U.S. patent application number 09/785246 was filed with the patent office on 2003-09-18 for (1 -> 3, 1 -> 4)-beta-glucanase of enhanced stability.
This patent application is currently assigned to BIOMOLECULAR RESEARCH INSTITUTE LTD. Invention is credited to Chen, Lin, Fincher, Geoffrey Bruce, Garrett, Thomas Peter John, Hoj, Peter Bordier, Varghese, Joseph Noozhumurry.
Application Number | 20030177530 09/785246 |
Document ID | / |
Family ID | 3777041 |
Filed Date | 2003-09-18 |
United States Patent
Application |
20030177530 |
Kind Code |
A1 |
Varghese, Joseph Noozhumurry ;
et al. |
September 18, 2003 |
(1 -> 3, 1 -> 4)-beta-glucanase of enhanced stability
Abstract
A modified cereal (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase is
produced by the method of single point substitution in a native
cereal (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase enzyme, whereby the
substitution: a) maintains enzyme specificity by conserving the
active site groove of the native cereal
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase enzyme; and b) effects
increased thermostability over the native cereal
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase enzyme by: i) replacing
glycine by proline or alanine in helices of the cereal
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase enzyme, in order to
stiffen the enzyme amino acid chain and reduce entropy of the
unfolded enzyme; ii) attaching negatively charged residues to
N-termini of helices in the native cereal
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase enzyme; iii) introducing
ion pairs into the native cereal (1.fwdarw.3,1.fwdarw.4)--
.beta.-glucanase enzyme, to increase binding energy in the folded
enzyme; iv) replacing lysine by arginine in the cereal
(1.fwdarw.3,1.fwdarw.4)-.be- ta.-glucanase enzyme, and thereby
preventing lysine glycation and increasing hydrogen bonding with
other parts of the enzyme; v) replacing, by glycine, an amino acid
in the native cereal (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase
enzyme in which the main chain torsion angle about the N and
C.sup..alpha. atoms is greater than 0.degree.; or vi) creating
cysteine pairs in the native cereal (1.fwdarw.3,1.fwdarw.4)-.-
beta.-glucanase enzyme which can form disulphide bonds across the C
and N terminals.
Inventors: |
Varghese, Joseph Noozhumurry;
(Melbourne, AU) ; Garrett, Thomas Peter John;
(Melbourne, AU) ; Fincher, Geoffrey Bruce;
(Melbourne, AU) ; Hoj, Peter Bordier; (Melbourne,
AU) ; Chen, Lin; (Melbourne, AU) |
Correspondence
Address: |
JACOBSON HOLMAN PLLC
400 SEVENTH STREET N.W.
SUITE 600
WASHINGTON
DC
20004
US
|
Assignee: |
BIOMOLECULAR RESEARCH INSTITUTE
LTD
|
Family ID: |
3777041 |
Appl. No.: |
09/785246 |
Filed: |
February 20, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09785246 |
Feb 20, 2001 |
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08584008 |
Jan 11, 1996 |
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6277615 |
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08584008 |
Jan 11, 1996 |
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PCT/AU94/00377 |
Jul 6, 1994 |
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Current U.S.
Class: |
800/284 ;
435/200; 435/320.1; 435/419; 435/69.1; 536/23.2 |
Current CPC
Class: |
C12N 15/8242 20130101;
C12N 9/244 20130101; C12N 9/2448 20130101; C12Y 302/01006 20130101;
C12Y 302/01073 20130101; A23K 10/14 20160501 |
Class at
Publication: |
800/284 ;
435/200; 435/69.1; 435/419; 435/320.1; 536/23.2 |
International
Class: |
A01H 005/00; C07H
021/04; C12N 009/24; C12P 021/02; C12N 005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 7, 1993 |
AU |
PL 9821 |
Claims
1. A plant (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase enzyme of
enhanced thermostability and/or pH stability, said enzyme being
modified by transfer of a non-homologous protein sequence of
different substrate specificity.
2. A plant (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase in which the
amino acid sequence of said enzyme: (a) is modified to comprise
structural elements of plant (1.fwdarw.3)-.beta.-glucanase, said
structural elements conferring improved heat stability; (b) is
modified at sites other than the active site to stabilise helices,
to increase binding energy of the folded protein, to increase
hydrogen bonding, and/or to prevent glycation; or (c) is modified
by creating cysteine pairs which can form disulphide bonds across
the C and N terminals.
3. A (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase according to claim 2,
in which two or more of the modifications (a) to (c) are
present.
4. A (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase according to claim 1
comprising the structural framework of the enzyme
(1.fwdarw.3)-.beta.-glu- canase and elements of the catalytic site
of (1.fwdarw.3,1.fwdarw.4)-.beta- .-glucanase.
5. An enzyme according to claim 4 additionally comprising
modification (b) and/or modification (c) of claim 2.
6. A (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase according to claim 1,
in which the amino acid sequence of
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase isoenzyme EII is modified
to comprise one or more substitutions selected from the group
consisting of: Ala 14 Ser Ala 15 Arg Thr 17 Asp Lys 23 Arg Lys 28
Arg Asn 36 Asp Gly 44 Arg Gly 45 Asn Gly 53 Asp Gly 53 Glu Lys 74
Arg Gln 78 Arg Ala 79 Pro Lys 82 Arg Ala 95 Asp Gly 97 Pro Phe 85
Tyr Lys 107 Arg Gly 111 Ala Gly 119 Pro Lys 122 Arg Ser 128 Arg Gly
133 Ala Gly 145 Asn Gly 152 Thr Pro 153 Asp Gln 156 Arg Asn 162 Gly
Gly 185 Asn Ala 191 Pro Gly 193 Ala Gly 199 Pro Ala 200 Gly Gly 202
Thr Gly 219 Glu Lys 220 Arg His 221 Ala Gly 223 Ala Ser 224 Pro Lys
227 Arg Gly 238 Ala Gly 239 Gln Ala 242 Gly Gly 260 Glu Pro 267 Arg
Gly 268 Glu Gly 286 Ala Gly 286 Asp Gln 289 Arg Met 298 Lys His 300
Pro subject to the proviso that the following ion pairs must both
be substituted:
9 Ala 15 Arg and Asn 36 Asp Thr 17 Asp and Met 298 Lys Ala 95 Asp
and Ser 128 Arg Pro 153 Asp and Gln 156 Arg Lys 227 Arg and Gly 268
Arg Gly 152 Thr and His 221 Ala.
