U.S. patent application number 09/753752 was filed with the patent office on 2002-05-16 for screening methods for enzymes and enzyme kits.
Invention is credited to Short, Jay M..
Application Number | 20020058254 09/753752 |
Document ID | / |
Family ID | 25529916 |
Filed Date | 2002-05-16 |
United States Patent
Application |
20020058254 |
Kind Code |
A1 |
Short, Jay M. |
May 16, 2002 |
Screening methods for enzymes and enzyme kits
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. Also disclosed is a process for identifying clones
of a recombinant library which express a protein with a desired
ctivity by screening a library of expression clones randomly
produced from DNA of at least one microorganism, said screeing
being effected on expression products of said clones to thereby
identify clones which express a protein with a desired activity.
Also disclosed is a process of screening clones having DNA from an
uncultivatedmicroorganism for a specified protein activity by
screening for a specified protein activity in a library of clones
prepared by (I) recovering DNA from a DNA population derived from
at least one uncultivated microorganism; and (ii) transforming a
host with recovered DNA to produce a library of clones which is
screened for the specified protein activity.
Inventors: |
Short, Jay M.; (Rancho Santa
Fe, CA) |
Correspondence
Address: |
LISA A. HAILE, PH.D.
GRAY CARY WARE & FREIDENRICH LLP
Suite 1600
4365 Executive Drive
San Diego
CA
92121
US
|
Family ID: |
25529916 |
Appl. No.: |
09/753752 |
Filed: |
January 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09753752 |
Jan 2, 2001 |
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08983367 |
Sep 30, 1998 |
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6168919 |
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09753752 |
Jan 2, 2001 |
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08657409 |
Jun 3, 1996 |
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5958672 |
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09753752 |
Jan 2, 2001 |
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08568994 |
Dec 7, 1995 |
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09753752 |
Jan 2, 2001 |
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08503606 |
Jul 18, 1995 |
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6004788 |
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Current U.S.
Class: |
435/6.16 ;
435/455; 435/6.19; 435/7.1 |
Current CPC
Class: |
C12N 15/1037 20130101;
C12N 15/1086 20130101; C12N 15/52 20130101; C12N 15/1093 20130101;
C12Q 1/00 20130101; C40B 40/02 20130101 |
Class at
Publication: |
435/6 ; 435/7.1;
435/455 |
International
Class: |
C12Q 001/68; G01N
033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 17, 1996 |
US |
PCT/US96/11854 |
Claims
What is claimed is:
1. A process for identifying clones of a recombinant library
produced from DNA derived from at least one uncultivated organism
which express a protein with a desired characteristic, comprising:
screening in the liquid phase a library of expression clones
randomly produced from DNA of at least one uncultivated organism,
said screening being effected on expression products of said clones
to thereby identify clones which express a protein with a desired
characteristic.
2. The method of claim 1, wherein the DNA is optionally modified or
mutagenized prior to formation of the recombinant library.
3. A process of screening clones having DNA recovered from an
uncultivated organism for a specified protein characteristic, which
process comprises: screening for a specified protein characteristic
in a library of clones prepared by (i) recovering DNA from a DNA
population derived from at least one uncultivated organism; and
(ii) transforming a host cell with the recovered DNA to produce a
library of clones which is screened for the specified protein
characteristic, wherein the DNA is optionally modified or
mutagenized prior to forming a library of clones.
Description
[0001] This invention relates to the field of preparing and
screening libraries of clones containing microbially derived DNA
and to protein, e.g. enzyme libraries and kits produced therefrom.
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] Naturally occurring assemblages of microorganisms often
encompass a bewildering array of physiological and metabolic
diversity. In fact, it has been estimated that to date less than
one percent of the world's organisms have been cultured. It has
been suggested that a large fraction of this diversity thus far has
been unrecognized due to difficulties in enriching and isolating
microorganisms in pure culture. Therefore, it has been difficult or
impossible to identify or isolate valuable enzymes from these
samples. These limitations suggest the need for alternative
approaches to characterize the physiological and metabolic
potential i.e. activities of interest of as-yet uncultivated
microorganisms, which to date have been characterized solely by
analyses of PCR amplified rRNA gene fragments, clonally recovered
from mixed assemblage nucleic acids.
[0004] In accordance with one aspect of 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.
[0005] More particularly, in accordance with this 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] In one aspect, the invention provides a process of screening
clones having DNA from an uncultivated microorganism for a
specified protein, e.g. enzyme, activity which process
comprises:
[0010] screening for a specified protein, e.g. enzyme, activity in
a library of clones prepared by
[0011] (i) recovering DNA from a DNA population derived from at
least one uncultivated microorganism and
[0012] (ii) transforming a host with recovered DNA to produce a
library of clones which are screened for the specified protein,
e.g. enzyme. activity.
[0013] The library is produced from DNA which is recovered without
culturing of an organism, particularly where the DNA is recovered
from an environmental sample containing microorganisms which are
not or cannot be cultured.
[0014] In a preferred embodiment of this aspect DNA is ligated into
a vector, particularly wherein the vector further comprises
expression regulatory sequences which can control and regulate the
production of a detectable enzyme activity from the ligated
DNA.
[0015] The f-factor (or fertility factor) in E. coli is a plasmid
which effects high frequency transfer of itself during conjugation
and less frequent transfer of the bacterial chromosome itself. To
archieve and stably propogate large DNA fragments from mixed
microbial samples, a particularly preferred embodiment is to use a
cloning vector containing an f-factor origin of replication to
generate genomic libraries that can be replicated with a high
degree of fidelity. When integrated with DNA from a mixed
uncultured environmental sample, this makes it possible to achieve
large genomic fragments in the form of a stable "environmental DNA
library."
[0016] In another preferred embodiment, double stranded DNA
obtained from the uncultivated DNA population is selected by:
[0017] converting the double stranded genomic DNA into single
stranded DNA:
[0018] recovering from the converted single stranded DNA single
stranded DNA which specifically binds, such as by hybridization, to
a probe DNA sequence: and
[0019] converting recovered single stranded DNA to double stranded
DNA.
[0020] The probe may be directly or indirectly bound to a solid
phase by which it is separated from single stranded DNA which is
not hybridized or otherwise specifically bound to the probe.
[0021] The process can also include releasing single stranded DNA
from said probe after recovering said hybridized or otherwise bound
single stranded DNA and amplifying the single stranded DNA so
released prior to converting it to double stranded DNA.
[0022] The invention also provides a process of screening clones
having DNA from an uncultivated microorganisms for a specified
protein, e.g. enzyme, activity which comprises screening for a
specified gene cluster protein product activity in the library of
clones prepared by: (i) recovering DNA from a DNA population
derived from at least one uncultivated microorganism; and (ii)
transforming a host with recovered DNA to produce a library of
clones with the screens for the specified protein, e.g. enzyme,
activity. The library is produced from gene cluster DNA which is
recovered without culturing of an organism, particularly where the
DNA gene clusters are recovered from an environmental sample
containing microorganisms which are not or cannot be cultured.
[0023] Alternatively, double-stranded gene cluster DNA obtained
from the uncultivated DNA population is selected by converting the
double-stranded genomic gene cluster DNA into single-stranded DNA;
recovering from the converted single-stranded gene cluster
polycistron DNA, single-stranded DNA which specifically binds, such
as by hybridization, to a polynucleotide probe sequence; and
converting recovered single-stranded gene cluster DNA to
double-stranded DNA.
[0024] These and other aspects of the present invention are
described with respect to particular preferred embodiments and will
be apparent to those skilled in the art from the teachings
herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 shows an overview of the procedures used to construct
an environmental library from a mixed picoplankion sample as
described in Example 3.
[0026] FIG. 2 is a schematic representation of one embodiment of
various tiers of chemical characteristics of an enzyme which may be
employed in the present invention as described in Example 4.
