U.S. patent application number 10/441602 was filed with the patent office on 2003-11-13 for enzyme kits and libraries.
Invention is credited to Short, Jay M..
Application Number | 20030211543 10/441602 |
Document ID | / |
Family ID | 24002794 |
Filed Date | 2003-11-13 |
United States Patent
Application |
20030211543 |
Kind Code |
A1 |
Short, Jay M. |
November 13, 2003 |
Enzyme kits and libraries
Abstract
Recombinant enzyme libraries and kits where a plurality of
enzymes are each characterized by different physical and/or
chemical characteristics and classified by common characteristics.
The characteristics are determined by screening of recombinant
enzymes expressed by a DNA library produced from various
microorganisms.
Inventors: |
Short, Jay M.; (Rancho Santa
Fe, CA) |
Correspondence
Address: |
DIVERSA CORPORATION
4955 DIRECTORS PLACE
SAN DIEGO
CA
92121
US
|
Family ID: |
24002794 |
Appl. No.: |
10/441602 |
Filed: |
May 19, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10441602 |
May 19, 2003 |
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09861267 |
May 18, 2001 |
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6566050 |
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09861267 |
May 18, 2001 |
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09467740 |
Dec 20, 1999 |
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09467740 |
Dec 20, 1999 |
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08503606 |
Jul 18, 1995 |
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6004788 |
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Current U.S.
Class: |
435/7.1 ;
435/455; 435/6.16; 435/91.2 |
Current CPC
Class: |
C12N 15/1093 20130101;
C12N 15/1086 20130101; C12Q 1/00 20130101; C12N 15/52 20130101 |
Class at
Publication: |
435/7.1 ; 435/6;
435/455; 435/91.2 |
International
Class: |
C12Q 001/68; G01N
033/53; C12P 019/34; C12N 015/85 |
Claims
What is claimed:
1. A method of screening clones having DNA recovered from a
plurality of species of organisms for a specified enzyme activity,
which method comprises: screening for a specified enzyme activity
in a library of clones prepared by (i) recovering DNA from a DNA
population derived from a plurality of species of organisms; and
(ii) transforming a host cell with the DNA of (i) to produce a
library of clones which is screened for the specified enzyme
activity.
2. The method of claim 1, wherein the DNA is amplified prior to
transforming the host cell.
3. The method of claim 1, wherein the DNA is ligated into a vector
prior to transforming the host cell.
4. The method of claim 3, wherein the vector comprises at least one
DNA sequence capable of regulating production of a detectable
enzyme activity from said DNA.
5. The method of claim 3, wherein the vector into which the DNA has
been ligated is used to transform a host cell.
Description
[0001] This invention relates to enzyme libraries and kits and to
the preparation thereof. More particularly, the present invention
is directed to recombinant enzyme expression libraries, recombinant
enzyme libraries and kits prepared therefrom which recombinant
enzymes are generated from DNA obtained from microorganisms.
[0002] Industry has recognized the need for new enzymes for a wide
variety of industrial applications. As a result, a variety of
microorganisms have been screened to ascertain whether or not such
microorganisms have a desired enzyme activity. If such
microorganism does have a desired enzyme activity, the enzyme is
then recovered from the microorganism.
[0003] In accordance with the present invention, there is provided
a novel approach for obtaining enzymes for further use, for
example, for packaging into kits for further research. In
accordance with the present invention, recombinant enzymes are
generated from microorganisms and are classified by various enzyme
characteristics. In this manner, the enzymes can be provided as
packaged enzyme screening kits, with enzymes in the kit being
grouped to have selected enzyme characteristics.
[0004] More particularly, in accordance with an aspect of the
present invention there is provided a recombinant expression
library which is comprised of a multiplicity of clones which are
capable of expressing recombinant enzymes. The expression library
is produced by recovering DNA from a microorganism, cloning such
DNA into an appropriate expression vector which is then used to
transfect or transform an appropriate host for expression of a
recombinant protein.
[0005] Thus, for example, genomic DNA may be recovered from either
a culturable or non-culturable organism and employed to produce an
appropriate recombinant expression library for subsequent
determination of enzyme activity.
[0006] In accordance with an aspect of the present invention, such
recombinant expression library may be prepared without prescreening
the organism from which the library is prepared for enzyme
activity.
[0007] Having prepared a multiplicity of recombinant expression
clones from DNA isolated from an organism, the polypeptides
expressed by such clones are screened for enzyme activity and
specified enzyme characteristics in order to identify and classify
the recombinant clones which produce polypeptides having specified
enzyme characteristics.
[0008] In accordance with a preferred aspect of the present
invention, the recombinant enzymes are characterized by both
physical and chemical characteristics and such chemical
characteristics are preferably classified in a tiered manner such
that recombinant enzymes having a chemical characteristic in common
are then classified by other chemical characteristics which may or
may not be more selective or specific chemical characteristic and
so on, as hereinafter indicated in more detail.
[0009] As hereinabove indicated, the recombinant enzymes are also
preferably classified by physical characteristics and one or more
tiers of the enzymes which are classified by chemical
characteristics may also be classified by physical classifications
or vice versa.