7. A (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase according to claim 4
in which amino acids in the loops forming the sides and bottom of
the active site cleft of (1.fwdarw.3)-.beta.-glucanase GII are
replaced by corresponding amino acids from
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase EII, as follows:
10 residue 8 Ile .fwdarw. Ser, residue 34 Phe .fwdarw. Ala, residue
208 Ala .fwdarw. Thr, residue 209 Met .fwdarw. Thr, residue 189-191
Gln-Pro-Gly .fwdarw. Asn-Ala-Ser residue 128-137
Ile-Arg-Phe-Asp-Glu-Val-Ala-Asn-Ser-Phe .fwdarw. Val-Ser-
Gln-Ala-Ile-Leu-Gly-Val-Phe-Ser (SEQ. ID NO: 1),
residue 171-179
Phe-Ala-Tyr-Arg-Asp-Asn-Pro-Gly-Ser.fwdarw.Leu-Ala-Trp-Ala-
-Tyr-Asn-Pro-Ser-Ala (SEQ. ID NO: 2) and residue 283-291
Thr-Gly-Asp-Ala-Thr-Glu-Arg-Ser-Phe.fwdarw.Asp-Ser-Gly-Val-Glu-Gln-Asn-Tr-
p (SEQ. ID NO: 3)
8. A (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase according to claim 6
comprising one or more of the following substitutions: Gly 53 Asp
Gly 53 Glu Thr 17 Asp; Met 298 Lys Ala 95 Asp; Ser 128 Arg Lys 122
Arg Lys 23 Arg Lys 74 Arg Gly 44 Arg Gly 223 Ala Ala 89 Pro Phe 85
Tyr
9. A (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase according to any one
of claims 6 to 8, additionally comprising the mutation 189-191
Gln-Pro-Gly-.fwdarw.Asn-Ala-Ser.
10. A (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase according to claim 6
comprising the substitution Lys 122.fwdarw.Arg and/or the
substitution Phe 85.fwdarw.Tyr.
11. A DNA molecule whose sequence encodes a
(1.fwdarw.3,1.fwdarw.4)-.beta.- -glucanase according to any one of
claims 1 to 10.
12. A plasmid comprising a DNA sequence according to claim 11.
13. An expression vector comprising a DNA sequence according to
claim 11.
14. A transgenic plant comprising a DNA sequence according to claim
11.
15. A transgenic plant according to claim 14, selected from the
group consisting of barley, wheat, rice, and maize.
16. A transgenic plant according to claim 15, which is barley.
17. A process selected from the group consisting of malting,
brewing and stockfeed processing, comprising the step of: (a) using
barley expressing the (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase of
any one of claims 1 to 10 as a starting material; or (b) adding the
(1.fwdarw.3,1.fwdarw.4)-.beta.-- glucanase of any one of claims 1
to 10 to a grain to be processed.
18. A composition for use in malting, brewing, or stockfeed
processing, comprising the (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase
of any one of claims 1 to 10, together with a carrier acceptable
for use in processing of beverages or of stockfeeds.
19. A beverage produced using a composition according to claim
18.
20. A stockfeed produced using a composition according to claim
18.
21. Grain produced by a transgenic plant according to claim 15.
22. Barley grain produced by a transgenic barley according to claim
16.
Description
BACKGROUND OF THE INVENTION
[0001] Barley quality encompasses a range of physical and chemical
attributes, depending on whether the grain is to be used in the
preparation of malt for brewing purposes, in the formulation of
stockfeed, or as a component of human foods. Currently,
specifications of barley quality are tailored primarily for the
malting and brewing industries, in which germinated barley (malt)
is the principal raw material. The quality specifications include
such parameters as grain size, dormancy, malt extract, grain
protein content, development of enzymes for starch degradation in
malt and (1.fwdarw.3,1.fwdarw.4)-.beta.- -glucan content. Malt
extract is a widely-used quality indicator. It is an estimate of
the percentage of malted grain that can be extracted with hot
water. Barley breeders and growers strive to produce grain with
high malt extract values, because greater extract percentages
provide higher levels of materials for subsequent fermentative
growth by yeast during brewing. Malt extract values are influenced
both by the composition of the ungerminated barley and by the speed
and extent of endosperm modification during malting. Given the
central role of cell walls as a potential barrier against the free
diffusion of starch- and protein-degrading enzymes from the
scutellum or from the aleurone to their substrates in cells of the
starchy endosperm, it is not surprising that wall composition and
the ability of the grain to rapidly produce enzymes that hydrolyse
wall constituents are important determinants of malt extract
values.
[0002] The major constituents of endosperm cell walls of barley are
the (1.fwdarw.3,1.fwdarw.4)-.beta.-glucans, which account for
approximately 70% by weight of the walls (Fincher, 1975). In the
germinating grain (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanases
function to depolymerise (1.fwdarw.3,1.fwdarw.4)-.beta.-glucans of
cell walls during endosperm mobilisation.
[0003] Total (1.fwdarw.3,1.fwdarw.4)-.beta.-glucan in ungerminated
barley grain is not highly correlated with malt extract (Henry
1986; Stuart et al, 1988). However, the residual
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucan in malted barley is highly
correlated, in a negative sense, with malt extract (Bourne et al,
1982; Henry 1986; Stuart et al, 1988), and this residual
polysaccharide reflects a combination of the initial
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucan levels in the barley and,
more importantly, the capacity of the grain to rapidly produce high
levels of (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase during malting
(Stuart et al, 1988). The (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase
potential of barley cultivars is also dependent on both genotype
and environment, although environmental conditions during grain
maturation appear to be particularly important in the development
of the enzymes (Stuart et al, 1988). Monoclonal antibodies specific
for barley (1.fwdarw.3,1.fwdarw.4)-- .beta.-glucanases have been
used in enzyme-linked immunosorbent assays (ELISA) that may be
useful for the quantitation of
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase levels in large numbers of
barley lines generated in breeding programs (H.O slashed.j et al,
1990). Furthermore, mutant barleys with altered
(1.fwdarw.3,1.fwdarw.4)-.beta.-g- lucan content (Aastrup 1983;
Molina-Cano et al, 1989) or
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase potential will be useful
in future studies on the effects of these components on malting
quality and may be valuable in breeding programmes.
