M13 mp9 vectors and the sequences determined. As required, dITP was
used to reduce compression artifacts (D. Mills et al., 1979, "Proc.
Nat. Acad. Sci. (U.S.A.)" 76:2232-2235). The restriction map for
the pNPRsubH1 fragment is shown in FIG. 9. The sequences of the
various fragments from restriction enzyme digests were compared and
an open reading frame spanning a codon sequence translatable into
the amino and carboxyl termini of the protease (P. Levy et al.,
Id.) was determined. An open reading frame is a DNA sequence
commencing at a known point which in reading frame (every three
nucleotides) does not contain any internal termination codons. The
open reading frame extended past the amino terminus to the end of
the 2400 by HindIII fragment. The 1300 bp BgIII - HindIII fragment
was prepared from pNPRsubRIb (which contained the 4300 bp EcoRI
fragment of .gamma.NPRG2) and cloned in M13 mp8. The sequence of
this fragment, which contained the portion of the neutral protease
leader region not encoded by the 2400 by fragment of pNPRsubH1, was
determined for 400 nucleotides upstream from the HindIII site.
The entire nucleotide sequence as determined for this neutral
protease gene, including the putative secretory leader and prepro
sequence, are shown in FIG. 10. The numbers above the line refer to
amino acid positions. The underlined nucleotides in FIG. 10 are
believed to constitute the ribosome binding (Shine-Dalgarno) site,
while the overlined nucleotides constitute a potential hairpin
structure presumed to be a terminator. The first 27-28 of the
deduced amino acids are believed to be the signal for the neutral
protease, with a cleavage point at ala-27 or ala-28. The "pro"
sequence of a proenzyme structure extends to the amino-terminal
amino acid (ala-222) of the mature, active enzyme.
A high copy plasmid carrying the entire neutral protease gene was
constructed by (FIG. 11) ligating the BG1II fragment of pNPRsubR1,
which contains 1900 bp (FIG. 9), with the PvuII - HindIII fragment
of pNPRsubH1, which contains 1400 bp. pBS42 (from Example 4 was
digested with BamHI and treated with bacterial alkaline phosphatase
to prevent plasmid recircularization. pNPRsubR1 was digested to
Bg1II, the 1900 bp fragment was isolated from gel electrophoresis
and ligated to the open BamHI sites of pBS42. The ligated plasmid
was used to transform E. coli ATCC 31446 by the calcium chloride
shock method (V. Hershfield et al., Id.), and transformed cells
selected by growth on plates containing LB medium with 12.5
.mu.g/ml chloramphenicol. A plasmid having the Bg1 II fragment in
the orientation shown in FIG. 11 was isolated from the
transformants and designated pNPRsubB1. pNPRsubB1 was digested
(linearized) with EcoRI, repaired to flush ends by Kienow treatment
and then digested with HindIII. The larger fragment from the
HindIII digestion (containing the sequence coding for the amino
terminal and upstream regions) was recovered.
The carboxyl terminal region of the gene was supplied by a fragment
from pNPRsubH1, obtained by digestion of pNPRsubH1 with PvuII and
HindIII and recovery of the 1400 bp fragment. The flush end PvuII
and the HindIII site of the 1400 bp fragment was ligated,
respectively, to the blunted EcoRI and the HindIII site of
pNPRsubB1, as shown in FIG. 11. This construct was used to
transform B. subtilis strain BG84 which otherwise excreted no
proteolytic activity by the assays described below. Transformants
were selected on plates containing LB medium plus 1.5 percent
carnation powdered nonfat milk and 5 .mu.g/ml chloramphenicol.
Plasmids from colonies that cleared a large halo were analyzed.
Plasmid pNPR10, incorporating the structural gene and flanking
regions of the neutral protease gene, was determined by restriction
analysis to have the structure shown in FIG. 11.
B. subtilis strain BG84 was produced by
N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis of B.
subtilis I168 according to the general technique of Adelberg et
al., 1965, "Biochem. Biophys. Res. Commun." 18:788-795. Mutagenized
strain I168 was plated on skim milk plates (without antibiotic).
Colonies producing a smaller halo were picked for further analysis.
Each colony was characterized for protease production on skim milk
plates and amylase production on starch plates. One such isolate,
which was partially protease deficient, amylase positive and
capable of sporulation, was designated BG77. The protease
deficiency mutation was designated prt-77. The prt-77 allele was
moved to a spoOA background by congression as described below to
produce strain BG84, a sporulation deficient strain.