[0027] FIG. 3 is a schematic representation of another embodiment
of various tiers of chemical characteristics of an enzyme which may
be employed in the present invention as described in Example 4.
[0028] FIG. 4 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 as described in Example 4.
[0029] FIG. 5 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 as described
in Example 4.
[0030] FIG. 6 shows the pH optima results produced by enzyme
ESL-001-01 in the experiments described in Example 5.
[0031] FIG. 7 shows the temperature optima results produced by
enzyme ESL-001-01 in the experiments described in Example 5.
[0032] FIG. 8 shows the organic solvent tolerance results produced
by enzyme ESL-001-01 in the experiments described in Example 5.
[0033] DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0034] 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.
[0035] 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 characteristics
or vice versa.
[0036] As used herein, the term "chemical characteristic" 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.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] Enzyme Tiers
[0048] Thus, as a representative but not limiting example, the
following are representative enzyme tiers:
[0049] 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.
[0050] 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.
[0051] Lipases and esterases both cleave the ester bond: the
distinction comes in whether the natural substrate is aggregated
into a membrane (lipases) or dispersed into solution
(esterases).
[0052] 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
[0053] 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
enantiomers (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.
[0054] TIER 5/ORTHOGONAL TIER/PHYSICAL CHARACTER TIER. 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.
[0055] Thus, in accordance with an aspect of the present invention,
an expression library is randomly produced from the DNA of a
microorganism, in particular, the genomic DNA or 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.
[0056] 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.
[0057] Similarly, the first tier or any other tier could be
physical characteristics and the next tier could he specified
chemical characteristics.
[0058] 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.
[0059] 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.
[0060] Preferably, the library is produced from DNA which is
recovered without culturing of an organism, particularly where the
DNA is recovered from an environmental sample containing
microorganisms which are not or cannot be cultured. Sources of
microorganism DNA as a starting material library from which DNA is
obtained are particularly contemplated to include environmental
samples, such as microbial samples obtained from Arctic and
Antarctic ice, water or permafrost sources, materials of volcanic
origin, materials from soil or plant sources in tropical areas,
etc. Thus, for example, genomic DNA may be recovered from either
uncultured or non-culturable organism and employed to produce an
appropriate library of clones for subsequent determination of
enzyme activity.
[0061] Bacteria and many eukaryotes have a coordinated mechanism
for regulating genes whose products are involved in related
processes. The genes are clustered, in structures referred to as
"gene clusters," on a single chromosome and are transcribed
together under the control of a single regulatory sequence,
including a single promoter which initiates transcription of the
entire cluster. The gene cluster, the promoter, and additional
sequences that function in regulation altogether are referred to as
an "operon" and can include up to 20 or more genes usually from 2
to 6 genes. Thus, a gene cluster is a group of adjacent genes that
are either identical or related, usually as to their function.
[0062] Some gene families consist of identical members. Clustering
is a prerequisite for maintaining identity between genes, although
clustered genes are not necessarily identical. Gene clusters range
from extremes where a duplication is venerated to adjacent related
genes to cases where hundreds of identical genes lie in a tandem
array. Sometimes no significance is discernable in a repetition of
a particular gene. A principal example of this is the expressed
duplicate insulin genes in some species. whereas a single insulin
gene is adequate in other mammalian species.
[0063] It is important to further research gene clusters and the
extent to which the full length of the cluster is necessary for the
expression of the proteins resulting therefrom. Further, gene
clusters undergo continual reorganization and, thus, the ability to
create heterogeneous libraries of gene clusters from, for example,
bacterial or other prokaryote sources is valuable in determining
sources of novel proteins. particularly including proteins, e.g.
enzymes, such as, for example, the polyketide synthases that are
responsible for the synthesis of polyketides having a vast array of
useful activities. Other types of proteins that are the product(s)
of gene clusters are also contemplated, including, for example,
antibiotics, antivirals, antitumor agents and regulatory proteins,
such as insulin.
[0064] Polyketides are molcules which are an extremely rich source
of bioactivities, including antibiotics (such as tetracyclines and
erythromycin), anti-cancer agents (daunomycin), immunosuppressants
(FK506 and rapamycin), and veterinary products (monensin). Many
polyketides (produced by polyketide synthases) are valuable as
therapeutic agents. Polyketide synthases are multifunctional
enzymes that catalyze the biosynthesis of a huge variety of carbon
chains differing in length and patterns of functionality and
cyclization. Polyketide synthase genes fall into gene clusters and
at least one type (designated type I) of polyketide synthases have
large size genes and enzymes, complicating genetic manipulation and
in vitro studies of these genes/proteins.
[0065] The ability to select and combine desired components from a
library of polyketide and postpolyketide biosynthesis genes for
generation of novel polyketides for study is appealing. Using the
method(s) of the present invention facilitates the cloning of novel
polyketide synthases, particularly when one uses the f-factor based
vectors, which facilitate cloning of gene clusters.
[0066] Preferably, the gene cluster DNA is ligated into a vector,
particularly wherein a vector further comprises expression
regulatory sequences which can control and regulate the production
of a detectable protein or protein-related array activity from the
ligated gene clusters. Use of vectors which have an exceptionally
large capacity for exogenous DNA introduction are particularly
appropriate for use with such gene clusters and are described by
way of example herein to include the f-factor (or fertility factor)
of E. coli. This f-factor of E. coli is a plasmid which affect
high-frequency transfer of itself during conjugation and is ideal
to achieve and stably propagate large DNA fragments, such as gene
clusters from mixed microbial samples.
[0067] The term "derived" or "isolated" means that material is
removed from its original environment (e.g., the natural
environment if it is naturally occurring). For example, a
naturally-occurring polynucleotide or polypeptide present in a
living animal is not isolated, but the same polynucleotide or
polypeptide separated from some or all of the coexisting materials
in the natural system, is isolated.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] The expression vector which 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.
[0072] 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.
[0073] The following outlines a general procedure for producing
expression libraries from both culturable and non-culturable
organisms.
[0074] CULTURABLE ORGANISMS
[0075] Obtain Biomass
[0076] DNA Isolation (CTAB)
[0077] Shear DNA (25 gauze needle)
[0078] Blunt DNA (Mung Bean Nuclease)
[0079] Methylate (Eco RI Methylase)
[0080] Ligate to Eco RI linkers (GGAATTCC)
[0081] Cut back linkers (Eco RI Restriction Endonuclease)
[0082] Size Fractionate (Sucrose Gradient)
[0083] Ligate to lambda vector (Lambda ZAP II and gt11)
[0084] Package (in vitro lambda packaging extract)
[0085] Plate on E. coli host and amplify
[0086] UNCULTURABLE ORGANISMS
[0087] Obtain cells
[0088] Isolate DNA (Various Methods)
[0089] Blunt DNA (Mung Bean Nuclease)
[0090] Ligate to adaptor containing a Not I site and conjugated to
magnetic beads
[0091] Ligate unconjugated adaptor to the other end of the DNA
[0092] Amplify DNA in a reaction which allows for high fidelity,
and uses adaptor sequences as primers
[0093] Cut DNA with Not I
[0094] Size fractionate (Sucrose Gradient or Sephacryl Column)
[0095] Ligate to lambda vector (Lambda ZAP II and gt11)
[0096] Package (in vitro lambda packaging extract)
[0097] Plate on E. Coli host and amplify
[0098] The probe DNA used for selectively recovering DNA of
interest from the DNA derived from the at least one uncultured
microorganism can be a full-length coding region sequence or a
partial coding region sequence of DNA for an enzyme of known
activity, a phylogenetic marker or other identified DNA sequence.