[0004] The ability of the (1.fwdarw.3;1.fwdarw.4)-.beta.-glucanases
[E.C. 3.2.1.73] to retain enzymic activity at elevated temperatures
(thermostability) is of extreme importance during the utilization
of barley in the malting and brewing industries. Malt quality, as
measured by the `malt extract` index, is highly dependent on the
ability of the grain to rapidly synthesize high levels of the
enzyme during germination (Stuart et al, 1988). High levels of
(1.fwdarw.3;1.fwdarw.4)-.beta.-gluca- nases are also desirable in
the brewing process, where residual
(1.fwdarw.3;1.fwdarw.4)-.beta.-glucans in malt extracts can
adversely effect wort and beer filtration due to their propensity
to form aqueous solutions of high viscosity. These residuals can
also contribute to the formation of certain hazes or precipitates
at elevated ethanol concentrations or low temperatures in the final
beer (Woodward and Fincher, 1983). The elevated temperatures used
during commercial malting and brewing lead to rapid and extensive
inactivation of these enzymes. The high temperatures (up to
85.degree.) of commercial kilning processes destroy greater than
60% of the enzyme activity and much of the remaining enzyme is
inactivated by the hot water used for malt extraction (Brunswick et
al, 1987), Loi et al, 1987). It is therefore highly desirable to
develop commercial strains of barley that express a thermostable
(1.fwdarw.3;1.fwdarw.4)-.beta.-glucanase enzyme, or to produce the
(1.fwdarw.3;1.fwdarw.4)-.beta.-glucanase enzymes exogenously as an
additive to be used in the brewing process.
[0005] Barley (1.fwdarw.3;1.fwdarw.4)-.beta.-glucans also pose
problems in the stockfeed industry. In poultry formulations
prepared from cereal grains, (1.fwdarw.3;1.fwdarw.4)-.beta.-glucans
significantly raise the viscosity of the gut contents of chickens.
This impairs digestion and slows growth rates, and results in
sticky faecal droppings that make hygienic handling of eggs and
carcases difficult (Fincher and Stone, 1986). This application
would require the enzyme to be stable at a range of pHs,
particularly in the pH region of the foregut. It would also be an
advantage for the enzyme to be sufficiently thermostable to
withstand the steam pelleting processes widely used in stockfeed
manufacture.
[0006] Thus it is envisaged that
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase of amino acid sequence
modified so as to provide enhanced thermostability and/or pH
stability will have a variety of industrial uses, either by means
of barley expressing the modified enzyme, or by addition of the
modified enzyme to barley being processed.
[0007] There has been considerable interest in inserting
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase genes into brewing yeasts,
in the expectation that low level, constitutive expression would
lead to the secretion of active enzyme and the depolymerisation of
residual (1.fwdarw.3,1.fwdarw.4)-.beta.-glucan during fermentation
(Hinchliffe, 1988). A barley
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase cDNA (Fincher et al, 1986)
fused with a mouse .alpha.-amylase signal peptide is expressed and
secreted from yeast under the direction of the yeast alcohol
dehydrogenase I gene promoter (Jackson et al, 1986). Although the
gene for isoenzyme EII has not yet been isolated, the availability
of almost full length CDNA for use as a probe means that such
isolation can readily be carried out using conventional
methods.
[0008] We have now determined the three dimensional structure of
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase isoenzyme EII and
(1.fwdarw.3)-.beta.-glucanase isgenzyme GII (E.C.3.2.1.39), and
have identified regions of the structures of these enzymes which
are candidates for modification in order to provide enhanced
thermal and pH stability, as well as suitable point mutations for
achieving such stabilisation. We have found that the 3-dimensional
structures of these two enzymes, which share only 50% sequence
homology, are remarkably similar in their structural framework, and
that their active sites are also surprisingly similar, despite the
difference in substrate specificity.
SUMMARY OF THE INVENTION
[0009] According to a first aspect, the invention provides a
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase of enhanced
thermostability and/or pH stability.
[0010] In a second aspect, the invention provides an isolated DNA
sequence encoding a (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase of
enhanced thermostability and/or pH stability, and plasmids,
expression vectors, and transgenic plants comprising said sequence.
Preferably the expression host is E. coli or Saccharomyces
cereviseae; preferably the transgenic plant is barley. It will be
clearly understood that barley grain from plants encoding the
improved enzyme is within the scope of this invention.
[0011] In a third aspect, the invention provides a method selected
from the group consisting of malting, brewing and stockfeed
processing, comprising the step of
[0012] a) using barley expressing the
(1.fwdarw.3,1.fwdarw.4)-.beta.-gluca- nase of this invention as a
starting material, or
[0013] b) adding (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase of this
invention to a grain to be processed.
[0014] In a fourth aspect, the invention provides a composition for
use in malting, brewing, or stockfeed processing, comprising the
improved (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase of the invention,
together with carriers acceptable for use in processing of
beverages or of stockfeeds.
DETAILED DESCRIPTION OF THE INVENTION
[0015] The invention will now be described in detail by way of
reference only to the following non-limiting examples, and to the
figures, in which
[0016] FIG. 1 shows a stereo view of the alpha carbon trace of the
polypeptide backbone of the EII and GII glucanase enzymes. The
heavy lines represent the EII enzyme and the lighter lines
represent the GII enzymes. The active site groove runs north to
south, and the C- and N-termini are indicated, as are the two
putative active site residues glutamic acids at residues 232 and
288 (using EII sequence numbers).
[0017] FIG. 2 shows the sequence comparison of the EII (lower line)
and GII (upper line) glucanase enzymes based on the 3-dimensional
structure, with the sequence given using the three letter code for
amino acids. Residue numbers at the start of each line are the
sequence numbers of the two enzymes. The secondary structure
elements of both enzymes are given above the GII sequence and below
the EII sequence (see text for notation used in the description of
the tertiary structure).
[0018] .alpha. represents alpha helices; .beta. represents beta
sheets; A and B represent additional alpha helices and beta sheets
to those of a typical .alpha./.beta. barrel.
[0019] FIG. 3 is a schematic drawing of the
(1.fwdarw.3,1.fwdarw.4)-.beta.- -glucanase EII enzyme. The elements
with arrow heads represent beta sheet structure and the elements
with a curled tape coil represent alpha helices. Some of the
smaller beta sheets are not drawn. Elsewhere the chain is
represented as a rope. The black dots represent amino acid
locations where thermostable mutants have been proposed (see
text).