TABLE A ______________________________________ Strain Relevant
Genotype origin ______________________________________ I168 trpC2
JH703 trpC2, pheA12, spoOA.DELTA.677 Trousdale et al..sup.a BG16
purB6, metB5, leuA8, lys-21, Pb 1665 hisA, thr-5 sacA321 BG77
trpC2, prt-77 NTG .times. I168 BG81 metB5, prt-77 BG16 DNA .times.
BG77 BG84 spoO.DELTA.677, prt-77 JH703 DNA .times. BG81
______________________________________ .sup.a "Mol. Gen. Genetics"
173:61 (1979)
BG84 was completely devoid of protease activity on skim milk plates
and does not produce detectable levels of either subtilisin or
neutral protease when assayed by measuring the change in absorbance
at 412 nm per minute upon incubation with 0.2 .mu.m/ml succinyl
(-L-ala-L-ala-L-pro-L-phe) p-nitroanilide (Vega) in 0.1M sodium
phosphate, pH 8, at 25.degree. C. BG84 was deposited in the ATCC as
deposit number 39382 on July 21, 1983. Samples for subtilisin assay
were taken from late logarithmic growth phase supernatants of
cultures grown in modified Schaeffer's medium (T. Leighton et al.,
1971, "J. Biol. Chem." 246:3189-3195).
EXAMPLE 9
Expression of the Neutral Protease Gene
BG84 transformed with pNPR10was inoculated into minimal medial
supplemented with 0.1 percent casein hydrolysate and 10 .mu.g
chloramphenicol and cultured for 16 hours. 0.1 ml of culture
supernatant was removed and added to a suspension of 1.4 mg/ml
Azocoll proteolytic substrate (Sigma) in 10 mM Tris-HCl, 100 mM
NaCl pH 6.8 and incubating with agitation. Undigested substrate was
removed by centrifugation and the optical density read at 505 nm.
Background values of an Azocoll substrate suspension were
subtracted. The amount of protease excreted by a standard
protease-expressing strain, BG16 was used to establish an arbitrary
level of 100. The results with BG16, and with BG84 transformed with
control and neutral protease gene-containing plasmids are shown in
Table B in Example 12 below. Transformation of the excreted
protease-devoid B. subtilis strain BG84 results in excretion of
protease activity at considerably greater levels than in BG16, the
wild-type strain.
EXAMPLE 10
Manufacture of an Inactivating Mutation of the Neutral Protease
Gene
The two RsaI bonded regions in the 2400 bp insert of pNPRsubH1,
totalling 527 bp, can be deleted in order to produce an incomplete
structural gene. The translational products of this gene are
enzymatically inactive. A plasmid having this deletion was
constructed as follows. pJH101 was cleaved by digestion with
HindIII and treated with bacterial alkaline phosphatase. The
fragments of the neutral protease gene to be incorporated into
linearized pJH101 were obtained by digesting pNPRsubH1 with HindIII
and RsaI, and recovering the 1200 bp HindIII-RsaI and 680 bp
RsaI-HindIII fragments by gel electrophoresis. These fragments were
ligated into linearized pJH101 and used to transform E. coli ATCC
31446. Transformants were selected on plates containing LB medium
and 20 .mu.g ampicillin/ml. Plasmids were recovered from the
transformants and assayed by restriction enzyme analysis to
identify a plasmid having the two fragments in the same orientation
as in the pNPRsubH1 starting plasmid. The plasmid lacking the
internal RsaI fragments was designated pNPRsubH1.DELTA..
EXAMPLE 11
Replacement of the Neutral Protease Gene with a Deletion Mutant
Plasmid pNPRsubh1.DELTA. was transformed into B. subtilis strain
BG2019 (the subtilisin deleted mutant from Example 6) and
chromosomal integrants were selected on skim milk plates. Two types
of Cm.sup.r transformants were noted, those with parental levels of
proteolysis surrounding the colony, and those with almost no zone
of proteolysis. Those lacking a zone of proteolysis were picked,
restreaked to purify individual colonies, and their protease
deficient character on skim milk plates confirmed. One of the
Cm.sup.r, proteolysis deficient colonies was chosen for further
studies (designated BG22034). Spontaneous Cm.sup.s revertants of
BG2034 were isolated by overnight growth in LB media containing no
Cm, plating for individual colonies, and replica plating on media
with and without Cm. Three Cm.sup.s revertants were isolated, two
of which were protease proficient, one of which was protease
deficient (designated BG2036). Hybridization analysis of BG2036
confirmed that the plasmid had been lost from this strain, probably
by recombination, leaving only the deletion fragments of subtilisin
and neutral protease.