The original DNA library can be preferably probed using mixtures of
probes comprising at least a portion of the DNA sequence encoding
the specified activity. These probes or probe libraries are
preferably single-stranded and the microbial DNA which is probed
has preferably been converted into single-stranded form. The probes
that are particularly suitable are those derived from DNA encoding
enzymes having an activity similar or identical to the specified
enzyme activity which is to be screened.
[0099] The probe DNA should be at least about 10 bases and
preferably at least 15 bases. In one embodiment, the entire coding
region may be employed as a probe. Conditions for the hybridization
in which DNA is selectively isolated by the use of at least one DNA
probe will be designed to provide a hybridization stringency of at
least about 50% sequence identity, more particularly a stringency
providing for a sequence identity of at least about 70%.
[0100] Hybridization techniques for probing a microbial DNA library
to isolate DNA of potential interest are well known in the art and
any of those which are described in the literature are suitable for
use herein, particularly those which use a solid phase-bound,
directly or indirectly bound, probe DNA for ease in separation from
the remainder of the DNA derived from the microorganisms.
[0101] Preferably the probe DNA is "labeled" with one partner of a
specific binding pair (i.e. a ligand) and the other partner of the
pair is bound to a solid matrix to provide ease of separation of
target from its source. The ligand and specific binding partner can
be selected from, in either orientation, the following: (1) an
antigen or hapten and an antibody or specific binding fragment
thereof: (2) biotin or iminobiotin and avidin or streptavidin; (3)
a sugar and a lectin specific therefor; (4) an enzyme and an
inhibitor therefor; (5) an apoenzyme and cofactor: (6)
complementary homopolymeric oligonucleotides; and (7) a hormone and
a receptor therefor. The solid phase is preferably selected from:
(1) a glass or polymeric surface; (2) a packed column of polymeric
beads; and (3) magnetic or paramagnetic particles.
[0102] The library of clones prepared as described above can be
screened directly for a desired, e.g. enzymatic, activity without
the need for culture expansion, amplification or other
supplementary procedures. However, in one preferred embodiment, it
is considered desirable to amplify the DNA recovered from the
individual clones such as by PCR.
[0103] Further, it is optional but desirable to perform an
amplification of the target DNA that has been isolated. In this
embodiment the selectively isolated DNA is separated from the probe
DNA after isolation. It is then amplified before being used to
transform hosts. The double stranded DNA selected to include as at
least a portion thereof a predetermined DNA sequence can be
rendered single stranded, subjected to amplification and reannealed
to provide amplified numbers of selected double stranded DNA.
Numerous amplification methodologies are now well known in the
art.
[0104] The selected DNA is then used for preparing a library for
screening by transforming a suitable organism. Hosts, particularly
those specifically identified herein as preferred, are transformed
by artificial introduction of the vectors containing the target DNA
by inoculation under conditions conducive for such
transformation.
[0105] As representative examples of expression vectors which may
be used there may be mentioned viral particles, baculovirus, phage,
plasmids, phagemids, cosmids, phosmids, bacterial artificial
chromosomes, viral DNA (e.g. vaccinia, adenovirus. foul pox virus,
pseudorabies and derivatives of SV40), P1-based artificial
chromosomes, yeast plasmids, yeast artificial chromosomes, and any
other vectors specific for specific hosts of interest (such as
bacillus, aspergillus, yeast, elc.) Thus, for example, the DNA may
be included in any one of a variety of expression vectors for
expressing a polypeptide. Such vectors include chromosomal,
nonchromosomal and synthetic DNA sequences. Large numbers of
suitable vectors are known to those of skill in the art, and are
commercially available. The following vectors are provided by way
of example: Bacterial: pQE70, pQE60, pQE-9 (Qiagen), psiX174,
pBluescript SK, pBluescript KS, pNH8A, pNH16a, pNH18A, pNH46A
(Stratagene); pTRC99a, pKK223-3, pKK233-3, pDR540, pRIT5
(Pharmacia); Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXT1, pSG
(Stratagene) pSVK3, pBPV, pMSG. pSVL (Pharmacia). However, any
other plasmid or vector may be used as long as they are replicable
and viable in the host.
[0106] A particularly preferred type of vector for use in the
present invention contains an f-factor origin of replication. The
f-factor (or fertility factor) in E. coli is a plasmid which
effects high frequency transfer of itself during conjugation and
less frequent transfer of the bacterial chromosome itself. A
particularly preferred embodiment is to use cloning vectors,
referred to as "fosmids" or bacterial artificial chromosome (BAC)
vectors. These are derived from the E. coli f-factor and are able
to stably integrate large segments of genomic DNA. When integrated
with DNA from a mixed uncultured environmental sample, this makes
it possible to achieve large genomic fragments in the form of a
stable "environmental DNA library."
[0107] The DNA derived from a microorganism(s) may be inserted into
the vector by a variety of procedures. In general, the DNA sequence
is inserted into an appropriate restriction endonuclease site(s) by
procedures known in the art. Such procedures and others are deemed
to be within the scope of those skilled in the art.
[0108] The DNA sequence in the expression vector is operatively
linked to an appropriate expression control sequence(s) (promoter)
to direct mRNA synthesis. Particular named bacterial promoters
include lac, lacZ, T3. T7, gpt, lambda P.sub.R, P.sub.L and trp.
Eukaryotic promoters include CMV immediate early. HSV thymidine
kinase, early and late SV40. LTRs from retrovirus, and mouse
metallothionein-I. Selection of the appropriate vector and promoter
is well within the level of ordinary skill in the art. The
expression vector also contains a ribosome binding site for
translation initiation and a transcription terminator. The vector
may also include appropriate sequences for amplifying expression.
Promoter regions can be selected from any desired gene using CAT
(chloramphenicol transferase) vectors or other vectors with
selectable markers.
[0109] In addition, the expression vectors preferably contain one
or more selectable marker genes to provide a phenotypic trait for
selection of transformed host cells such as dihydrofolate reductase
or neomycin resistance for eukaryotic cell culture, or such as
tetracycline or ampicillin resistance in E. coli.
[0110] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived
from a highly-expressed gene to direct transcription of a
downstream structural sequence. Such promoters can be derived from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), .alpha.-factor, acid phosphatase, or heat shock
proteins. among others. The heterologous structural sequence is
assembled in appropriate phase with translation initiation and
termination sequences, and preferably, a leader sequence capable of
directing secretion of translated protein into the periplasmic
space or extracellular medium.
[0111] The DNA selected and isolated as hereinabove described is
introduced into a suitable host to prepare a library which is
screened for the desired enzyme activity. The selected DNA is
preferably already in a vector which includes appropriate control
sequences whereby selected DNA which encodes for an enzyme may be
expressed, for detection of the desired activity. The host cell can
be a higher eukaryotic cell, such as a mammalian cell, or a lower
eukaryotic cell, such as a yeast cell, or the host cell can be a
prokaryotic cell, such as a bacterial cell. Introduction of the
construct into the host cell can be effected by transformation,
calcium phosphate transfection. DEAE-Dextran mediated transfection,
DMSO or electroporation (Davis, L. Dibner, M. Battey, I. Basic
Methods in Molecular Biology, (1986)).
[0112] As representative examples of appropriate hosts, there may
be mentioned: bacterial cells, such as E. coli, Bacillus,
Streptomyces, Salmonella tryphimurium; fungal cells, such as yeast:
insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells
such as CHO. COS or Bowes melanoma: adenoviruses; plant cells, etc.
The selection of an appropriate host is deemed to be within the
scope of those skilled in the art from the teachings herein.
[0113] Host cells are genetically engineered (transduced or
transformed or transfected) with the vectors. The engineered host
cells can be cultured in conventional nutrient media modified as
appropriate for activating promoters, selecting transformants or
amplifying genes. The culture conditions, such as temperature, pH
and the like, are those previously used with the host cell selected
for expression, and will be apparent to the ordinarily skilled
artisan.