[0020] FIG. 4 is a schematic drawing of the
(1.fwdarw.3)-.beta.-glucanase GII enzyme. The elements with arrow
head represent beta sheet structure and the elements with a curled
tape coil represent alpha helices. Some of the smaller beta sheets
are not drawn. Elsewhere the chain is represented as a rope. The
black dots represent amino acids locations around the active site
groove which confer the specific activity of the enzymes. It is
proposed to modify these amino acids to change the specificity of
the GII enzyme into that of the EII enzyme.
[0021] FIG. 5 shows a comparison between stability of
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase isoenzyme EII with that of
(1.fwdarw.3)-.beta.-glucanase isoenzyme GII at pH 3.5.
[0022] FIG. 6 compares the stabilities of
(1.fwdarw.3,1.fwdarw.4)-.beta.-g- lucanase isoenzymes EII with that
of (1.fwdarw.3)-.beta.-glucanase isoenzyme GII at 50.degree..
[0023] FIG. 7 compares the stabilities of
(1.fwdarw.3,1.fwdarw.4)-.beta.-g- lucanase isoenzyme EII with that
of (1.fwdarw.3)-.beta.-glucanase isoenzyme GII at increasing
temperatures.
[0024] FIG. 8 compares the stabilities of wildtype
(1.fwdarw.3,1.fwdarw.4)- -.beta.-glucanase isoenzyme EII and mutant
H300P on heating for 15 minutes.
[0025] FIG. 9 compares the stabilities of wildtype
(1.fwdarw.3,1.fwdarw.4)- -.beta.-glucanase isoenzyme EII and mutant
H300P at 48.degree. C.
[0026] FIG. 10 compares the stabilities of wildtype
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase isoenzyme EII and mutant
H300P during mashing at 55.degree. C.
[0027] The (1.fwdarw.3;1.fwdarw.4)-.beta.-glucanases catalyse the
hydrolysis of (1.fwdarw.4)-.beta.-glucosyl linkages in
(1.fwdarw.3;1.fwdarw.4)-.beta.-glucans, only where the alucosyl
residue is substituted at the C(O)3 position, as follows: 1
[0028] The glucosyl residues are represented by G, (1.fwdarw.3)-
and (1.fwdarw.4)-.beta.-linkages by 3 and 4, respectively, and the
reducing terminus (red) of the polysaccharide chain is indicated.
Thus the enzymes have an absolute requirement for adjacent
(1.fwdarw.3)- and (1.fwdarw.4)-.beta.-linked glucosyl residues in
their substrates. The (1.fwdarw.3)-.beta.-glucanases [EC 3.2.1.39]
are able to hydrolyse the single (1.fwdarw.3)-.beta.-linkages found
in (1.fwdarw.3;1.fwdarw.4)-.bet- a.-glucans, but can catalyse the
hydrolysis of (1.fwdarw.3)-.beta.-glucosy- l linkages in
(1.fwdarw.3)-.beta.-glucans, as follows: 2
[0029] Arrows indicate the hydrolysis of
(1.fwdarw.3)-.beta.-linkages between glucosyl residues (G).
[0030] Furthermore it is known that the
(1.fwdarw.3)-.beta.-glucanase isoenzyme GII is more thermostable,
pH stable and protease resistant than the
(1.fwdarw.3;1.fwdarw.4)-.beta.-glucanase EII enzyme. Thus using the
three dimensional structures of these enzymes, we can create more
stable forms of the (1.fwdarw.3;1.fwdarw.4)-.beta.-glucanase by the
following methods:
[0031] (a) transferring the structural elements that generate the
heat stability of the (1.fwdarw.3)-.beta.-glucanase, on to the
(1.fwdarw.3;1.fwdarw.4)-.beta.-glucanase.
[0032] (b) modifying the (1.fwdarw.3;1.fwdarw.4)-.beta.-glucanase
using general principles of protein structure and stability
(Matthews, 1987).
[0033] (c) engineering a thermostable or pH stable
(1.fwdarw.3;1.fwdarw.4)- -.beta.-glucanase enzyme by transforming
the (1.fwdarw.3)-.beta.-glucanase into the
(1.fwdarw.3;1.fwdarw.4)-.beta.-glucanase. This is done by
transferring elements of the catalytic site of the
(1.fwdarw.3;1.fwdarw.4)-.beta.-glucanase enzyme on to the
(1.fwdarw.3)-.beta.-glucanase enzyme.
[0034] (d) engineering a thermostable
(1.fwdarw.3,1.fwdarw.4)-.beta.-gluca- nase and
(1.fwdarw.3)-.beta.-glucanase by creating cysteine pairs which can
form disulphide bonds across the C and N terminals.
[0035] A combination of two or more of these methods may be
used.
[0036] For each of these methods knowledge of the protein
structures is an important prerequisite. This knowledge enables us
to separate differences between the two enzymes which govern
substrate specificity from those for thermal and pH stability. It
also enables us to predict which kind of changes to the sequence
which will enhance the stability of the secondary structure
elements. Random mutagenesis of glucanase genes will invariably
reduce the stability of the protein by disrupting its structure, or
may cause inactivation of the enzyme. This is due to the inability
of current methods to predict protein folding and catalytic
activity from amino acid sequence information alone.
EXAMPLE 1
Determination of the 3-Dimensional Structure of the Glucanase
Enzymes
[0037] We have determined the 3-dimensional structure of
(1.fwdarw.3;1.fwdarw.4)-.beta.-glucanase isoenzyme EII (hereafter
called EII) and (1.fwdarw.3)-.beta.-glucanase isoenzyme GII
(hereafter called GII) to Sigh resolution (2.2 .ANG.) by X-ray
crystallographic techniques described by Blundell and Johnson
(1979).
[0038] In Appendix 3 we have set out the 3-dimensional coordinates
and mean thermal vibration parameters (isotropic B values) of the
two enzymes, as determined from the crystallographic refinement of
the X-ray diffraction data obtained from single crystals of each
enzyme.
[0039] The EII and GII glucanase structures have essentially
identical .alpha./.beta. barrel folds (FIG. 1). Minor perturbations
are found in the loops mainly at positions where there are sequence
insertions and deletions. A sequence comparison is set out in FIG.
2. The active site groove, which runs along the full length of the
upper surface of the molecule perpendicular to the barrel axis, is
almost identical in the central region of the groove, and different
in detail towards the ends of the groove. The carboxylate groups of
the two putative active site glutamates (Chen et al, 1993) are
positioned in an identical way some 7 .ANG. apart. Also around
these residues are a ring of residues which are totally conserved
in all plant (1.fwdarw.3)-.beta.-glucanases known (Xu et al, 1992
and sequences from the Genbank database). Details of the structure,
which is a novel type of .alpha./.beta. barrel are given below.