EXAMPLE 12
Phenotype of Strains Lacing Functional Subtilisin and Neutral
Protease
The growth, sporulation and expression of proteases was examined in
strains lacking a functional gene for either the neutral or
alkaline protease or both. The expression of proteases was examined
by a zone of clearing surrounding a colony on a skim milk plate and
by measurement of the protease levels in liquid culture
supernatants (Table B). A strain (BG2035) carrying the subtilisin
gene deletion, and showed a 30 percent reduction level of protease
activity and a normal halo on milk plates. Strain BG2043, carrying
the deleted neutral protease gene and active subtilisin gene, and
constructed by transforming BG16 (Ex. 8). with DNA from BG2036
(Example 11), showed an 80 percent reduction in protease activity
and only a small halo on the milk plate.
TABLE B ______________________________________ Effect of protease
deletions on protease expression and sporulation. Protease Percent
Genotype.sup.a activity.sup.b Sporulation
______________________________________ BG16 Wild type 100 40 BG2035
apr.DELTA.684 70 20 BG2043 aprE.DELTA.522 20 20 BG2054
apr.DELTA.684, nprE.DELTA.522 ND 45 BG84(pBS42) spoOA.DELTA.677,
prt-77 ND -- BG84(pNPR10) spoOA.DELTA.677, prt-77 3000 --
______________________________________ .sup.a Only the loci
relevant to the protease phenotype are shown. .sup.b Protease
activity is expressed in arbitrary units, BG16 was assigned a level
of 100. ND indicates the level of protease was not detectable in
the assay used.
Strain BG2054, considered equivalent to BG2036 (Example 11) in that
it carried the foregoing deletions in both genes, showed no
detectable protease activity in this assay and no detectable halo
on milk plates. The deletion of either or both of the protease
genes had no apparent effect on either growth or sporulation.
Strains carrying these deletions had normal growth rates on both
minimal glucose and LB media. The strains sporulated at frequencies
comparable to the parent strain BG16. Examination of morphology of
these strains showed no apparent differences from strains without
such deletions.
EXAMPLE 13
Site-specific Saturation Mutagenesis of the B. Amyloliquefaciens
Subtilisin Gene at Position 222; Preparation of the Gene for
Cassette Insertion
pS4-5, a derivative of pS4 made according to Wells et al., "Nucleic
Acids Res.", 1983, 11:7911-7924 was digested with EcoRI and BamHI,
and the 1.5 kb EcoRIBamHI fragment recovered. This fragment was
ligated into replicative form M-13 mp9 which had been digested with
EcoRI and BamHI (Sanger et al., 1980, "J. Mol. Biol." 143 161-178.
Messing et al, 1981, "Nucleic Acids Research", 9, 304-321. Messing,
J. and Vieira, J. (1982) Gene 19, 269-276). The M-13 mp9 phage
ligations, designated M-13 mp9 SUBT, were used to transform E. coli
strain JM101 and single stranded phage DNA was prepared from a two
mL overnight culture. An oligonucleotide primer was synthesized
having the sequence 5'-GTACAACGGTACCTCACGCACGCTGCAGGAGCGGCTGC-3'.
This primer conforms to the sequence of the subtilis gene fragment
encoding amino acids 216-232 except that the 10 bp of codons for
amino acids 222-225were deleted, and the codons for amino acids
220, 227 and 228 were mutated to introduce a KpnI site 5' to the
met-222 codon and a PstI site 3' to the met.times.222 codon. See
FIG. 12. Substituted nucleotides are denoted by asterisks, the
underlined codons in line 2 represent the new restriction sites and
the scored sequence in line 4 represents the inserted
oligonucleotides. The primer (about 15 .mu.M) was labelled with
[.sup.32 p] by incubation with [.gamma..sup.32 p]-ATP (10 .mu.L in
20 .mu.L reaction)(Amersham 5000 Ci/mmol, 10218) and T.sub.4
polynucleotide kinase (10 units) followed by non-radioactive ATP
(100 .mu.M) to allow complete phosphorylation of the mutagenesis
primer. The kinase was inactivated by heating the phosphorylation
mixture at 68.degree. C. for 15 min.