[0010] As used herein, the term "chemical characteristics" of a
recombinant enzyme refers to the substrate or chemical
functionality upon which the enzyme acts and/or the catalytic
reaction performed by the enzyme, e.g., the catalytic reaction may
be hydrolysis (hydrolases) and the chemical functionality may be
the type of bond upon which the enzyme acts (esterases cleave ester
bonds) or may be the particular type of structure upon which the
enzyme acts (a glycosidase which acts on glycosidic bonds). Thus,
for example, a recombinant enzyme which acts on glycosidic bonds
may, for example, be chemically classified in accordance with the
tiered system as: Tier 1: hydrolase; Tier 2: acetal bonds; Tier 3:
glycosidase.
[0011] As used herein, a "physical characteristic" with respect to
a recombinant enzyme means a property (other than a chemical
reaction) such as pH; temperature stability; optimum temperature
for catalytic reaction; organic solvent tolerance; metal ion
selectivity; detergent sensitivity, etc.
[0012] In an embodiment of the invention, in which a tiered
approach is employed for classifying the recombinant enzymes by
chemical and/or physical characteristics, the enzymes at one or
more of the chemical characteristic tiers may also be classified by
one or more physical characteristics and vice versa. In a preferred
embodiment, the enzymes are classified by both physical and
chemical characteristics, e.g., the individual substrates upon
which they act as well as physical characteristics.
[0013] Thus, for example, as a representative example of the manner
in which a recombinant enzyme may be classified in accordance with
the present invention, a recombinant enzyme which is a protease (in
this illustration Tier 1 is hydrolase; Tier 2 is amide (peptide
bond) that may be further classified in Tier 3 as to the ultimate
site in the amino acid sequence where cleavage occurs; e.g., anion,
cation, large hydrophobic, small hydrophobic. Each of the
recombinant enzymes which has been classified by the side chain in
Tier 3 may also be further classified by physical characteristics
of the type hereinabove indicated.
[0014] In this manner, it is possible to select from the
recombinant library, enzymes which have a specified chemical
characteristic in common, e.g., all endopeptidases (which act on
internal peptide bonds) and which have a specified physical
characteristic in common, e.g., all act optimally at a pH within a
specified range.
[0015] As hereinabove indicated, a recombinant enzyme library
prepared from a microorganism is preferably classified by chemical
characteristics in a tiered approach. This may be accomplished by
initially testing the recombinant polypeptides generated by the
library in a low selectivity screen, e.g., the catalytic reaction
performed by the enzyme. This may be conveniently accomplished by
screening for one or more of the six IUB classes; Oxidoreductases;
transferases; hydrolases; lyases, isomerases, ligases.
[0016] The recombinant enzymes which are determined to be positive
for one or more of the IUB classes may then be rescreened for a
more specific enzyme activity.
[0017] Thus, for example, if the recombinant library is screened
for hydrolase activity, then those recombinant clones which are
positive for hydrolase activity may be rescreened for a more
specialized hydrolase activity, i.e., the type of bond on which the
hydrolase acts. Thus, for example, the recombinant enzymes which
are hydrolases may be rescreened to ascertain those hydrolases
which act on one or more specified chemical functionalities, such
as: (a) amide (peptide bonds), i.e., proteases; (b) ester bonds,
i.e., esterases and lipases; (c) acetals, i.e., glycosidases,
etc.
[0018] The recombinant enzymes which have been classified by the
chemical bond on which they act may then be rescreened to determine
a more specialized activity therefor, such as the type of substrate
on which they act.
[0019] Thus, for example, those recombinant enzymes which have been
classified as acting on ester bonds (lipases and esterases) may be
rescreened to determine the ability thereof to generate optically
active compounds, i.e., the ability to act on specified substrates,
such as meso alcohols, meso diacids, chiral alcohols, chiral acids,
etc.
[0020] For example, the recombinant enzymes which have been
classified as acting on acetals may be rescreened to classify such
recombinant enzymes by a specific type of substrate upon which they
act, e.g., (a) P1 sugar such as glucose, galactose, etc., (b)
glucose polymer (exo-, endo- or both), etc.
[0021] Enzyme Tiers
[0022] Thus, as a representative but not limiting example, the
following are representative enzyme tiers:
[0023] TIER 1. Divisions are based upon the catalytic reaction
performed by the enzyme, e.g., hydrolysis, reduction, oxidation,
etc. The six IUB classes will be used: Oxidoreductase,
Transferases, Hydrolases, Lyases, Isomerases, Ligases.
[0024] TIER 2. Divisions are based upon the chemical functionality
undergoing reaction, e.g., esters, amides, phosphate diesters,
sulfate mono esters, aldehydes, ketones, alcohols, acetals, ketals,
alkanes, olefins, aromatic rings, heteroaromatic rings, molecular
oxygen, enols, etc.
[0025] Lipases and esterases both cleave the ester bond; the
distinction comes in whether the natural substrate is aggreagated
into a membrane (lipases) or dispersed into solution
(esterases).
[0026] TIER 3. Divisions and subdivisions are based upon the
differences between individual substrate structures which are
covalently attached to the functionality undergoing reaction as
defined in Tier 2. For example acetal hydrolysis: is the acetal
part of glucose or galactose; or is the acetal the .alpha. or
.beta. anomer? These are the types of distinctions made in TIER 3.
The divisions based upon substrate specificity are unique to each
particular enzyme reaction; there will be different substrate
distinctions depending upon whether the enzyme is, for example, a
protease or phosphatase.