[0040] In FIG. 2 elements of the secondary structure have been
identified alongside the sequence alignment of the two enzymes. We
shall refer to the beta barrel strands as .beta..sub.i and the
major (longest) helices connecting the beta strands as
.alpha..sub.1, where i goes from 1 to 8. Minor .beta. sheet and
.alpha. helices are referred to as B.sub.i and A.sub.i,
respectively if they appear after the strand .beta..sub.i and
before .beta..sub.i+1, and a further subscript a or b, if more than
one occur.
[0041] Looking at the glucanase tertiary structure from above, down
the barrel axis (the long axis of the elliptical barrel running
east west), the active site groove runs north to south on the upper
face of the molecule, as shown in FIGS. 3 and 4.
[0042] The N-terminal starts under the molecule entering the east
side of the barrel as .beta.1 and emerges on the upper surface and
the heads back towards the bottom surface as .alpha..sub.1
(traversing the outside of the molecule) to meet .beta..sub.2,
where this motif is repeated for strands .beta..sub.2 to
.beta..sub.4, building the upper half of a conventional
.alpha./.beta. barrel (note that for the third .alpha./.beta. loop
there are two helices).
[0043] The lower half of the barrel has more elaborate secondary
structural elements, not previously observed in other
.alpha./.beta. barrel structures. There is what could be called a
subdomain built around the helix .alpha..sub.6. This helix runs
perpendicular to the groove axis and at the southern end of the
groove and is supported by three two stranded antiparallel .beta.
sheet `fingers` (B.sub.5 on the upper surface, B.sub.7 on the
underneath surface and B.sub.6 at the southern end of the groove)
and three small helices (A.sub.5 at the western side and A.sub.6a
and A.sub.6a at the eastern side of the groove). This subdomain,
which forms a platform for the residues making up the lower half of
the groove, is different in detail (possibly arising from the
difference in specificity) between the EII and GII enzymes; for
example the helix A.sub.5 is missing in GII.
[0044] The C-terminal strand, consisting of some 30 residues,
starts after the strand .beta..sub.8, and has an unusual turn which
involves a cis peptide bond between residues Phe 275 and Ala 276 (a
cis proline could not accommodate this type of turn). This turn
allows the loop of residues from 276 to 286 to position the
glutamate at 288, which is in a small helical turn .alpha..sub.8,
at the appropriate orientation to act as a catalytic acid group.
The C-terminal strand then finds its way down to the underside of
the molecule between the helices .alpha..sub.1 and .alpha..sub.7 to
within 4.2 .ANG. from the N-terminus.
EXAMPLE 2
Identification of Sites of Contact with Substrate
[0045] In order to observe which amino acids in the
substrate-binding groove contacted the substrate, the structure of
glucanase GII was determined after soaking crystals with 1.fwdarw.3
linked oligosaccharides. Three sites were found where glucose units
of monomer or disaccharides bind to the protein The coordinates of
these sites are listed in Appendix 2. This establishes the
orientation of the substrate within the groove, and that some of
the proposed changes to GII are important for substrate
binding.
EXAMPLE 3
Proposed Modification of the
(1.fwdarw.3,1.fwdarw.4)-.beta.-Glucanase of Barley to Increase the
Thermostability of the Enzyme
[0046] The following amino acid changes are proposed for enhancing
the thermostability of (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase
EII, based on the 3-dimensional structure of the EII and GII
enzymes. Some of the changes proposed involve substituting the GII
amino acids that could be responsible for stabilizing that protein.
These substitutions are based on the principle that the proposed
changes will not alter the specificity of the enzyme (leave the
active site groove unaltered), and where changes would not lead to
deleterious changes in the 3-dimensional structure of the protein.
Where possible glycines have been replaced by prolines or alanines
in helices (Matthews et al, 1987) in order to stiffen the amino
acid chain and reduce the entropy of the unfolded protein.
Negatively charged residues have been attached to the N-termini of
helices to stabilise them (Nicholson et al, 1988, Eijsink et al,
1992). Ion pairs have been introduced to increase the binding
energy of the folded protein, and lysines changed. to arginines to
prevent glycation and improve stability (Mrabet et al, 1992) by
increasing the hydrogen-bonding with other parts of the protein. EI
and EII refer to the isozymes of
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase and GI to GVI refer to the
isozymes of the (1.fwdarw.3)-.beta.-glucanase (Xu et al, 1992). The
location of these substitutions are shown on FIG. 3. The mutation
is described using the following notation: eg. the mutation Ala 14
Ser represents the mutation of the Alanine residue to a Serine at
position 14 in the amino acid sequence (FIG. 3). The conventional 3
letter code for amino acids is used.
1 Mutation comments Ala 14 Ser as in GII, GV, GVI to stabilise
helix .alpha..sub.1 Ala 15 Arg as in GII, GIV, GV ion pair with Asp
36 at end of groove Thr 17 Asp as in GII to form ion pair with Met
298 Lys in GII Lys 23 Arg as in GI to GIV, H-bond to O46 Lys 28 Arg
Asn 36 Asp as in GI, GII, GIV, GVI, EI, to stabilise helix
.alpha..sub.2, ibid Gly 44 Arg as in GI, GII, GV, GVI Gly 45 Asn as
in GII, solvent exposed Gly 53 Asp as in GI, GII, GIII, GV, forms a
stable ion pair with Arg 31 Gly 53 Glu Lys 74 Arg as in GI, GV Gln
78 Arg as in GI, GII Ala 79 Pro as in GI, GII, GVI, surface residue
Lys 82 Arg Ala 95 Asp as in GIII, ion pair with Arg 128 at end of
groove, Asn in GII Gly 97 Pro Phe 85 Tyr OH of Tyr H-bonds to O 76
Lys 107 Arg as in GI, GII, GIII, GIV Gly 111 Ala as in GII, helix
residue Gly 119 Pro Lys 122 Arg conserved in all except GVI, H-bond
to O 161 and O 120 Ser 128 Arg as in GI to GV Gly 133 Ala as in
GII, on the lip of the groove, could have packing problems here
with Thr 144 Gly 145 Asn different conformation in GII Gly 152 Thr
as in GII, His 221 will clash with Thr so need to change His to Ala
Pro 153 Asp as in GII, see below for ion pair Gln 156 Arg as in
GII, need Pro 153 Asp for ion pair Asn 162 Gly Gly 185 Asn as in
GII, stabilised by Asp 183 Ala 191 Pro as in GII, buried (near
surface) Gly 193 Ala wrong dihedrals for a Pro Gly 199 Pro as in
GI, GII has a different loop conformation solvated, so could be
modified. Ala 200 Gly Gly 202 Thr as in GII, H-bond to Thr 194 and
Arg 197 space for Pro here. Gly 219 Glu as in GI to GVI, ion pair
with Arg 265 might need Glu 266 Lys Lys 220 Arg as in GI H-bonds to
O139 His 221 Ala as in GII, ibid Gly 223 Ala as in GII (buried) Ser
224 Pro as in GI to GV Lye 227 Arg as in GI, GIV, GV, ion pair with
Glu 268 Gly 238 Ala as in GI, GII, GIV, GV, could clash with Asn
290 Gly 239 Gln as in GIII wrong dihedrals form a Pro Ala 242 Gly
Gly 260 Glu ion pair with Arg 261 or Pro Pro 267 Arg as in GII Gly
268 Glu as in GII, could for ion pair with Arg 227 (peptide flipped
wrt GII) Gly 286 Ala as in GII or Asp to stabilise helix .alpha.7
Gln 289 Arg as in GII, GIV, GV Met 298 Lys as in GI, GII, GIV, GV,
ibid His 300 Pro as in GI to GV
[0047] Of the above proposed modification the following ion pairs
have to be substituted at the same time.