The primer was hybridized to M-13 mp9 SUBT as modified from Norris
et al., 1983, "Nucleic Acids Res." 11, 5103-5112 by combining 5
.mu.L of the labelled mutagenesis primer (.about.3 .mu.M), .about.1
.mu.g M-13 mp9 SUBT template, 1 .mu.L of 1 .mu.M M-13 sequencing
primer (17-mer), and 2.5 .mu.L of buffer (0.3M Tris pH 8, 40 mM
MgCl.sub.2, 12 mM EDTA, 10 mM DTT, 0.5 mg/ml BSA). The mixture was
heated to 68.degree. C. for 10 minutes and cooled 10 minutes at
room temperature. To the annealing mixture was added 3.6 .mu.L of
0.25 mM dGTP, dCTP, dATP, and dTTP, 1.25 .mu.L of 10 mM ATP, 1
.mu.L ligase (4 units) and 1 .mu.L Klenow (5 units). The primer
extension and ligation reaction (total volume 25 .mu.l) proceeded 2
hours at 14.degree. C. The Klenow and ligase were inactivated by
heating to 68.degree. C. for 20 min. The heated reaction mixture
was digested with BamH1 and EcoRI and an aliquot of the digest was
applied to a 6 percent polyacrylamide gel and radioactive fragments
were visualized by autoradiography. This showed the [.sup.32 p]
mutagenesis primer had indeed been incorporated into the EcoRIBamH1
fragment containing the now mutated subtilisin gene.
The remainder of the digested reaction mixture was diluted to 200
.mu.L with 10 mM Tris, pH 8, containing 1 mM EDTA, extracted once
with a 1:1 (v:v), phenol/chloroform mixture, then once with
chloroform, and the aqueous phase recovered. 15 .mu.L of 5M
ammonium acetate (pH 8) was added along with two volumes of ethanol
to precipitate the DNA from the aqueous phase. The DNA was pelleted
by centrifugation for five minutes in a microfuge and the
supernatant was discarded. 300 .mu.L of 70 percent ethanol was
added to wash the DNA pellet, the wash was discarded and the pellet
lyophilized.
pBS42 from example 4 above was digested with BamH1 and EcoRI and
purified on an acrylamide gel to recover the vector. 0.5.mu.g of
the digested vector, 50 .mu.M ATP and 6 units ligase were dissolved
in 20 .mu.l of ligation buffer. The ligation went overnight at
14.degree. C. The DNA was transformed into E. coli 294 rec.sup.+
and the transformants grown in 4 ml of LB medium containing 12.5
.mu.g/ml chloramphenicol. Plasmid DNA was prepared from this
culture and digested with KpnI, EcoRI and BamHI. Analysis of the
restriction fragments showed 30-50 percent of the molecules
contained the expected KpnI site programmed by the mutagenesis
primer. It was hypothesized that the plasmid population not
including the KpnI site resulted from M-13 replication before
bacterial repair of the mutagenesis site, thus producing a
heterogenous population of KpnI.sup.+ and KnpI.sup.- plasmids in
some of the transformants. In order to obtain a pure culture of the
KpnI.sup.+ B plasmid, the DNA was transformed a second time into E.
coli to clone plasmids containing the new KpnI site. DNA was
prepared from 16 such transformants and six were found to contain
the expected KpnI site.
Preparative amounts of DNA were made from one of these six
transformants (designated p.DELTA.222) and restriction analysis
confirmed the presence and location of the expected KpnI and PstI
sites. 40 .mu.g of p.DELTA.222 were digested in 300 .mu.L of KpnI
buffer plus 30 .mu.L KpnI (300 units) for 1.5 h at 37.degree. C.
The DNA was precipitated with ethanol, washed with 70 percent
ethanol, and lyophilized. The DNA pellet was taken up in 200 .mu.L
HindIII buffer and digested with 20 .mu.L (500 units) PstI for 1.5
h at 37.degree. C. The aqueous phase was extracted with
phenol/CHCl.sub.3 and the DNA precipitated with ethanol. The DNA
was dissolved in water and purified by polyacrylamide gel
electrophoresis. Following electroelution of the vector band (120 v
for 2 h at 0.degree. C. in 0.1 times TBE (Maniatis et al., Id.))
the DNA was purified by phenol/CHCl.sub.3 extraction, ethanol
precipitation and ethanol washing.