[0114] The recombinant enzymes in the library which are classified
as described herein 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.
[0115] 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.
[0116] 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.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] 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.
[0121] The library may be screened for a specified enzyme activity
by procedures known in the art. For example, the enzyme activity
may be screened for one or more of the six IUB classes;
oxidoreductases, transferases, hydrolases, lyases. isomerases and
ligases. 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.
[0122] Alternatively, the library may be screened for a more
specialized enzyme activity. For example, instead of generically
screening for hydrolase activity, the library may be screened for a
more specialized activity, i.e. the type of bond on which the
hydrolase acts. Thus, for example, the library may be screened 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.
[0123] The clones which are identified as having the specified
enzyme activity may then be sequenced to identify the DNA sequence
encoding an enzyme having the specified activity. Thus, in
accordance with the present invention it is possible to isolate and
identify; (i) DNA encoding an enzyme having a specified enzyme
activity, (ii) enzymes having such activity (inlcuding the amino
acid sequence thereof) and (iii) produce recombinant enzymes having
such activity.
[0124] The screening for enzyme activity 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.
[0125] 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.
[0126] 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:
[0127] pH optima
[0128] <3
[0129] 3-6
[0130] 6-9
[0131] 9-12
[0132] >12
[0133] temperature optima
[0134] >90.degree. C.
[0135] 75-90.degree. C.
[0136] 60-75.degree. C.
[0137] 45-60.degree. C
[0138] 30-45.degree. C.
[0139] 15-30.degree. C.
[0140] 0-15.degree. C.
[0141] temperature stability
[0142] half-life at:
[0143] 90.degree. C.
[0144] 75.degree. C.
[0145] 60.degree. C.
[0146] 45.degree. C.
[0147] organic solvent tolerance
[0148] water miscible
[0149] (DMF)
[0150] 90%
[0151] 75%
[0152] 45%
[0153] 30%
[0154] water immiscible
[0155] hexane
[0156] toluene
[0157] metal ion selectivity
[0158] EDTA -10 mM
[0159] Ca.sup.+2-1 mM
[0160] Mg.sup.+2-100 .mu.M
[0161] Mn.sup.+2-10 .mu.M
[0162] Co.sup.+3-10 .mu.M
[0163] detergent sensitivity
[0164] neutral (triton)
[0165] anionic (deoxycholate)
[0166] cationic (CHAPS)
[0167] 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:
[0168] 1. Lipase/Esterase
[0169] a. Enantioselective hydrolysis of esters
(lipids)/thioesters
[0170] 1) Resolution of racemic mixtures
[0171] 2) Synthesis of optically active acids or alcohols from
meso-diesters
[0172] b. Selective syntheses
[0173] 1) Regiospecific hydrolysis of carbohydrate esters
[0174] 2) Selective hydrolysis of cyclic secondary alcohols
[0175] c. Synthesis of optically active esters, lactones, acids,
alcohols
[0176] 1) Transesterification of activated/nonactivated esters
[0177] 2) Interesterification
[0178] 3) Optically active lactones from hydroxyesters
[0179] 4) Regio- and enantioselective ring opening of
anhydrides
[0180] d. Detergents
[0181] e. Fat/Oil conversion
[0182] f. Cheese ripening
[0183] 2. Protease
[0184] a. Ester/amide synthesis
[0185] b. Peptide synthesis
[0186] c. Resolution of racemic mixtures of amino acid esters
[0187] d. Synthesis of non-natural amino acids
[0188] e. Detergents/protein hydrolysis
[0189] 3. Glycosidase/Glycosvl transferase
[0190] a. Sugar/polymer synthesis
[0191] b. Cleavage of glycosidic linkages to form mono, di-and
oligosaccharides
[0192] c. Synthesis of complex oligosaccharides
[0193] d. Glycoside synthesis using UDP-galactosyl transferase
[0194] e. Transglycosvlation of disaccharides, glycosyl fluorides,
aryl galactosides
[0195] f. Glycosyl transfer in oligosaccharide synthesis
[0196] g. Diastereoselective cleavage of
.beta.-glucosylsulfoxides
[0197] h. Asymmetric glycosylations
[0198] i. Food processing
[0199] j. Paper processing
[0200] 4. Phosphatase/Kinase
[0201] a. Synthesis/hydrolysis of phosphate esters
[0202] 1) Regio-, enantioselective phosphorylation
[0203] 2) Introduction of phosphate esters
[0204] 3) Synthesize phospholipid precursors
[0205] 4) Controlled polynucleotide synthesis
[0206] b. Activate biological molecule
[0207] c. Selective phosphate bond formation without protecting
groups
[0208] 5. Mono/Dioxygenase
[0209] a. Direct oxyfunctionalization of unactivated organic
substrates
[0210] b. Hydroxylation of alkane, aromatics, steroids
[0211] c. Epoxidation of alkenes
[0212] d. Enantioselective sulphoxidation
[0213] e. Regio- and stereoselective Bayer-Villiger oxidations
[0214] 6. Haloperoxidase
[0215] a. Oxidative addition of halide ion to nucleophilic
sites
[0216] b. Addition of hypohalous acids to olefinic bonds
[0217] c. Ring cleavage of cyclopropanes
[0218] d. Activated aromatic substrates converted to ortho and para
derivatives
[0219] e. 1.3 diketones converted to 2-halo-derivatives
[0220] f. Heteroatom oxidation of sulfur and nitrogen containing
substrates
[0221] g. Oxidation of enol acetates, alkynes and activated
aromatic rings
[0222] 7. Lignin peroxidase/Diarvlpropane peroxidase
[0223] a. Oxidative cleavage of C--C bonds
[0224] b. Oxidation of benzylic alcohols to aldehvdes
[0225] c. Hydroxylation of benzylic carbons
[0226] d. Phenol dimerization
[0227] e. Hydroxylation of double bonds to form diols
[0228] f. Cleavage of lignin aldehydes
[0229] 8. Epoxide hydrolase
[0230] a. Synthesis of enantiomerically pure bioactive
compounds
[0231] b. Regio- and enantioselective hydrolysis of epoxide
[0232] c. Aromatic and olefinic epoxidation by monooxygenases to
form epoxides
[0233] d. Resolution of racemic epoxides
[0234] e. Hydrolysis of steroid epoxides
[0235] 9. Nitrile hydratase/nitrilase
[0236] a. Hydrolysis of aliphatic nitriles to carboxamides
[0237] b. Hydrolysis of aromatic, heterocyclic, unsaturated
aliphatic nitriles to corresponding acids
[0238] c. Hydrolysis of acrylonitrile
[0239] d. Production of aromatic and carboxamides, carboxylic acids
(nicotinamide, picolinamide, isonicotinamide)
[0240] e. Regioselective hydrolysis of acrylic dinitrile
[0241] f. .alpha.-amino acids from .alpha.-hydroxynitriles
[0242] 10. Transaminase
[0243] a. Transfer of amino groups into oxo-acids
[0244] 11. Amidase/Acylase
[0245] a. Hydrolysis of amides, amidines, and other C--N bonds
[0246] b. Non-natural amino acid resolution and synthesis
[0247] 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
Production of Expression Library
[0248] The following describes a representative procedure for
preparing an expression library for screening by the tiered
approach of the present invention.
[0249] One gram of Thermococcus GU5L5 cell pellet was lysed and the
DNA isolated by literature procedures (Current Protocols in
Molecular Biology, 2.4.1, 1987). 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' E. coli strain
according to the manufacturer. The pBluescript.RTM. phagemids were
excised from the lambda library, and grown in E. coli DHIOB F' kan,
according to the method of Hay and Short (Hav and Short. J.
Strategies, 5:16, 1992). 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 .mu.L 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 phagemid with a unique DNA
insert.