2 Ala 15 Arg and Asn 36 Asp Thr 17 Asp and Met 298 Lys Ala 95 Asp
and Ser 128 Arg Pro 153 Asp and Gln 156 Arg Lys 227 Arg and Gly 268
Arg Gly 152 Thr and His 221 Ala
[0048] It will be clearly understood that, subject to this
requirement for concurrent substitution of ion pairs, combinations
of two or more of the proposed modifications may be used.
[0049] An additional class of mutations is proposed in which the
main chain torsion angle about the N and C.alpha. atoms is greater
than 0.degree.. In this case a replacement by a Gly residue is
energetically more favourable, particularly at the C terminal of an
.alpha.-helix (Aurora et al., 1994). These mutations are:
3 Asn 162 Gly as in GI, GII, GV, GVI, EI, terminus of helix
.alpha.5 Ala 200 Gly as in GIII, GIV, GV, GIV, main chain torsion
angles Ala 242 Gly Main chain torsion angles Met 298 Gly Main chain
torsion angles
EXAMPLE 4
Proposed Modification of the (1.fwdarw.3)-.beta.-Glucanase of
Barley to Alter its Catalytic Activity to that of
(1.fwdarw.3,1.fwdarw.4)-.beta.-Gl- ucanase and Increase the
Thermostability and pH Stability of the Enzyme
[0050] As mentioned before the most noticeable feature of both the
GII and EII enzymes is a deep groove across one face of the
molecule. This appears to be the substrate binding site. Using
structural information from both the GII and EII enzymes it is
possible to determine which amino acid residues are likely to
control substrate specificity. Furthermore, as these two enzymes
are very similar in structure it is possible to graft the loops
from one enzyme on to the more heat and pH stable framework of the
other to change the specificity.
[0051] We propose replacing the GII loops which form the sides and
bottom of the cleft by the corresponding amino acids from the EII
enzyme. These changes are as follows:
4 residue 8 Ile.fwdarw.Ser, residue 34 Phe.fwdarw.Ala, residue 208
Ala.fwdarw.Thr, residue 209 Met.fwdarw.Thr, residue 213
Val.fwdarw.Phe residue 128-137 Ile-Arg-Phe-Asp- (SEQ. ID NO:1)
Glu-Val-Ala-Asn-Ser-Phe.fwdarw. Val-Ser-Gln-Ala-Ile-Leu-
Gly-Val-Phe-Ser, residue 171-179 Phe-Ala-Tyr-Arg- (SEQ. ID NO:2)
Asp-Asn-Pro-Gly-Ser.fwda- rw. Leu-Ala-Trp-Ala-Tyr-Asn- Pro-Ser-Ala
and residue 283-291 Thr-Gly-Asp-Ala- (SEQ. ID NO:3)
Thr-Glu-Arg-Ser-Phe.fwdarw. Asp-Ser-Gly-Val-Glu-Gln- Asn-Trp
[0052] Some or all of these changes are necessary. The skilled
person will readily be able to test the effectiveness of the
substitutions.
[0053] Again combinations of two or more of these proposed
modifications may be used.
[0054] Doan and Fincher (1992) showed that relative to the EI
enzyme, EII is more thermostable because of the carbohydrate at
residue 190. We propose to introduce a carbohydrate attachment site
into the modified GII enzyme to enhance the thermostability. The
mutations required are 189-191 Gln-Pro-Gly.fwdarw.Asn-Ala-Ser
[0055] FIG. 4 is a schematic drawing of the GII enzyme structure
showing locations of the proposed mutations.
EXAMPLE 5
Construction of Mutant Glucanases
[0056] Construction of the proposed mutant glucanases may be
effected using the polymerase chain reaction (PCR)-based megaprimer
method (Sarkar & Sommers, 1990), and single site mutants of the
isozymes EI and EII have already been produced in this way by one
of us (Doan and Fincher, 1992). Briefly, for each site mutant or
short series of adjacent mutations one oligonucleotide is
synthesised which contains the complementary sequence required for
the mutation(s) and sufficient flanking regions to anneal to the
wild type cDNA. This oligonucleotide is extended against the cDNA
template with a DNA polymerase. PCR is used to amplify the mutant
section of cDNA, and then this is inserted back into the plasmid
containing the original cDNA. For multiple mutations this process
is repeated to produce the final construct. Alternatively,
commercially-available site directed mutagenesis kits based on the
unique site elimination method (Deng and Nickoloff, 1992) can be
used.
[0057] We currently have the cDNAs for the EII and GII enzymes
which form the starting points for the mutagenesis (Doan and
Fincher, 1992; H.O slashed.j et al, 1989). For the purposes of
demonstrating improved stability or altered specificity of these
enzymes and for production of the enzymes in quantity, the proteins
can then be expressed in E. coli (Wynn et al, 1992) using the
plasmid ET or other vectors or in insect cells (e.g. Sf9 cells)
using a Baculovirus system (Doan & Fincher, 1992). A person
skilled in the art will be aware of a variety of other suitable
expression systems. For example, yeast would be a suitable host,
and such an engineered yeast could be used directly in the brewing
process. The availability of the gene encoding
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucanas- e isoenzyme EI and near
full-length cDNAs for isoenzymes EI and EII (Slakeski et al, 1990)
presents an opportunity to accelerate or enhance
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase development in germinated
grain through gene technology. Increased enzyme activity might be
achieved by several means, for example, by splicing more efficient
promoters onto the gene, by altering the existing promoter to
enhance expression levels, by the use of translational enhancers,
or by increasing the copy number of the genes.