Although p.DELTA.222 could be digested to completion (>98
percent) by either KnpI or Pst1 separately, exhaustive double
digestion was incomplete (<<50 percent). This may have
resulted from the fact that these sites were so close (10 bp) that
digestion by KpnI allowed "breathing" of the DNA in the vicinity of
the PstI site, i.e., strand separation or fraying. Since PstI will
only cleave double stranded DNA, strand separation could inhibit
subsequent PstI digestion.
EXAMPLE 14
Ligation of Oligonucleotide Casettes into the Subtilisin Gene
10 .mu.M of four complementary oligonucleotide pools (A-D, Table C
below) which were not 5' phosphorylated were annealed in 20 .mu.l
ligase buffer by heating for five minutes at 68.degree. C. and then
cooling for fifteen minutes at room temperature. 1 .mu.M of each
annealed oligonucleotide pool, .about.0.2 .mu.g KpnI and
PstI-digested p.DELTA.222 obtained in Example 13, 0.5 mM ATP,
ligase buffer and 6 units T.sub.4 DNA ligase in 20 .mu.L total
volume was reacted overnight at 14.degree. C. to ligate the pooled
cassettes in the vector. A large excess of cassettes
(.about.300.times.0 over the p.DELTA.222 ends) was used in the
ligation to help prevent intramolecular KpnI-KpnI ligation. The
reaction was diluted by adding 25 .mu.L of 10 mM Tris pH 8
containing 1 mM EDTA. The mixture was reannealed to avoid possible
cassette concatemer formation by heating to 68.degree. C. for five
minutes and cooling for 15 minutes at room temperature. The
ligation mixtures from each pool were transformed separately into
E. coli 294 rec.sup.+ cells. A small aliquot from each
transformation mixture was plated to determine the number of
independent transformants. The large number of transformants
indicated a high probability of multiple mutagenesis. The rest of
the transformants (.about.200-400 transformants) were cultured in 4
ml of LB medium plus 12.5 .mu.g chloramphenicol/ml. DNA was
prepared from each transformation pool (A-D). This DNA was digested
with KnpI, .about.0.1 .mu.g was used to retransform E coli
rec.sup.+ and the mixture was plated to isolate individual colonies
from each pool. Ligation of the cassettes into the gene and
bacterial repair upon transformation destroyed the KnpI and PstI
sites. Thus, only p.DELTA.222 was cut when the transformant DNA was
digested with KpnI. The cut plasmid would not transform E. coli.
Individual transformants were grown in culture and DNA was prepared
from 24 to 26 transformants per pool for direct plasmid sequencing.
A synthetic oligonucleotide primer having the sequence
5'-GAGCTTGATGTCATGGC-3' was used to prime the dideoxy sequencing
reaction. The mutants which were obtained are described in Table C
below.
Two codon+222 mutants (i.e., gln and ile) were not found after the
screening described. To obtain these a single 25 mer
oligonucleotide was synthesized for each mutant corresponding to
the top oligonucleotide strand in FIG. 12. Each was phosphorylated
and annealed to the bottom strand of its respective
nonphosphorylated oligonucleotide pool (i.e., pool A for gln and
pool D for ile). This was ligated into KnpI and PstI digested
p.DELTA.222 and processed as described for the original
oligonucleotide pools. The frequency of appearance for single
mutants obtained in this way was 2/8 and 0/7 for gln and ile,
respectively. To avoid this apparent bias the top strand was
phosphorylated and annealed to its unphosphorylated complementary
pool. The heterophosphorylated cassette was ligated into cut
p.DELTA.222 and processed as before. The frequency of appearance of
gln and ile mutants was now 7/7 and 7/7, respectively.