[0027] TIER 4. Divisions are based on which of the two possible
enantiomeric products the enzyme produces. This is a measure of the
ability of the enzyme to selectively react with one of the two
enantimoers (kinetic resolution), or the ability of the enzyme to
react with a meso difunctional compound to selectively generate one
of the two enantiomeric reaction products.
[0028] TIER 5/ORTHOGONAL TIER/PHYSICAL CHARACTER TIER.
[0029] The fifth tier is orthogonal to the other tiers. It is based
on the physical properties of the enzymes, rather than the chemical
reactions, per se: The Fifth Tier forms a second dimension with
which to classify the enzymes. The Fifth Tier can be applied to any
of the other Tiers, but will most often be applied to the Third
Tier.
[0030] Thus, in accordance with an aspect of the present invention,
an expression library is randomly produced from the DNA of
microorganism, in particular, the genomic DNA of cDNA of the
microorganism and the recombinant proteins or polypeptides produced
by such expression library are screened to classify the recombinant
enzymes by different enzyme characteristics. In a preferred
embodiment, the recombinant proteins are screened for one or more
particular chemical characteristics and the enzymes identified as
having such characteristics are then rescreened for a more specific
chemical characteristic and this rescreening may be repeated one or
more times. In addition, in a preferred embodiment, the recombinant
enzymes are also screened to classify such enzymes by one or more
physical characteristics. In this manner, the recombinant enzymes
generated from the DNA of a microorganism are classified by both
chemical and physical characteristics and it is therefore possible
to select recombinant enzymes from one or more different organisms
that have one or more common chemical characteristics and/or one or
more common physical characteristics. Moreover, since such enzymes
are recombinant enzymes, it is possible to produce such enzymes in
desired quantities and with a desired purity.
[0031] The tiered approach of the present invention is not limited
to a tiered approach in which, for example, the tiers are more
restrictive. For example, the tiered approach is also applicable to
using a tiered approach in which, for example, the first tier is
"wood degrading" enzymes. The second chemical tier could then, for
example, be the type of enzyme which is a "wood degrading"
enzyme.
[0032] Similarly, the first tier or any other tier could be
physical characteristics and the next tier could be specified
chemical characteristics.
[0033] Thus, the present invention is generally applicable to
providing recombinant enzymes and recombinant enzyme libraries
wherein various enzymes are classified by different chemical and/or
physical characteristics.
[0034] The microorganisms from which the recombinant libraries may
be prepared include prokaryotic microorganisms, such as Eubacteria
and Archaebacteria, and lower eukaryotic microorganisms such as
fungi, some algae and protozoa. The microorganisms may be cultured
microorganisms or uncultured microorganisms obtained from
environmental samples and such microorganisms may be extremophiles,
such as thermophiles, hyperthermophiles, psychrophiles,
psychrotrophs, etc.
[0035] The recombinant enzymes in the library which are classified
as hereinabove described may or may not be sequenced and may or may
not be in a purified form. Thus, in accordance with the present
invention, it is possible to classify one or more of the
recombinant enzymes before or after obtaining the sequence of the
enzyme or before or after purifying the enzyme to essential
homogeneity.
[0036] The screening for chemical characteristics may be effected
on individual expression clones or may be initially effected on a
mixture of expression clones to ascertain whether or not the
mixture has one or more specified enzyme activities. If the mixture
has a specified enzyme activity, then the individual clones may be
rescreened for such enzyme activity or for a more specific
activity. Thus, for example, if a clone mixture has hydrolase
activity, then the individual clones may be recovered and screened
to determine which of such clones has hydrolase activity.
[0037] The present invention is also directed to preparing and
providing enzyme kits for use in further screening and/or research.
Thus, in accordance with an aspect of the invention, a reagent
package or kit is prepared by placing in the kit or package, e.g.,
in suitable containers, at least three different recombinant
enzymes with each of the at least three different recombinant
enzymes having at least two enzyme characteristics in common. In a
preferred embodiment, one common characteristic is a chemical
characteristic or property and the other common characteristic is a
physical characteristic or property; however, it is possible to
prepare kits which have two or more chemical characteristics or
properties in common and no physical characteristics or property in
common and vice versa.
[0038] Since, in accordance with the present invention, it is
possible to provide a recombinant enzyme library from one or more
microorganisms which is classified by a multiplicity of chemical
and/or physical properties, a variety of enzyme kits or packages
can be prepared having a variety of selected chemical and/or
physical characteristics which can be formulated to contain three
or more recombinant enzymes in which at least three and preferably
all of the recombinant enzymes have in common at least one chemical
characteristic and have in common at least one physical
characteristic. The kit should contain an appropriate label
specifying such common characteristics.
[0039] In one embodiment, at least three recombinant enzymes in the
kit have in common the most specific chemical characteristic
specified on the label. The term "label" is used in its broadest
sense and includes package inserts or literature associated or
distributed in conjunction with the kit or package. Thus, for
example, if the kit is labeled for a specific substrate (one of the
Tier 3 examples above), then for example, at least three of the
enzymes in the kit would act on such substrate.
[0040] The kits will preferably contain more than three enzymes,
for example, five, six or more enzymes and in a preferred
embodiment at least three and preferably a majority and in some
cases all of the recombinant enzymes in the kit will have at least
two enzyme properties or characteristics in common, as hereinabove
described.