[0058] Two more steps are required for the mutant enzymes to be
incorporated into barley and expressed in a spatially and
temporally appropriate manner. These are construction of a barley
glucanase gene with the appropriate control of expression, and the
insertion of the gene into a viable barley plant. The sequence the
EII gene, including the promoter regions and the coding region and
the signal peptide has been determined (Wolf, 1991). Thus for
correct expression of the mutant glucanases we will replace a
portion of this gene by the corresponding portion of a mutant cDNA
using the above methods. It is expected that transformation of
barley, that is to regenerate a fertile transgenic barley plant,
will be possible in the near future. Foreign or manipulated DNA can
be integrated into the barley genome in a stable form (Lazzeri et
al, 1991) and fertile plants can be regenerated from single
protoplasts (Jahne et al, 1991a, b). Among the cereals related to
barley, rice can now be routinely transformed, and transformation
of both wheat and maize has been reported. Methods for effecting
transformation of monocotyledonous plants such as barley using
biolistic techniques are widely used, and whole plants of
transgenic barley have been grown. Barley has recently been
transformed using the biolistic microprojectile gun procedure (Wan
and Lemaux, 1994).
EXAMPLE 6
[0059] i) Stability of GII and EII at pH 3.5
[0060] (1.fwdarw.3)-.beta.-glucanase isoenzyme GII (9.2 .mu.g/ml)
and (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase isoenzyme EII (0.23
mg/ml) were incubated in 100 mM sodium acetate buffer at pH 3.5 in
the presence of bovine serum albumin at 37.degree. C. (0.5 mg/ml)
Residual enzyme activities (A.sub.t) were determined and compared
to the initial activity at t=0 (A.sub.o). The results are
illustrated in FIG. 5. GII shows markedly greater stability with
time at pH 3.5 than does EII. (Note: at pH 4.3 the enzymes differ
only slightly in their stability and exhibit only minimal loss of
activity; data not shown).
[0061] ii) Stabilities of GII and EII at 50.degree. C.
[0062] (1.fwdarw.3)-.beta.-glucanase isoenzyme GII (16 .mu.g/ml)
and (1.fwdarw.3;1.fwdarw.4)-.beta.-glucanase isoenzyme EII (19
.mu.g/ml) were incubated in 50 mm sodium acetate buffer at pH 5.0
in the presence of bovine serum albumin (1 mg/ml) at 50.degree. C.
Residual enzyme activities (A.sub.t) were determined and compared
to the initial activity at t=0 (A.sub.o). The results are
illustrated in FIG. 6. GII is very much more stable at 50.degree.
C. than is EII.
[0063] iii) Stabilities of GII and EII at Increasing
Temperatures
[0064] (1.fwdarw.3)-.beta.-glucanase isoenzyme GII (16 .mu.g/ml)
and (1.fwdarw.3;1.fwdarw.4)-.beta.-glucanase isoenzyme EII (19
.mu.g/ml) were incubated in 50 mM sodium acetate buffer at pH 5.0
at the indicated temperature for 15 min. Residual enzyme activities
(A.sub.t) were determined and compared to the initial activity at
t=0) (A.sub.o). The results are illustrated in FIG. 7. EII is
stable only up to 40.degree. C., while GII is stable up to
50.degree. C.
EXAMPLE 7
Size-directed Mutagenesis
[0065] Of the possible mutations listed in Example 3, the following
alterations were considered to be the most likely to improve
stability. The alterations are based on:
5 1. creation of ion pairs: Gly 53 Asp Gly 53 Glu Thr 17 Asp; Met
298 Lys Ala 95 Asp; Ser 128 Arg 2. removal of potential glycation
sites: Lys 122 Arg Lys 23 Arg Lys 74 Arg 3. reduction in entropy of
unfolded state: Gly 44 Arg Gly 223 Ala Ala 79 Pro 4. hydrophobic
effects: Phe 85 Tyr
[0066] Site-directed mutagenesis was carried out by the unique
restriction enzyme site elimination procedure using a U.S.E.
Mutagenesis Kit (Pharmacia) with double-stranded plasmid DNA as a
template. Appropriate mutagenic primers were designed to generate
the mutations and were synthesized on a standard DNA synthesizer.
All oligonucleotide primers were phosphorylated at their 5'-end
before use, and the mutagenesis procedure was performed essentially
as prescribed by the manufacturer. Mutants were confirmed by
dideoxynucleotide sequencing using a Sequence version 2.0
sequencing Kit (U.S. Biochemical Co.).
6 The following EII mutants were produced and confirmed by sequence
analysis: Lys 74 Arg Gly 44 Arg Phe 85 Arg Gly 53 Glu Lys 122 Arg
Lys 23 Arg Ala 79 Pro In addition, we have also made the following
mutants: Gly 223 Ala Gly 53 Asp
EXAMPLE 8
Expression of Mutant Enzymes in E. coli
[0067] The mutant cDNA inserts in the expression plasmid pMAL-c2
were transformed in E. coli DH5.sup..alpha. cells, and grown
overnight at 37.degree. C. in LB containing 0.2% glucose and 100
.mu.g/ml ampicillin. Aliquots of the cell suspension were
sub-cultured into the same medium and grown at 37.degree. C. with
vigorous shaking to an optical density at 600 nm of 0.5, induced
for 3 h with 1 mM isophenyl-.beta.-thiogalactoside and lysed with
lysozyme treatment and freeze/thawing. After removal of cell debris
by centrifugation, enzyme activity was measured either in the
unpurified extract or following purification.