EXAMPLE 2
Preparation of a Mammalian DNA Library
[0250] The following outlines the procedures used to generate a
gene library from a sample of the exterior surface of a whale bone
found at 1240 meters depth in the Santa Catalina Basin during a
dive expedition.
[0251] Isolate DNA.
[0252] IsoQuick Procedure as per manufacturer's instructions.
[0253] Shear DNA
[0254] 1. Vigorously push and pull DNA through a 25 G double-hub
needle and 1-cc syringes about 500 times.
[0255] 2. Check a small amount (0.5 .mu.g) on a 0.8% agarose gel to
make sure the majority of the DNA is in the desired size range
(about 3-6 kb).
[0256] Blunt DNA
[0257] 1. Add:
1 H.sub.2O to a final volume of 405 .mu.l 45 .mu.l 10X Mung Bean
Buffer 2.0 .mu.l Mung Bean Nuclease (150 u/.mu.l)
[0258] 2. Incubate 37.degree. C., 15 minutes.
[0259] 3. Phenol/chloroform extract once.
[0260] 4. Chloroform extract once.
[0261] 5. Add 1 ml ice cold ethanol to precipitate.
[0262] 6. Place on ice for 10 minutes.
[0263] 7. Spin in microfuge, high speed, 30 minutes.
[0264] 8. Wash with 1 ml 70% ethanol.
[0265] 9. Spin in microfuge, high speed, 10 minutes and dry.
[0266] Methylate DNA
[0267] 1. Gently resuspend DNA in 26 .mu.l TE.
[0268] 2. Add:
2 4.0 .mu.l 10X EcoR I Methylase Buffer 0.5 .mu.l SAM (32 mM) 5.0
.mu.l EcoR I Methylase (40 u/.mu.l)
[0269] 3. Incubate 37.degree., 1 hour.
[0270] Insure Blunt Ends
[0271] 1. Add to the methylation reaction:
3 5.0 .mu.l 100 mM MgCl.sub.2 8.0 .mu.l dNTP mix (2.5 mM of each
dGTP, dATP, dTTP, dCTP) 4.0 .mu.l Klenow (5 u/.mu.l)
[0272] 2. Incubate 12.degree. C., 30 minutes.
[0273] 3. Add 450 .mu.l IX STE.
[0274] 4. Phenol/chloroform extract once.
[0275] 5. Chloroform extract once.
[0276] 6. Add 1 ml ice cold ethanol to precipitate and place on ice
for 10 minutes.
[0277] 7. Spin in microfuge, high speed. 30 minutes.
[0278] 8. Wash with 1 ml 70% ethanol.
[0279] 9. Spin in microfuge, high speed, 10 minutes and dry.
[0280] Linker Ligation
[0281] 1. Gently resuspend DNA in 7 .mu.l Tris-EDTA (TE).
[0282] 2. Add:
4 14 .mu.l Phosphorylated EcoR I linkers (200 ng/.mu.l) 3.0 .mu.l
10X Ligation Buffer 3.0 .mu.l 10 mM rATP 3.0 .mu.l T4 DNA Ligase (4
Wu/.mu.l)
[0283] 3. Incubate 4.degree. C., overnight.
[0284] EcoRI Cutback
[0285] 1. Heat kill ligation reaction 68.degree. C. 10 minutes.
[0286] 2. Add:
5 237.9 .mu.l H.sub.2O 30 .mu.l 10X EcoR I Buffer 2.1 .mu.l EcoR I
Restriction Enzyme (100 u/.mu.l)
[0287] 3. Incubate 37.degree. C., 1.5 hours.
[0288] 4. Add 1.5 .mu.l 0.5 M EDTA.
[0289] 5. Place on ice.
[0290] Sucrose Gradient (2.2 ml) Size Fractionation
[0291] 1. Heat sample to 65.degree. C., 10 minutes.
[0292] 2. Gently load on 2.2 ml sucrose gradient.
[0293] 3. Spin in mini-ultracentrifuge, 45 K. 20.degree. C. 4 hours
(no brake).
[0294] 4. Collect fractions by puncturing the bottom of the
gradient tube with a 20 G needle and allowing the sucrose to flow
through the needle. Collect the first 20 drops in a Falcon 2059
tube then collect 10 1-drop fractions (labelled 1-10). Each drop is
about 60 .mu.l in volume.
[0295] 5. Run 5 .mu.l of each fraction on a 0.8% agarose gel to
check the size.
[0296] 6. Pool fractions 1-4 (-10-1.5 kb) and, in a separate tube,
pool fractions 5-7 (about 5-0.5 kb).
[0297] 7. Add 1 ml ice cold ethanol to precipitate and place on ice
for 10 minutes.
[0298] 8. Spin in microfuge, high speed, 30 minutes.
[0299] 9. Wash with 1 ml 70% ethanol.
[0300] 10. Spin in microfuge, high speed, 10 minutes and dry.
[0301] 11. Resuspend each in 10 .mu.l TE buffer.
[0302] Test Ligation to Lambda Arms
[0303] 1. Plate assay to get an approximate concentration. Spot 0.5
.mu.l of the sample on agarose containing ethidium bromide along
with standards (DNA samples of known concentration). View in UV
light and estimate concentration compared to the standards.
Fraction 1-4=>1.0 .mu.g/.mu.l. Fraction 5-7=500 .mu.g/.mu.l. 2.
Prepare the following ligation reactions (5 .mu.l reactions) and
incubate 4.degree. C. overnight:
6 Lambda 10X arms T4 DNA Ligase 10 mM (gt11 and Insert Ligase (4
Sample H.sub.2O Buffer rATP ZAP) DNA Wu/.mu.) Fraction 1-4 0.5
.mu.l 0.5 .mu.l 0.5 .mu.l 1.0 .mu.l 2.0 .mu.l 0.5 .mu.l Fraction
5-7 0.5 .mu.l 0.5 .mu.l 0.5 .mu.l 1.0 .mu.l 2.0 .mu.l 0.5 .mu.l
[0304] Test Package and Plate
[0305] 1. Package the ligation reactions following manufacturer's
protocol. Package 2.5 .mu.l per packaging extract (2 extracts per
ligation).
[0306] 2. Stop packaging reactions with 500 .mu.l SM buffer and
pool packaging that came from the same ligation.
[0307] 3 Titer 1.0 .mu.l of each on appropriate host
(OD.sub.600=1.0) [XLI-Blue MRF for ZAP and Y1088 for gt11]
[0308] Add 200 .mu.l host (in mM MgSO.sub.4) to Falcon 2059
tubes
[0309] Inoculate with 1 .mu.l packaged phage
[0310] Incubate 37.degree. C., 15 minutes
[0311] Add about 3 ml 48.degree. C. top agar
[0312] [50 ml stock containing 150 .mu.l IPTG (0.5 M) and 300 .mu.l
X-GAL (350 mg/ml)]
[0313] Plate on 100 mm plates and incubate 37.degree. C.,
overnight.
[0314] 4. Efficiency results:
[0315] gt 11. 1.7.times.10.sup.4 recombinants with 95%
background
[0316] ZAP 11:4.2.times.10.sup.4 recombinants with 66%
background
[0317] Contaminants in the DNA sample may have inhibited the
enzymatic reactions, though the sucrose gradient and organic
extractions may have removed them. Since the DNA sample was
precious, an effort was made to "fix" the ends for cloning:
[0318] Re-Blunt DNA
[0319] 1. Pool all left over DNA that was not ligated to the lambda
arms (Fractions 1-7) and add H.sub.2O to a final volume of 12
.mu.l. Then add:
7 143 .mu.l H.sub.2O 20 .mu.l 10X Buffer 2 (from Stratagene's cDNA
Synthesis Kit) 23 .mu.l Blunting dNTP (from Stratagene's cDNA
Synthesis Kit) 2.0 .mu.l Pfu (from Stratagene"s cDNA Synthesis
[0320] 2. Incubate 72.degree. C., 30 minutes.