The data in Table C demonstrate a bias in the frequency of mutants
obtained from the pool. This probably resulted from unequal
representation of oligonucleotides in the pool. This may have been
caused by unequal coupling of the particular trimers over the
mutagenesis codon in the pool. Such a bias problem could be
remedied by appropriate adjustment of trimer levels during
synthesis to reflect equal reaction. In any case, mutants which
were not isolated in the primary screen were obtained by
synthesizing a single strand oligonucleotide representing the
desired mutation, phosphorylating both ends, annealing to the pool
of non-phosphorylated complementary strands and ligating into the
cassette site. A biased heteroduplex repair observed for the
completely unphosphorylated cassette may result from the fact that
position 222 is closer to the 5' end of the upper strand than it is
to the 5' end of the lower strand (see FIG. 12). Because a gap
exists at the unphosphorylated 5' ends and the mismatch bubble in
the double stranded DNA is at position 222, excision repair of the
top strand gap would more readily maintain a circularly hybridized
duplex capable of replication. Consistent with this hypothesis is
the fact that the top strand could be completely retained by
selective 5' phosphorylation. In this case only the bottom strand
contained a 5' gap which could promote excision repair. This method
is useful in directing biased incorporation of synthetic
oligonucleotide strands when employing mutagenic oligonucleotide
cassettes.
EXAMPLE 15
Site-Specific Mutagenesis of the Subtilisin Gene at Position
166
The procedure of Examples 13-14 was followed in substantial detail,
except that the mutagenesis primer differed (the 37 mer shown in
FIG. 13 was used), the two restriction enzymes were SacI and XmaIII
rather than PstI and KpnI and the resulting constructions differed,
as shown in FIG. 13.
Bacillus strains excreting mutant subtilisins at position 166 were
obtained as described below in Example 16. The mutant subtilisins
exhibiting substitutions of ala, asp, gln, phe, his, lys, asn, arg,
and val for the wild-type residue were recovered.
EXAMPLE 16
Preparation of Mutant Subtilisin Enzymes
B. subtilis strain BG2036 obtained by the method of Example 11 was
transformed by the plasmids of Examples 14, 15 or 20 and by pS4-5
as a control. Transformants were plated or cultured in shaker
flasks for 16 to 48 h at 37.degree. C. in LB media plus 12.5
.mu.g/ml chloramphenicol. Mutant enzymatically active subtilisin
was recovered by dialyzing cell broth against 0.01M sodium
phosphate buffer, pH 6.2. The dialyzed broth was then titrated to
pH 6.2 with 1N HCl and loaded on a 2.5.times.2 cm column of CM
cellulose (CM-52 Whatman). After washing with 0.01M sodium
phosphate, pH 6.2, the subtilisins (except mutants at position
+222) were eluted with the same buffer made 0.08N in NaCl. The
mutant subtilisins at position +222 were each eluted with 0.1M
sodium phosphate, pH 7.0. The purified mutant and wild type enzymes
were then used in studies of oxidation stability, Km, Kcat, Kcat/Km
ratio, pH optimum, and changes in substrate specificity.
TABLE C ______________________________________ Oligonucleotide Pool
Organization and Frequency of Mutants Obtained Pool Amino Acids
Codon-222.sup.a Frequency.sup.b
______________________________________ A asp GAT 2/25 met ATG 3/25
cys TGT 13/25 arg AGA 2/25 gln GAA 0/25 unexpected mutants.sup.a
5/25 B leu CTT 1/25 pro CCT 3/25 phe TTC 6/25 tyr TAC 5/25 his CAC
1/25 unexpected mutants 9/25 C glu GAA 3/17 ala GCT 3/17 thr ACA
1/17 lys AAA 1/17 asn AAC 1/17 unexpected mutants 8/17 D gly GGC
1/23 trp TGG 8/23 ile ATC 0/23 ser AGC 1/23 val GTT 4/23 unexpected
mutants 9/23 ______________________________________ .sup.a Codons
were chosen based on frequent use in the closed subtilisin gene
sequence (Wells et al., 1983, id.). .sup.b Frequency was determined
from single track analysis by direct plasmid sequencing. .sup.c
Unexpected mutants generally comprised double mutants with changes
in codons next to 222 or at the points of ligation. These were
believed t result from impurities in the obigonucleotide pools
and/or erroneous repair of the gapped ends.
EXAMPLE 17
Mutant Subtilisin Exhibiting Improved Oxidation Stability
Subtilisins having cysteine and alanine substituted at the 222
position for wild-type methionine (Example 16) were assayed for
resistance to oxidation by incubating with various concentrations
of sodium nypochloride (Clorox Bleach).