[0068] The following EII mutants have been expressed in E. coli and
the expressed proteins have been confirmed to be of the correct
size:
[0069] Lys 122 Arg
[0070] Phe 85 Tyr
[0071] Gly 44 Arg
EXAMPLE 9
Purification of Recombinant Fusion Proteins
[0072] For the purification of the wild-type enzyme, crude extract
from 1 litre culture was diluted 10-fold with 15 mM Tris-Hcl
buffer, pH 8.0 and applied at a flow rate of 2.5 ml/min to a
DEAE-Sepharose Fast Flow (Pharmacia) column (3.times.11.5 cm)
equilibrated with 25 mM Tris-HCl buffer, pH 8.0. After washing the
column exhaustively, bound proteins were eluted with a linear 0-250
mM NaCl gradient in 1.2 litre equilibration buffer. Fractions
containing significant enzyme activity were pooled, desalted and
adjusted to 25 mM NaAc, pH 5.0. After exhaustive washing, bound
proteins were eluted with a linear 0-200 mM NaCl gradient in 1
litre equilibration buffer. The fractions containing pure protein
were pooled to give 5.0 mg active fusion protein.
[0073] Mutant enzymes were all purified by a single ion-exchange
chromatography step employing a shallow salt gradient elution. The
crude extract from 4 to 5 litre culture was diluted 10 fold with 15
mM Tris-HCl (pH 8.0) and applied at a flow rate of 2.5-3.0 ml/min
to a DEAE-Sepharose column (5.times.21 cm) equilibrated with 12.5
mM Tris-HCl (pH 8.5). After exhaustive washing, bound proteins were
eluted with a 1.9 litre linear 0-80 mM NaCl gradient at a flow rate
of 2.0 ml/min. Fractions containing pure fusion protein were
located by SDS-PAGE, pooled, concentrated and adjusted to 2.5 mM
sodium acetate (pH 5.0) by ultrafiltration before clarification by
centrifugation.
EXAMPLE 10
Activity of Expressed Enzymes
[0074] (1.fwdarw.3,1.fwdarw.4)-.beta.-Glucanase activity was
measured viscometrically at 40.degree. C., using 5 mg/ml barley
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucan in 50 mM sodium acetate pH
5.0 as substrate. A unit of activity is defined as the amount of
enzyme causing an increase of 1.0 in the reciprocal specific
viscosity (.DELTA.1/.eta..sub.sp) per minute. Specific activity is
expressed as the activity per mg protein.
[0075] The activities of the following mutant enzymes have been
measured and compared with the activity of the expressed wild type
enzyme:
7 Lys 122 Arg activity same as wild type Phe 85 Pyr activity
approx. 70% of wild type Gly 44 Arg activity very low
EXAMPLE 11
Thermostability Assays
[0076] Aliquots of wild type or mutant fusion proteins were diluted
with 50 mM sodium acetate buffer, pH 5.5 and incubated at
temperatures ranging from 40.degree. C. to 60.degree. C. for 15
min. Samples incubated at 0.degree. C. were used as controls.
Residual enzyme activity was determined viscometrically with 550
.mu.l (1.fwdarw.3,1.fwdarw.4)-.beta.-- glucan substrate, as
described for Example 10.
[0077] References listed herein are identified on the following
pages.
[0078] It will be apparent to the person skilled in the art that
while the invention has been described in some detail for the
purposes of clarity and understanding, various modifications and
alterations to the embodiments and methods described herein may be
made without departing from the scope of the inventive concept
disclosed in this specification.
EXAMPLE 12
Increased Thermostability of Isoenzyme EII by Site-Directed
Mutagenesis
[0079] Stability of (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase
isoenzyme EII (mutant H300P)
[0080] The cDNA encoding (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase
isoenzyme EII was subjected to site-directed mutagenesis using the
unique site elimination method (Deng and Nickoloff, 1992), to
generate mutant H300P. The mutagenesis procedure was performed
using a modified pET-3a vector containing the wild type
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase isoenzyme EII cDNA as a
template, which enables the rapid purification of expressed foreign
proteins using a nickel-based affinity resin (Hochuli et al, 1987).
The expressed mutant H300P showed an increase in the T.sub.50 value
(the temperature at which only 50% of the initial activity remains)
of approximately 3.8.degree. C., after heating for 15 minutes at
various temperatures. This is illustrated in FIG. 8.
[0081] An additional test for increased thermostability was
provided by following the residual activity (A.sub.t) of wild type
isoenzyme EII and the corresponding mutant H300P over time at
48.degree. C. The results are shown in FIG. 9. Finally, as a
further indication of increased thermostability in a commercial
context, activity of the wild type and mutant
(1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase isoenzyme EII was measured
over time in a simulated mashing experiment at 55.degree. C.
Briefly, mashing conditions were simulated by stirring malted,
dried barley grain in water at 55.degree. C. for 40 minutes to
inactivate any endogenous (1.fwdarw.3,1.fwdarw.4)-.beta.-glucanase
activity, and then wild type or mutant H300P enzyme was added to
the mash and residual activity (A.sub.t) was monitored over time.
The results are shown in FIG. 10.
EXAMPLE 13
Further Mutants Expected to Enhance Thermostability
[0082]
8 Met 7 Val as GI GII GIII, allow loop 7-12 to pack tighter against
C-terminus Ala 9 Gly as GII GIII GV GVI, allow loop 7-12 to pack
tighter against C-terminus Ala 15 Pro as GIII GVI Met 21 Leu as
GI-GVI, prevent close contact with Met 298 (or Lys) Phe 22 Tyr as
GI-GVI, buried H-bond with Val 30 Asn 25 Lys as GI-GIV, cover
hydrophobic patch Gly 26 Asn as GV, GVI, rigidify helix capping
residue Gly 240 Ala rigidify loop Asn 279 Asp stronger H-bonds Ser
285 Pro rigidify loop Val 287 Pro rigidify loop Asn 290 His as GI
GIV, His would pack tighter Phe 294 Tyr could H-bond to Asn 25 OD1
Asn 297 Asp as GI GII GVI, tighter H-bond in loop Met 298 Gly Main
chain torsion angles suit Gly Val 301 Ala as GI-GIII, change water
structure 307 Asn extend C terminus to make a salt bridge with Lys
28 Ala 176 Arg and Gly 286 Asp ion pair Ser 237 Phe and Asn 279 Ser
close packed bridge across or Trp C-terminal tail
[0083] As the N and C termini are close to each other it would be
possible to tie down the C terminus by linking the ends together.
The shortest linker with a structurally reasonable conformation is
Ala-Ala-Gly (or Gly-Pro-Gly or combinations). As helix a6 and
strand b7 are buried in the protein, new N and C termini at Val 226
and Gly 223 will not reduce the thermostability of the protein.
Furthermore the new termini could form an ion pair.
[0084] References
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Sequence CWU 1
1
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