[0041] The recombinant enzymes in the kits may have two or more
enzymes in a single container or individual enzymes in individual
containers or various combinations thereof.
[0042] As hereinabove indicated, the expression library may be
produced from environmental samples in which case DNA may be
recovered without culturing of an organism or the DNA may be
recovered from a cultured organism.
[0043] In preparing the expression library genomic DNA may be
recovered from either a cultured organism or an environmental
sample (for example, soil) by various procedures. The recovered or
isolated DNA is then fragmented into a size suitable for producing
an expression library and for providing a reasonable probability
that desired genes will be expressed and screened without the
necessity of screening an excessive number of clones. Thus, for
example, if the average genome fragment produced by shearing is 4.5
kbp, for a 1.8 Mbp genome about 2000 clones should be screened to
achieve about a 90% probability of obtaining a particular gene. In
some cases, in particular where the DNA is recovered without
culturing, the DNA is amplified (for example by PCR) after
shearing.
[0044] The sized DNA is cloned into an appropriate expression
vector and transformed into an appropriate host, preferably a
bacterial host and in particular E. coli. Although E. coli is
preferred, a wide variety of other hosts may be used for producing
an expression library.
[0045] The expression vector that is used is preferably one which
includes a promoter which is known to function in the selected host
in case the native genomic promoter does not function in the
host.
[0046] As representative examples of expression vectors which may
be used for preparing an expression library, there may be mentioned
phage, plasmids, phagemids cosmids, phosmids, bacterial artificial
chromosomes, P1-based artificial chromosomes, yeast artificial
chromosomes, and any other vectors specific for specific hosts of
interest (such as bacillus, aspergillus, yeast, etc.) The vector
may also include a tag of a type known in the art to facilitate
purification.
[0047] The following outlines a general procedure for producing
expression libraries from both culturable and non-culturable
organisms.
[0048] Culturable Organisms
[0049] Obtain Biomass
[0050] DNA Isolation (CTAB)
[0051] Shear DNA (25 gauge needle)
[0052] Blunt DNA (Mung Bean Nuclease)
[0053] Methylate (EcoR I Methylase)
[0054] Ligate to EcoR I linkers (GGAATTCC)
[0055] Cut back linkers (EcoR I Restriction Endonuclease)
[0056] Size Fractionate (Sucrose Gradient)
[0057] Ligate to lambda vector (Lambda ZAP II and gt11)
[0058] Package (in vitro lambda packaging extract)
[0059] Plate on E. coli host and amplify
[0060] Unculturable Organisms
[0061] Obtain cells
[0062] Isolate DNA (Various Methods)
[0063] Blunt DNA (Mung Bean Nuclease)
[0064] Ligate to adaptor containing a Not I site and conjugated to
magnetic beads
[0065] Ligate unconjugated adaptor to the other end of the DNA
[0066] Amplify DNA in a reaction which allows for high fidelity,
and uses adaptor sequences as primers
[0067] Cut DNA with Not I
[0068] Size fractionate (Sucrose Gradient or Sephacryl Column)
[0069] Ligate to lambda vector (Lambda ZAP II and gt11)
[0070] Package (in vitro lambda packaging extract)
[0071] Plate on E. coli host and amplify
[0072] The expression libraries may be screened for one or more
selected chemical characteristics. Selected representative chemical
characteristics are described below but such characteristics do not
limit the present invention. Moreover, the expression libraries may
be screened for some or all of the characteristics. Thus, some of
the chemical characteristics specified herein may be determined in
all of the libraries, none of the libraries or in only some of the
libraries.
[0073] The recombinant enzymes may also be tested and classified by
physical properties. For example, the recombinant enzymes may be
classified by physical properties such as follows:
[0074] pH Optima
[0075] <3
[0076] 3-6
[0077] 6-9
[0078] 9-12
[0079] >12
[0080] Temperature Optima
[0081] >90.degree. C.
[0082] 75-90.degree. C.
[0083] 60-75.degree. C.
[0084] 45-60.degree. C.
[0085] 30-45.degree. C.
[0086] 15-30.degree. C.
[0087] 0-15.degree. C.
[0088] Temperature Stability
[0089] half-life at:
[0090] 90.degree. C.
[0091] 75.degree. C.
[0092] 60.degree. C.
[0093] 45.degree. C.
[0094] Organic Solvent Tolerance
[0095] water miscible
[0096] (DMF)
[0097] 90%
[0098] 75%
[0099] 45%
[0100] 30%
[0101] water immiscible
[0102] hexane
[0103] toluene
[0104] metal ion selectivity
[0105] EDTA-10 mM
[0106] Ca.sup.+2-1 mM
[0107] Mg.sup.+2-100 TM
[0108] Mn.sup.+2-10 TM
[0109] Co.sup.+3-10 TM
[0110] Detergent Sensitivity
[0111] neutral (triton)
[0112] anionic (deoxycholate)
[0113] cationic (CHAPS)
[0114] The recombinant enzymes of the libraries and kits of the
present invention may be used for a variety of purposes and the
present invention by providing a plurality of recombinant enzymes
classified by a plurality of different enzyme characteristics
permits rapid screening of enzymes for a variety of applications.