To a total volume of 400 .mu.l of 0.1M, pH 7, NaPO.sub.4 buffer
containing the indicated bleach concentrations (FIG. 14) sufficient
enzyme was added to give a final concentration of 0.016 mg/ml of
enzyme. The solutions were incubated at 25.degree. C. for 10 min.
and assayed for enzyme activity as follows: 120 .mu.l of either
ala+222 or wild type, or 100 .mu.l of the cys+222 incubation
mixture was combined with 890 .mu.l 0.1M tris buffer at pH 8.6 and
10 .mu.l of a sAAPFpN (Example 18) substrate solution (20 mg/ml in
DMSO). The rate of increase in absorbance at 410 nm due to release
of p-nitroaniline (Del Mar, E.G., et al., 1979 "Anal. Biochem." 99,
316-320) was monitored. The results are shown in FIG. 14. The
alanine substitution produced considerably more stable enzyme than
either the wild-type enzyme or a mutant in which a labile cysteine
residue was substituted for methionine. Surprisingly, the alanine
substitution did not substantially interfere with enzyme activity B
against the assay substrate, yet conferred relative oxidation
stability on the enzyme. The serine+222 mutant also exhibited
improved oxidation stability.
EXAMPLE 18
Mutant Subtilisins Exhibiting Modified Kinetics and Substrate
Specificity
Various mutants for glycine+166 were screened for modified Kcat, Km
and Kcat/Km ratios. Kinetic parameters were obtained by analysis of
the progress curves of the reactions. The rate of reaction was
measured as a function of substrate concentration. Data was
analyzed by fitting to the Michaelis-Menton equation using the
non-linear regression algorithm of Marquardt (Marquardt, D. W.
1963, "J. Soc. Ind. Appl. Math." 11, 431-41). All reactions were
conducted at 25.degree. C. in 0.1M tris buffer, pH 8.6, containing
benzoyl-L-Valyl-Glycyl-L-Arginyl-p-nitroanilide (BVGRpN; Vega
Biochemicals) at initial concentrations of 0.0025M to 0.00026M
(depending on the value of Km for the enzyme of
interest--concentrations were adjusted in each measurement so as to
exceed Km) or
succinyl-L-Alanyl-L-Alanyl-L-Prolyl-L-Phenylalanyl-p-nitro-anilide
(sAAPFpN; Vega Biochemicals) at initial concentrations of 0.0010M
to 0.00028M (varying as described for BVGRpN).
The results obtained in these experiments were as follows:
TABLE D ______________________________________ Substrate Enzyme
Kcat (s.sup.-1) Km (M) Kcat/Km
______________________________________ sAAPFpN gly - 166 37 1.4
.times. 10.sup.-4 3 .times. 10.sup.5 (wild type) ala + 166 19 2.7
.times. 10.sup.-5 7 .times. 10.sup.5 asp + 166 3 5.8 .times.
10.sup.-4 5 .times. 10.sup.3 glu + 166 11 3.4 .times. 10.sup.-4 3
.times. 10.sup.4 phe + 166 3 1.4 .times. 10.sup.-5 2 .times.
10.sup.5 hys + 166 15 1.1 .times. 10.sup.-4 1 .times. 10.sup.5 lys
+ 166 15 3.4 .times. 10.sup.-5 4 .times. 10.sup.5 asn + 166 26 1.4
.times. 10.sup.-4 2 .times. 10.sup.5 arg + 166 19 6.2 .times.
10.sup.-5 3 .times. 10.sup.5 val + 166 1 1.4 .times. 10.sup.-4 1
.times. 10.sup.4 BVGRpN Wild Type 2 1.1 .times. 10.sup.-3 2 .times.
10.sup.3 asp + 166 2 4.1 .times. 10.sup.-5 5 .times. 10.sup.4 glu +
166 2 2.7 .times. 10.sup.-5 7 .times. 10.sup.4 asn + 166 1 1.2
.times. 10.sup.-4 8 .times. 10.sup.3
______________________________________
The Kcat/Km ratio for each of the mutants varied from that of the
wild-type enzyme. As a measure of catalytic efficiency, these
ratios demonstrate that enzymes having much higher activity against
a given substrate can be readily designed and selected by screening
in accordance with the invention herein. For example, A166 exhibits
over 2 times the activity of the wild type on sAAPFpN.