[0321] 3. Phenol/chloroform extract once.
[0322] 4. Chloroform extract once.
[0323] 5. Add 20 .mu.L 3 M NaOAc and 400 .mu.l ice cold ethanol to
precipitate.
[0324] 6. Place at -20.degree. C., overnight.
[0325] 7. Spin in microfuge, high speed, 30 minutes.
[0326] 8. Wash with 1 ml 70% ethanol.
[0327] 9. Spin in microfuge, high speed, 10 minutes and dry.
[0328] (Do NOT Methylate DNA Since it was Already Methylated in the
First Round of Processing)
[0329] Adaptor Ligation
[0330] 1. Gently resuspend DNA in 8 .mu.l EcoR I adaptors (from
Stratagene's cDNA Synthesis Kit).
[0331] 2. Add:
8 1.0 .mu.l 10X Ligation Buffer 1.0 .mu.l 10 mM rATP 1.0 .mu.l T4
DNA Ligase (4 Wu/.mu.l)
[0332] 3. Incubate 4.degree. C. 2 days.
[0333] (Do NOT Cutback Since Using ADAPTORS This Time. Instead,
Need to Phosphervlate)
[0334] Phosphorvlate Adaptors
[0335] 1. Heat kill ligation reaction 70.degree. C. 30 minutes.
Add:
9 1.0 .mu.l 10X Ligation Buffer 2.0 .mu.l 10 mM rATF 6.0 .mu.l
H.sub.2O 1.0 .mu.l PNK (from Stratagene's cDNA Synthesis Kit).
[0336] Kit).
[0337] 3. Incubate 37.degree. C., 30 minutes.
[0338] 4. Add 31 .mu.l H.sub.2O and 5 .mu.l 10.times.STE.
[0339] 5. Size fractionate on a Sephacryl S-500 spin column (pool
fractions 1-3).
[0340] 6. Phenol/chloroforrn extract once.
[0341] 7. Chloroform extract once.
[0342] 8. Add ice cold ethanol to precipitate.
[0343] 9. Place on ice, 10 minutes.
[0344] 10. Spin in microfuge, high speed, 30 minutes.
[0345] 11. Wash with 1 ml 70% ethanol.
[0346] 12. Spin in microfuge, high speed. 10 minutes and dry.
[0347] 13. Resuspend in 10.5 .mu.l TE buffer.
[0348] Do Not Plate Assay. INSTEAD, Ligate directly to Arms as
Above Except Use 2.5 .mu.l of DNA and No Water.
[0349] Package and Titer as Above.
[0350] Efficiency results:
10 gt11: 2.5 .times. 10.sup.6 recombinants with 2.5% background ZAP
II: 9.6 .times. 10.sup.5 recombinants with 0% background
[0351] Amplification of Libraries (5.0.times.10.sup.5 Recombinants
From Each Library)
[0352] 1. Add 3.0 ml host cells (OD.sub.600=1.0) to two 50 ml
conical tube.
[0353] 2. Inoculate with 2.5.times.10.sup.5 pfu per conical
tube.
[0354] 3. Incubate 37.degree. C., 20 minutes.
[0355] 4. Add top agar to each tube to a final volume of 45 ml.
[0356] 5. Plate the tube across five 150 mm plates.
[0357] 6. Incubate 37.degree. C., 6-8 hours or until plaques are
about pin-head in size.
[0358] 7. Overlay with 8-10 ml SM Buffer and place at 4.degree. C.
overnight (with gentle rocking if possible).
[0359] Harvest Phage
[0360] 1. Recover phage suspension by pouring the SM buffer off
each plate into a 50-ml conical tube.
[0361] 2. Add 3 ml chloroform, shake vigorously and incubate at
room temperature, 15 minutes.
[0362] 3. Centrifuge at 2K rpm, 10 minutes to remove cell
debris.
[0363] 4. Pour supernatant into a sterile flask, add 500 .mu.l
chloroform.
[0364] 5. Store at 4.degree. C.
[0365] Titer Amplified Library
[0366] 1. Make serial dilutions:
[0367] 10.sup.-5=1 .mu.l amplified phage in 1 ml SM Buffer
[0368] 10.sup.-6=1 .mu.l of the 10.sup.-3 dilution in 1 ml SM
Buffer
[0369] 2. Add 200 .mu.l host (in 10 mM MgSO.sub.4) to two
tubes.
[0370] 3. Inoculate one with 10 .mu.l 10.sup.-6 dilution
(10.sup.-5).
[0371] 4. Inoculate the other with 1 .mu.l 10.sup.-6 dilution
(10.sup.-6).
[0372] 5. Incubate 37.degree. C., 15 minutes.
[0373] 6. Add about 3 ml 48.degree. C. top agar.
[0374] [50 ml stock containing 150 .mu.l IPTG (0.5M) and 375 .mu.l
X-GAL (350 mg/ml)]
[0375] 7. Plate on 100 mm plates and incubate 37.degree. C.,
overnight.
[0376] 8. Results:
11 gt11: 1.7 .times. 10.sup.11/ml ZAP II: 2.0 .times.
10.sup.10/ml
[0377] Excise The ZAP II Library to Create The pBluescript
library.
EXAMPLE 3
Preparation of an Uncultivated Prokarvotic DNA Library
[0378] FIG. 1 shows an overview of the procedures used to construct
an environmental library from a mixed picoplankton sample. The goal
was to construct a stable, large insert DNA library representing
picoplankton genomic DNA.
[0379] Cell Collection and Preparation of DNA.
[0380] Agarose plugs containing concentrated picoplankton cells
were prepared from samples collected on an oceanographic cruise
from Newport, Oregon to Honolulu. Hi. Seawater (30 liters) was
collected in Niskin bottles, screened through 10 Am Nitex, and
concentrated by hollow fiber filtration (Amicon DC10) through
30,000 MW cutoff polysulfone filters. The concentrated
bacterioplankton cells were collected on a 0.22 .mu.m, 47 mm
Durapore filter, and resuspended in 1 ml of 2.times.STE buffer (1M
NaCl, 0. IM EDTA, 10 mM Tris, pH 8.0) to a final density of
approximately 1.times.10.sup.10 cells per ml. The cell suspension
was mixed with one volume of 1% molten Seaplaque LMP agarose (FMC)
cooled to 40.degree. C., and then immediately drawn into a 1 ml
syringe. The syringe was sealed with parafilm and placed on ice for
10 min. The cell-containing agarose plug was extruded into 10 ml of
Lysis Buffer (10 mM Tris pH 8.0, 50 mM NaCl, 0.1 M EDTA, 1%
Sarkosyl, 0.2% sodium deoxycholate, a mg/ml lysozyme) and incubated
at 37.degree. C. for one hour. The agarose plug was then
transferred to 40 mis of ESP Buffer (1% Sarkosyl, 1 mg/ml
proteinase-K, in 0.5M EDTA), and incubated at 55.degree. C. for 16
hours. The solution was decanted and replaced with fresh ESP
Buffer, and incubated at 55.degree. C. for an additional hour. The
agarose plugs were then placed in 50 mM EDTA and stored at
4.degree. C. shipboard for the duration of the oceanographic
cruise.
[0381] One slice of an agarose plug (72 .mu.l) prepared from a
sample collected off the Oregon coast was dialyzed overnight at
4.degree. C. against 1 mL of buffer A (100 mM NaCl, 10 mM Bis Tris
Propane-HCl, 100 .mu.g/ml acetylated BSA: pH 7.0 @ 25.degree. C.)
in a 2 mL microcentrifuge tube. The solution was replaced with 250
.mu.l of fresh buffer A containing 10 mM MgCl.sub.2 and 1 mM DTT
and incubated on a rocking platform for 1 hr at room temperature.