Thus, for example, the present invention permits assembly of enzyme
kits which contain a plurality of enzymes which are capable of
operating on a specific bond or a specific substrate at specified
conditions to thereby enable screening of enzymes for a variety of
applications. As representative examples of such applications,
there may be mentioned:
[0115] 1 Lipase/Esterase
[0116] a. Enantioselective hydrolysis of esters
(lipids)/thioesters
[0117] 1) Resolution of racemic mixtures
[0118] 2) Synthesis of optically active acids or alcohols from
meso-diesters
[0119] b. Selective syntheses
[0120] 1) Regiospecific hydrolysis of carbohydrate esters
[0121] 2) Selective hydrolysis of cyclic secondary alcohols
[0122] c. Synthesis of optically active esters, lactones, acids,
alcohols
[0123] 1) Transesterification of activated/nonactivated esters
[0124] 2) Interesterification
[0125] 3) Optically active lactones from hydroxyesters
[0126] 4) Regio- and enantioselective ring opening of
anhydrides
[0127] d. Detergents
[0128] e. Fat/Oil conversion
[0129] f. Cheese ripening
[0130] Protease
[0131] a. Ester/amide synthesis
[0132] b. Peptide synthesis
[0133] c. Resolution of racemic mixtures of amino acid esters
[0134] d. Synthesis of non-natural amino acids
[0135] e. Detergents/protein hydrolysis
[0136] Glycosidase/Glycosyl Transferase
[0137] a. Sugar/polymer synthesis
[0138] b. Cleavage of glycosidic linkages to form mono, di- and
oligosaccharides
[0139] c. Synthesis of complex oligosaccharides
[0140] d. Glycoside synthesis using UDP-galactosyl transferase
[0141] e. Transglycosylation of disaccharides, glycosyl fluorides,
aryl galactosides
[0142] f. Glycosyl transfer in oligosaccharide synthesis
[0143] g. Diastereoselective cleavage of -glucosylsulfoxides
[0144] h. Asymmetric glycosylations
[0145] i. Food processing
[0146] j. Paper processing
[0147] Phosphatase/Kinase
[0148] a. Synthesis/hydrolysis of phosphate esters
[0149] 1) Regio-, enantioselective phosphorylation
[0150] 2) Introduction of phosphate esters
[0151] 3) Synthesize phospholipid precursors
[0152] 4) Controlled polynucleotide synthesis
[0153] b. Activate biological molecule
[0154] c. Selective phosphate bond formation without protecting
groups
[0155] Mono/Dioxygenase
[0156] a. Direct oxyfunctionalization of unactivated organic
substrates
[0157] b. Hydroxylation of alkane, aromatics, steroids
[0158] c. Epoxidation of alkenes
[0159] d. Enantioselective sulphoxidation
[0160] e. Regio- and steroselective Bayer-Villiger oxidations
[0161] Haloperoxidase
[0162] a. Oxidative addition of halide ion to nucleophilic
sites
[0163] b. Addition of hypohalous acids to olefinic bonds
[0164] c. Ring cleavage of cyclopropanes
[0165] d. Activated aromatic substrates converted to ortho and para
derivatives
[0166] e. 1.3 diketones converted to 2-halo-derivatives
[0167] f. Heteroatom oxidation of sulfur and nitrogen containing
substrates
[0168] g. Oxidation of enol acetates, alkynes and activated
aromatic rings
[0169] Lignin peroxidase/Diarylpropane Peroxidase
[0170] a. Oxidation cleavage of C-C bonds
[0171] b. Oxidation of benzylic alcohols to adlehydes
[0172] c. Hydorxylation of benzylic carbons
[0173] d. Phenol dimerization
[0174] e. Hydroxylation of double bonds to form diols
[0175] f. Cleavage of lignin aldehydes
[0176] Epoxide Hydrolase
[0177] a. Synthesis of enantiomerically pure bioactive
compounds
[0178] b. Regio- and enantioselective hydrolysis of epoxide
[0179] c. Aromatic and olefinic epoxidation by monooxygenases to
form epoxides
[0180] d. Resoltuion of racemic epoxides
[0181] e. Hydrolysis of steroid epoxides
[0182] Nitrile Hydratase/nitrilase
[0183] a. Hydrolysis of aliphatic nitriles to carboxamides
[0184] b. Hydrolysis of aromatic, heterocyclic, unsaturated
aliphatic nitriles to corresponding acids
[0185] c. Hydrolysis of acrylonitrile
[0186] d. Production of aromatic and carboxamides, carboxylic acids
(nicotinamide, picolinamide, isonicotinamide)
[0187] e. Regioselective hydrolysis of acrylic dinitrile
[0188] f. .alpha.-amino acids form .alpha.-hydroxynitriles
[0189] 10 Transaminase
[0190] a. Transfer of amino groups into oxo-acids
[0191] 11 Amidase/Acylase
[0192] a. Hydrolysis of amides, amidines, and other C-N bonds
[0193] b. Non-natural amino acid resolution and synthesis
DESCRIPTION OF THE DRAWINGS
[0194] FIG. 1 is a schematic representation of one embodiment of
various tiers of chemical characteristics of an enzyme which may be
employed in the present invention.
[0195] FIG. 2 is a schematic representation of another embodiment
of various tiers of chemical characteristics of an enzyme which may
be employed in the present invention.
[0196] FIG. 3 is a schematic representation of a further embodiment
of various tiers of chemical characteristics of an enzyme which may
be employed in the present invention.
[0197] FIG. 4 is a schematic representation of a still further
embodiment of various tiers of chemical characteristics of an
enzyme which may be employed in the present invention.