This data also demonstrates changes in substrate specificity upon
mutation of the wild type enzyme. For example, the Kcat/Km ratio
for the D166 and E166 mutants is higher than the wild type enzyme
with the BVGpN substrate, but qualitatively opposite results were
obtained upon incubation with sAAPFpN. Accordingly, the D166 and
E166 mutants were relatively more specific for BVGRpN than for
sAAPFpN.
EXAMPLE 19
Mutant Subtilisin Exhibiting Modified pH-Activity Profile
The pH profile of the Cys+222 mutant obtained in Example 16 was
compared to that of the wild type enzyme. 10 .mu.l of 60 mg/ml
sAAPFpN in DMSO, 10 .mu.l of Cys+222 (0.18 mg/ml) or wild type (0.5
mg/ml) and 980 .mu.l of buffer (for measurements at pH 6.6, 7.0 and
7.6, 0.1M NaPO.sub.4 buffer; at pH 8.2, 8.6 and 9.2, 0.1M tris
buffer; and at pH 9.6 and 10.0, 0.1M glycine buffer), after which
the initial rate of change in absorbance at 410 nm per minute was
measured at each pH and the data plotted in FIG. 15. The Cys+222
mutant exhibits a sharper pH optimum than the wild type enzyme.
EXAMPLE 20
Site-Specific Mutagenesis of the Subtilisin Gene at Position
169
The procedure of Examples 13-14 was followed in substantial detail,
except that the mutagenesis primer differed (the primer shown in
FIG. 16 was used), the two restriction enzymes were KpnI and EcoRV
rather than PstI and KpnI and the resulting constructions differed,
as shown in FIG. 16.
Bacillus strains excreting mutant subtilisins at position 169 were
obtained as described below in Example 16. The mutant subtilisins
exhibiting substitutions of ala and ser for the wild-type residue
were recovered and assayed for changes in kinetic features. The
assay employed SAAPFpN at pH 8.6 in the same fashion as set forth
in Example 18. The results were as follows:
TABLE E ______________________________________ Enzyme Kcat
(s.sup.-1) Km (M) Kcst/Km ______________________________________
ala + 169 58 7.5 .times. 10.sup.-5 8 .times. 10.sup.5 ser + 169 38
8.5 .times. 10.sup.-5 4 .times. 10.sup.5
______________________________________
EXAMPLE 21
Alterations in Specific Activity on a Protein Substrate
Position 166 mutants from Examples 15 and 16 were assayed for
alteration of specific activity on a naturally occurring protein
substrate. Because these mutant proteases could display altered
specificity as well as altered specific activity, the substrate
should contain sufficient different cleavage sites i.e., acidic,
basic, neutral, and hydrophobic, so as not to bias the assay toward
a protease with one type of specificity. The substrate should also
contain no derivitized residues that result in the masking of
certain cleavage sites. The widely used substrates such as
hemoglobin, azocollogen, azocasein, dimetnyl casein, etc., were
rejected on this basis. Bovine casein, .alpha.and .alpha..sub.2
chains, was chosen as a suitable substrate.
A 1 percent casein (w/v) solution was prepared in a 100 mM Tris
buffer, pH 8.0, 10 mM EDTA. The assay protocol is as follows:
790 .mu.l 50 mM Tris pH 8.2
100 .mu.l 1 percent casein (Sigma) solution
10 .eta.l test enzyme (10-200 .mu.g).
This assay mixture was mixed and allowed to incubate at room
temperature for 20 minutes. The reaction was terminated upon the
addition of 100 .mu.l 100 percent trichloroacetic acid, followed
by. incubation for 15 minutes at room temperature. The precipitated
protein was pelleted by centrifugation and the optical density of
the supernatant was determined spectrophotometrically at 280 nm.
The optical density is a reflection of the amount of
unprecipitated, i.e., hydrolyzed, casein in the reaction mixture.
The amount of casein hydrolysed by each mutant protease was
compared to a series of standards containing various amounts of the
wild type protease, and the activity is expressed as a percentage
of the corresponding wild type activity. Enzyme activities were
converted to specific activity by dividing the casein hydrolysis
activity by the 280 nm absorbance of the enzyme solution used in
the assay.
All of the mutants which were assayed showed less specific activity
on casein than the wild type with the exception of Asn+166 which
was 26 percent more active on casein than the wild type. The mutant
showing the least specific activity was ile+166 at 0.184 of the
wild type activity.
* * * * *