The solution was then changed to 250 .mu.l of the same buffer
containing 4 U of Sau3AI (NEB), equilibrated to 37.degree. C. in a
water bath, and then incubated on a rocking platform in a
37.degree. C. incubator for 45 min. The plug was transferred to a
1.5 ml microcentrifuge tube and incubated at 68.degree. C. for 30
min to inactivate the protein, e.g. enzyme, and to melt the
agarose. The agarose was digested and the DNA dephosphorylased
using Gelase and HK-phosphatase (Epicentre), respectively according
to the manufacturer s recommendations. Protein was removed by
gentle phenol/chloroform extraction and the DNA was ethanol
precipitated, pelleted, and then washed with 70% ethanol. This
partially digested DNA was resuspended in sterile H.sub.2O to a
concentration of 2.5 ng/.mu.l for ligation to the pFOSI vector.
[0382] PCR amplification results from several of the agarose plugs
(data not shown) indicated the presence of significant amounts of
archaeal DNA. Quantitative hybridization experiments using rRNA
extracted from one sample, collected at 200 m of depth off the
Oregon Coast, indicated that planktonic archaea in (this assemblage
comprised approximately 4.7% of the total picoplankton biomass
(this sample corresponds to "PACI "-200 m in Table 1 of DeLong et
al., high abundance of Archaea in Antarctic marine picoplankton,
Nature, 371:695-698, 1994). Results from archaeal-biased rDNA PCR
amplification performed on agarose plug lysates confirmed the
presence of relatively large amounts of archaeal DNA in this
sample. Agarose plugs prepared from this picoplankton sample were
chosen for subsequent fosmid library preparation. Each 1 ml agarose
plug from this site contained approximately 7.5.times.10.sup.5
cells, therefore approximately 5.4.times.10.sup.5 cells were
present in the 72 .mu.l slice used in the preparation of the
partially digested DNA.
[0383] Vector arms were prepared from pFOSI as described (Kim et
al. Stable propagation of casmid sized human DNA inserts in an F
factor based vector. Nucl. Acids Res., 20:10832-10835. 1992).
Briefly, the plasmid was completely digested with AstII,
dephosphorylated with HK phosphatase, and then digested with BamHI
to generate two arms, each of which contained a cos site in the
proper orientation for cloning and packaging ligated DNA between
3545 kbp. The partially digested picoplankton DNA was ligated
overnight to the PFOS1 arms in a 15 .mu.l ligation reaction
containing 25 ng each of vector and insert and 1 U of T4 DNA ligase
(Boehringer-Mannheim). The ligated DNA in four microliters of this
reaction was ill vitro packaged using the Gigapack XL packaging
system (Stratagene), the fosmid particles transfected to E. coli
strain DHIOB (BRL), and the cells spread onto LB.sub.cm15 plates.
The resultant fosmid clones were picked into 96-well microliter
dishes containing LB.sub.cm15 supplemented with 7% glycerol.
Recombinant fosmids, each containing ca. 40 kb of picoplankton DNA
insert, yielded a library of 3.552 fosmid clones, containing
approximately 1.4.times.10.sup.8 base pairs of cloned DNA. All of
the clones examined contained inserts ranging from 38 to 42 kbp.
This library was stored frozen at -80.degree. C. for later
analysis.
EXAMPLE 4
Enzymatic Activity Assay
[0384] The following is a representative example of a procedure for
screening an expression library prepared in accordance with Example
2. In the following, the chemical characteristic Tiers are as
follows:
[0385] Tier 1: Hydrolase
[0386] Tier 2: Amide. Ester and Acetal
[0387] 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.
[0388] Tier 4: The two possible enantiomeric products which the
enzyme may produce from a substrates
[0389] 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.
[0390] Screening for Tier 1-hydrolase: Tier 2-amide.
[0391] 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 clones from
each of the eleven source library plates. The Condensed Plate was
grown for 2h at 37.degree. C. and then used to inoculate two white
96-well Dynatech microtiter daughter plates containing in each well
250 .mu.L of LB Amp/Meth, glycerol. The original condensed plates
was 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 AM with 50 mM pH 7.5 Hepes
buffer containing 0.6 mg/mL of the detergent dodecyl maltoside.
1
[0392] 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.
[0393] These 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
[0394] 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.
[0395] These data indicated that the active clone was in plate
[0396] 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.
[0397] 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.
[0398] As shown in FIG. 2, 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. 2, 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.
[0399] As shown in FIGS. 3 and 4, 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. 3 and 4, 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. 3 and 4, R.sub.2 represents the alcohol portion of
the ester and R.sub.1 represents the acid portion of the ester.
[0400] As shown in FIG. 5, 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. 5, the various classes of Tier 3 are followed by a
parenthetical code which identifies the substrates of Table 4 which
are used in identifying such specificities of Tier 3.
[0401] 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
[0402] For each substrate which is turned over the
enantioselectivity value, E, is determined according to the
equation below: 1 E = ln [ ( 1 - c ( 1 + ee p ) ] ln [ ( 1 - c ( 1
- ee p ) ]
[0403] where ee.sub.P=the enantiomeric excess (ee) of the
hydrolyzed product and c=the percent conversion of the reaction.
See Wong and Whitesides. Enzymes in Synthetic Organic Chemistry,
1994, Elsevier, Tarrytown, N.Y., pgs. 9-12.
[0404] The enantiomeric excess is determined by either chiral high
performance liquid chromatography (HPLC) or chiral capillary
electrophoresis (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 is added
and the increase in fluorescence monitored versus time until 50% of
the substrate is consumed or the reaction stops, which ever comes
first.
[0405] Enantioselectivity was determined for one of the esterases
identified as follows. For the reaction to form
(transesterification) or breakdown (hydrolysis) .alpha.-methyl
benzyl acetate, the enantioselectivity of the enzyme was obtained
by determining: ee.sub.C (the enantiomeric excess (ee) of the
unreacted substrate), ee.sub.P (the ee of the hydrolyzed product),
and c (the percent conversion of the reaction). The enantiomeric
excess was by determined chiral high performance gas chromatography
(GC). Chromatography conditions were as follows:
[0406] Sample Preparation: Samples were filtered through a 0.2
.mu.m, 13 mm diameter PTFE filter.
[0407] Column: Supelco .beta.-DEX 120, 0.25 mm ID. 30 m, 0.25 Am
d.sub.f.
[0408] Oven: 90.degree. C. for 1 min, then 90.degree. C. to
150.degree. C at 5.degree. C./min.
[0409] Carrier Gas: Helium, 1 mL/min for 2 min then 1 mL/min, to 3
mL/min at 0.2 mL/min.
[0410] Detector: FID, 300.degree. C.
[0411] Injection: 1 .mu.L (1 mM substrate in reaction solvent),
split (1:75), 200.degree. C.
[0412] The transesterification reaction was performed according to
the procedure described in: Organic solvent tolerance. Water
immiscible solvents. See below.
[0413] Transesterification with Enzyme ESL-001-01 gave the
following results:
12 Solvent % ee.sub.s % ee.sub.p % c n-heptane 10.9 44.3 19.8
toluene 3.2 100 3.1
[0414] The hydrolysis reaction was performed as follows: Fifty
.mu.L of a 10 mM solution of .alpha.-methyl benzyl acetate in 10%
aqueous DMSO (v/v) was added to 200 .mu.L of 100 mM, pH 6.9
phosphate buffer. To this solution was added 250 AL of Enzyme
ESL-001-01(2 mg/mL in 100 mM, pH 6.9 phosphate buffer) and the
reaction heated at 70.degree. C. for 15 min. The reaction was
worked up according to the following procedure: remove 250 .mu.L of
hydrolysis reaction mixture and add to a 1 mL Eppendorf tube. Add
250 AL of ethyl acetate and shake vigorously for 30 seconds. Allow
phases to separate for 15 minutes. Pipette off 200 .mu.L of top
organic phase and filter through a 0.2 .mu.m. 4 mm diameter PTFE
filter. Analyze by chiral GC as above.