[0198] The invention will be further described with reference to
the following examples; however, the scope of the present invention
is not to be limited thereby. Unless otherwise specified, all parts
are by weight.
EXAMPLE 1
[0199] Production of Expression Library.
[0200] The following describes a representative procedure for
preparing an expression library for screening by the tiered
approach of the present invention.
[0201] One gram of Themococcus GU5L5 cell pellet was lysed and the
DNA isolated by literature procedures.sup.1. Approximately 100
.mu.g of the isolated DNA was resuspended in TE buffer and
vigorously passed through a 25 gauge double-hubbed needle until the
sheared fragments were in the size range of 0.5-10.0 Kb (3.0 Kb
average). The DNA ends were "polished" or blunted with Mung Bean
Nuclease (300 units, 37.degree. C., 15 minutes), and EcoRI
restriction sites in the target DNA protected with EcoRI Methylase
(200 units, 37.degree. C., 1 hour). EcoRI linkers [GGAATTCC] were
ligated to the blunted/protected DNA using 10 pmole ends of linkers
to 1 pmole end of target DNA. The linkers were cut back with EcoRI
restriction endonuclease (200 units, 37.degree. C., 1.5 hours) and
the DNA size fractionated by sucrose gradient (Maniatis, T.,
Fritsch, E. F., and Sambrook, J. Molecular Cloning, Cold Spring
Harbor Press, New York, 1982). The prepared target DNA was ligated
to the Lambda ZAP.RTM. II vector (Stratagene), packaged using in
vitro lambda packaging extracts and grown on XL1-Blue MRF's E. coli
strain according to the manufacturer. The pBluescript.RTM.
phagemids were excised from the lambda library, and grown in E.
coli DH10B F' kan, according to the method of Hay and Short.sup.2.
The resulting colonies were picked with sterile toothpicks and used
to singly inoculate each of the wells of 11 96-well microtiter
plates (1056 clones in all). The wells contained 250 TL of LB media
with 100 .mu.g/mL ampicillin, 80 .mu.g/mL methicillin, and 10% v/v
glycerol (LB Amp/Meth, glycerol). The cells were grown overnight at
37.degree. C. without shaking. This constituted generation of the
"Source Library"; each well of the Source Library thus contained a
stock culture of E. coli cells, each of which contained a
pBluescript.RTM. phagemid with a unique DNA insert. .sup.1 Current
Protocols in Molecular Biology (1987) 2.4.1. .sup.2 Hay, B. and
Short, J. Strategies. 1992, 5, 16.
EXAMPLE 2
[0202] The following is a representative example of a procedure for
screening an expression library prepared in accordance with Example
1. In the following, the chemical characteristic Tiers are as
follows:
[0203] Tier 1: Hydrolase
[0204] Tier 2: Amide, Ester and Acetal
[0205] Tier 3: Divisions and subdivisions are based upon the
differences between individual substrates which are covalently
attached to the functionality of Tier 2 undergoing reaction; as
well as substrate specificity.
[0206] Tier 4: The two possible enantiomeric products which the
enzyme may produce from a substrate.
[0207] Although the following example is specifically directed to
the above mentioned tiers, the general procedures for testing for
various chemical characteristics is generally applicable to
substrates other than those specifically referred to in this
Example.
[0208] Screening for Tier 1-hydrolase; Tier 2-amide.
[0209] The eleven plates of the Source Library were used to
multiply inoculate a single plate (the "Condensed Plate")
containing in each well 200 .mu.L of LB Amp/Meth, glycerol. This
step was performed using the High Density Replicating Tool (HDRT)
of the Beckman Biomek with a 1% bleach, water, isopropanol, air-dry
sterilization cycle in between each inoculation. Each well of the
Condensed Plate thus contained 11 different pBluescript.RTM. clones
from each of the eleven source library plates. The Condensed Plate
was grown for 2 h at 37.degree. C. and then used to inoculate two
white 96-well Dynatech microtiter daughter plates containing in
each well 250 TL of LB Amp/Meth, glycerol. The original condensed
plates were incubated at 37.degree. C. for 18 h then stored at
-80.degree. C. The two condensed daughter plates were incubated at
37.degree. C. also for 18 h. The condensed daughter plates were
then heated at 70.degree. C. for 45 min. to kill the cells and
inactivate the host E. coli enzymes. A stock solution of 5 mg/mL
morphourea phenylalanyl-7-amino-4-trifluoromethyl coumarin
(MuPheAFC, the `substrate`) in DMSO was diluted to 600 .mu.M with
50 mM pH 7.5 Hepes buffer containing a 0.6 mg/mL of the detergent
dodecyl maltoside. 1
[0210] Fifty .mu.L of the 600 .mu.M MuPheAFC solution was added to
each of the wells of the white condensed plates with one 100 .mu.L
mix cycle using the Biomek to yield a final concentration of
substrate of .about.100 .mu.M. The fluorescence values were
recorded (excitation=400 nm, emission=505 nm) on a plate reading
fluorometer immediately after addition of the substrate (t=0). The
plate was incubated at 70.degree. C. for 100 min, then allowed to
cool to ambient temperature for 15 additional minutes. The
fluorescence values were recorded again (t=100). The values at t=0
were subtracted from the values at t=100 to determine if an active
clone was present.
[0211] This data indicated that one of the eleven clones in well G8
was hydrolyzing the substrate. In order to determine the individual
clone which carried the activity, the eleven source library plates
were thawed and the individual clones used to singly inoculate a
new plate containing LB Amp/Meth, glycerol. As above, the plate was
incubated at 37.degree. C. to grow the cells, heated at 70.degree.
C. to inactivate the host enzymes, and 50 .mu.L of 600 .mu.M
MuPheAFC added using the Biomek. Additionally three other
substrates were tested. The methyl umbelliferone heptanoate, the
CBZ-arginine rhodamine derivative, and fluorescein-conjugated
casein (.about.3.2 mol fluorescein per mol of casein). 2
[0212] The umbelliferone and rhodamine were added as 600 .mu.M
stock solutions in 50 .mu.L of Hepes buffer. The fluorescein
conjugated casein was also added in 50 .mu.L at a stock
concentration of 20 and 200 mg/mL. After addition of the substrates
the t=0 fluorescence values were recorded, the plate incubated at
70.degree. C., and the t=100 min. values recorded as above.
[0213] This data indicated that the active clone was in plate 2.
The arginine rhodamine derivative was also turned over by this
activity, but the lipase substrate, methyl umbelliferone
heptanoate, and protein, fluorescein-conjugated casein, did not
function as substrates.
[0214] Based on the above data the Tier 1 classification is
`hydrolase` and the Tier 2 classification is amide bond. There is
no cross reactivity with the Tier 2-ester classification.
[0215] As shown in FIG. 1, a recombinant clone from the library
which has been characterized in Tier 1 as hydrolase and in Tier 2
as amide may then be tested in Tier 3 for various specificities. In
FIG. 1, the various classes of Tier 3 are followed by a
parenthetical code which identifies the substrates of Table 1 which
are used in identifying such specificities of Tier 3.
[0216] As shown in FIGS. 2 and 3, a recombinant clone from the
library which has been characterized in Tier 1 as hydrolase and in
Tier 2 as ester may then be tested in Tier 3 for various
specificities. In FIGS. 2 and 3, the various classes of Tier 3 are
followed by a parenthetical code which identifies the substrates of
Tables 2 and 2 which are used in identifying such specificities of
Tier 3. In FIGS. 2 and 3, R.sub.2 represents the alcohol portion of
the ester and R.sub.1 represents the acid portion of the ester.
[0217] As shown in FIG. 4, a recombinant clone from the library
which has been characterized in Tier 1 as hydrolase and in Tier 2
as acetal may then be tested in Tier 3 for various specificities.
In FIG. 4, the various classes of Tier 3 are followed by
parenthetical code which identifies the substrates of Table 4 which
are used in identifying such specificities of Tier 3.
[0218] Enzymes may be classified in Tier 4 for the chirality of the
product(s) produced by the enzyme. For example, chiral amino esters
may be determined using at least the following substrates: 3
[0219] For each substrate which is turned over the
enantioselectivity value, E, is determined according to the
equation below.sup.3: .sup.3 Wong and Whitesides, Enzymes in
Synthetic Organic Chemistry, 1994, Elsevier, Tarrytown, N.Y., pp.
9-12. 1 E = ln [ ( 1 - c ( 1 + ee p ) ] ln [ ( 1 - c ( 1 - ee p )
]
[0220] where ee.sub.p=the enantiomeric excess (ee) of the
hydrolyzed product and c=the percent conversion of the
reaction.
[0221] The enantiomeric excess is determined by either chiral high
performance liquid chromatography (HPLC) or chiral capillary
clectrophoresis (CE). Assays are performed as follows: two hundred
.mu.L of the appropriate buffer is added to each well of a 96-well
white microtiter plate, followed by 50 .mu.L of partially or
completely purified enzyme solution; 50 .mu.L of substrate as added
and the increase in fluorescence monitored versus time until 50% of
the substrate is consumed or the reaction stops, which ever comes
first.
EXAMPLE 3
[0222] This Example 3 describes procedures for testing for certain
physical characteristics of recombinant clones of a library.
[0223] pH Optima.
[0224] Two hundred .mu.L of the appropriate pH and ionic strength,
temperature equilibrated buffer.sup.4 is added to each well of a
96-well white microtiter plate, followed by 50 .mu.L of partially
or completely purified enzyme solution; 50 .mu.L of substrate at
5.times. K.sub.M (final concentration) or the solubility limit,
which ever is lower, is added to initiate the reaction and the
increase in fluorescence monitored versus time. The rate .sup.4
Ellis, K. J. and Morrison, J. F. Methods in Enzymology, 1982, 87,
405. of reaction at a defined temperature is determined under
initial velocity (linear) conditions. .sup.4 Ellis, K. J. and
Morrison, J. F. Methods in Enzymology, 1982, 87, 405.
[0225] Temperature Optima.
[0226] Two hundred .mu.L of the optimal pH and ionic strength,
temperature equilibrated buffer is added to each well of a 96-well
white microtiter plate, followed by 50 .mu.L of partially or
completely purified enzyme solution; 50 .mu.L of substrate at
5.times. K.sub.M or the solubility limit, which ever is lower, is
added to initiate the reaction and the increase in fluorescence
monitored versus time. The temperature of the incubation is varied.
The rate of reaction is determined under initial velocity (linear)
conditions.
[0227] Temperature Stability.
[0228] Reactions are performed as described for Temperature optima
except that substrate is added at selected time points during the
incubation and the initial velocity determined. The decrease in
activity (if present) is fit to a single exponential function to
determine the rate and half-life of inactivation.
[0229] Metal Ion Selectivity.
[0230] Two hundred .mu.L of the optimal pH and ionic strength,
temperature equilibrated buffer is added to each well of a 96-well
white microtiter plate, followed by 50 .mu.L of partially or
completely purified enzyme solution; 50 .mu.L of substrate at
5.times. K.sub.M or the solubility limit, which ever is lower, and
the desired metal ion or EDTA at 6.times. the indicated
concentration is added to initiate the reaction, and the increase
in fluorescence monitored versus time. The rate of reaction is
determined under initial velocity (linear) conditions.
[0231] Detergent Sensitivity.
[0232] Two hundred .mu.L of the optimal pH and ionic strength,
temperature equilibrated buffer is added to each well of a 96-well
white microtiter plate, followed by 50 .mu.L of partially of
completely purified enzyme solution; 50 .mu.L of substrate at
5.times. K.sub.M or the solubility limit, which ever is lower, and
the desired detergent at 6.times. the indicated concentration is
added to initiate the reaction, and the increase in fluorescence
monitored versus time. The rate of reaction is determined under
initial velocity (linear) conditions.
[0233] Organic Solvent Tolerance.
[0234] Water Miscible.
[0235] Two hundred .mu.L of buffer and dimethylformamide (DMF)
mixture (see table below) is added to each well of a 96-well white
microtiter plate, followed by 30 .mu.L of partially or completely
purified enzyme solution; 50 .mu.L of substrate at 5.times.
K.sub.M, or the solubility limit, which ever is lower in 100% DMF
is added to initiate the reaction, and the increase in fluorescence
monitored versus time. The rate of reaction is determined under
initial velocity (linear) conditions.
1 .mu.L DMF .mu.L Buffer % DMF Final 200 0 90 175 25 75 130 70 60
85 115 45 40 160 30
[0236] Water Immiscible.
[0237] Two hundred .mu.L of hexane or toluene is added to each well
of a 96-well white microtiter plate, followed by 50 .mu.L of
partially or completely purified enzyme solution; 50 .mu.L of
substrate at 5.times. K.sub.M or the solubility limit, which ever
is lower in buffer is added to initiate the reaction, and the
increase in fluorescence monitored versus time. The reaction is
shaken at 100 RPM. The rate of reaction is determined under initial
velocity (linear) conditions.
[0238] Numerous modifications and variations of the present
invention are possible in light of the above teachings; therefore,
within the scope of the claims, the invention may be practiced
other than as particularly described.
2 TABLE 1 A2 Fluorescein conjugated casein (3.2 mol fluorescein/mol
casein) CBZ-Ala-AMC t-BOC-Ala-Ala-Asp-AMC succinyl-Ala-Gly-Leu-AMC
CBZ-Arg-AMC CBZ-Met-AMC morphourea-Phc-AMC t-BOC = 1-butoxy
carbonyl, CBZ = carbonyl benzyloxy AMC = 7-amino-4-methyl coumarin
4 5 6 AD3 Fluorescein conjugated casein t-BOC-Ala-Ala-Asp-AFC
CBZ-Ala-Ala-Lys-AFC succinyl-Ala-Ala-Phe-AFC
succinyl-Ala-Gly-Leu-AFC AFC = 7-amino-4-trifluoromethyl coumarin.)
AE3 Fluorescein conjugated casein AF3 t-BOC-Ala-Ala-Asp-AFC
CBZ-Asp-AFC AG3 CBZ-Ala-Ala-Lys-AFC CBZ-Arg-AFC AH3
succinyl-Ala-Ala-Phe-AFC CBZ-Phe-AFC CBZ-Trp-AFC AI3
succinyl-Ala-Gly-Leu-AFC CBZ-Ala-AFC CBZ-Sewr-AFC
[0239]
3TABLE 2 L2 7 8 9 10 11 12 13 14 LA3 15 16 17 18 LB3 19 20 21 22
LD3 23 LE3 24 LF3 25 LG3 26
[0240]
4 TABLE 3 LII3 27 28 29 30 LI3 31 32 33 34 35 36 LJ3 37 38 39 40 41
42 LK3 43 LL3 44 45 LM3 46 LN3 47 LO3 48
[0241]
5TABLE 4 49 4-methyl umbelliferone where R = G2 .beta.-D-galactose
.beta.-D-glucose .beta.-D-glucuronide GB3 .beta.-D-cellotrioside
.beta.B-cellobiopyranoside GC3 .beta.-D-galactose
.alpha.-D-galactose GD3 .beta.-D-glucose .alpha.-D-glucose GE3
.beta.-D-glucuronide GI3 .beta.-D-N,N-diacetylchitobiose GJ3
.beta.-D-fucose .alpha.-L-fucose .beta.-L-fucose GK3
.beta.-D-mannose .beta.-D-mannose non-Umbelliferyl substrates GA3
amylose [polyglucan .alpha.1,4 linkages], amylopectin [polyglucan
branching .alpha.1,6 linkages] GF3 xylan [poly 1,4-D-xylan] GG3
amylopectin, pullulan GH3 sucrose, fructofuranoside
* * * * *