[0415] Hydrolysis with Enzvme ESL-001-01 gave the following
results:
13 % ee.sub.s % ee.sub.p % c 100 0.7 99.3
EXAMPLE 5
Testing for Physical Characteristics of a Recombinant Clone
[0416] This example describes procedures for testing for certain
physical characteristics of a recombinant clone of a library.
[0417] pH Optima.
[0418] Two hundred .mu.L of 4-methy 1-umbelliferyl-2
,2-dimethyl-4-pentenoate was added to each well of a 96-well
microtiter plate and serially diluted from column 1 to 12. Fifty
.mu.L of the appropriate 5.times.pH buffer was added to each row of
the plate so that reaction rate in eight different pH's were tested
on a single plate. Twenty .mu.L of Enzyme ESL-001-01 (1:3000
dilution of a 1 mg/mL stock solution) was added to each well to
initiate the reaction. The increase in absorbance at 370 nm at
70.degree. C. was monitored to determine the rate of reaction; the
rate versus substrate concentration fit to the Michaelis-Menten
equation to determine VMAX at each pH.
[0419] Enzyme ESL-001-01 gave the results shown in FIG. 6.
[0420] Temperature Optima.
[0421] To a one mL thermostatted cuvette was added 930 .mu.L of 50
mM, pH 7.5 Hepes buffer. After temperature equilibration 50 AL of
Enzyme ESL-001-01 (1:8000 dilution of a 1 mg/mL stock solution in
Hepes buffer) and 20 .mu.L of 5 mM 4-methyl-umbelliferyl-heptanoate
containing 30 mg/mL dodecyl maltoside. The rate of increase in
absorbance at 370 nm was measured at 10, 20, 30, 40, 50, 60, 70,
80,and 90.degree. C.
[0422] Enzyme ESL-001-01 Gave the Results Shown in FIG. 7.
ability.
[0423] Temperature Stability.
[0424] One mL samples of Enzyme ESL-001-01 (1:4000 dilution of a 1
mg/mL stock solutions in Hepes buffer) were incubated at 70, 80,
and 90.degree. C. At selected time points 25 .mu.L aliquots were
removed and assayed as above in a 96 well microtiter plate with 200
.mu.L of 100 .mu.M 4-methylumbelliferyl palmitate and 0.6 mg/mL
dodecyl maltoside. This data was used to determine the half life
for inactivation of the enzyme.
[0425] Enzyme ESL-001-01 gave the following results:
14 Temperature Half Life 90 23 min. 80 32 min. 70 110 h
[0426] Organic Solvent Tolerance.
[0427] Water Miscible Solvents (Dimethylsulfoxide (DMSO) and
Tetrahydro Furan (THF)).
[0428] Thirty .mu.L of of 1 mM 4-methyl-umbelliferyl-butyrate in
the organic solvent was added to the wells of a 96-well miciotiter
plate. Two hundred forty .mu.L of buffer and organic solvent
mixture (see table below) were added the wells of the plate,
followed by 30 .mu.L of an Enzyme ESL-001-01 (1:50,000 dilution of
a 1 mg/mL stock solution in 50 mM, pH 6.9 MOPS buffer) and
incubation at 70.degree. C. The increase in fluorescence (EX=360
nm, EM=440 nm) was monitored versus time to determine the relative
activities.
15 .mu.L Organic Solvent .mu.L Buffer % Organic Solvent Final 240 0
90 195 45 75 150 90 60 120 120 50 90 150 40 60 180 30 30 210 20 0
240 10
[0429] Enzyme ESL-001-01 Ol gave the results shown in FIG. 8.
[0430] Water Immiscible Solvents (n-heptane, toluene)
[0431] One mL of the solvent was added to a vial containing 1 mg of
lyophilized Enzyme ESL-001-01 and a stir bar. Ten .mu.L of 100 mM
1-phenethyl alcohol and 10 .mu.L of 100 mM vinyl acetate were added
to the vial and the vial stirred in a heating block at 70.degree.
C. for 24 h. The sample was filtered through a 0.2 .mu.m. 4 mm
diameter PTFE filter and analyzed by chiral GC as above. See
previous section for data.
[0432] Specific Activity
[0433] The specific activity was determined using 100 .mu.M
4-methyl umbelliferyl heptanoate at 90.degree. C. in pH 6.9 MOPS
buffer. The specific activity obtained for Enzyme ESL-001-01 was
1662 .mu.mol/min mg.
EXAMPLE 6
Testing for Substrate Specificity of a Recombinant Clone
[0434] This example describes procedures for testing for substrate
specificity of a recombinant clone of a library.
[0435] Substrate Fingerprint.
[0436] One and one quarter millimolar solutions containing 1 mg/mL
of dodecyl maltoside in 50 mM pH 6.9 MOPS buffer of each of the
following substrates were prepared:
[0437] 4-methyl umbelliferyl acetate (A)
[0438] 4-methyl umbelliferyl propanoate (B)
[0439] 4-methyl umbelliferyl butyrate (C)
[0440] 4-methyl umbelliferyl heptanoate (D)
[0441] 4-methyl umbelliferyl .alpha.-methyl butyrate (E)
[0442] 4-methyl umbelliferyl .beta.-methylcrotonoate (F)
[0443] 4-methyl umbelliferyl 2,2-dimethyl-4-pentenoate (G)
[0444] 4-methyl umbelliferyl adipic acid monoester (H)
[0445] 4-methyl umbelliferyl 1,4-cycylohexane dicarboxylate (1)
[0446] 4-methyl umbelliferyl benzoate (M)
[0447] 4-methyl umbelliferyl p-trimethyl ammonium cinnamate (N)
[0448] 4-methyl umbelliferyl 4-guanidinobenzoate (0)
[0449] 4-methyl umbelliferyl .alpha.-methyl phenyl acetate (P)
[0450] 4-methyl umbelliferyl .alpha.-methoxy phenyl acetate (Q)
[0451] 4-methyl umbelliferyl palmitate (S)
[0452] 4-methyl umbelliferyl stearate (T)
[0453] 4-methyl umbelliferyl oleate (U)
[0454] 4-methyl umbelliferyl elaidate (W).
[0455] Two hundred .mu.L of each of the above solutions were added
to the wells of a 96 well microtiter plate, followed by 50 .mu.L of
Enzyme ESL-001-01 (1:2000 dilution of a 1 mg/mL stock solution in
MOPS buffer) and incubation at 70.degree. C. for 20 min. The
fluorescence (EX=360 nm, EM=440 nm) was measured and fluorescence
due to nonenzymatic hydrolysis was subtracted. Table 5 shows the
relative fluorescence of each of the above substrates.
[0456] 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.
16TABLE 1 A2 Fluorescein conjugated casein (32 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-Phe-AMC t-BOC = t-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
[0457]
17TABLE 2 7 8 9 10 11 12 13 14
[0458]
18TABLE 3 15 16 17 18 19 20 21 22
[0459]
19TABLE 4 23 wherein R = 4-methyl umbelliferone G2
.beta.-D-galactose .beta.-D-glucose .beta.-D-glucoronide GB3
.beta.-D-cellotrioside .beta.-B-cellobiopyranoside GC3
.beta.-D-galactose .alpha.-D-galactose GD3 .beta.-D-glucose
.alpha.-D-glucose GE3 .beta.-D-glucoronide GI3
.beta.-D-N,N-diacetylchitobiose GJ3 .beta.-D-fucose
.alpha.-L-fucose .beta.-L-fucose GK3 .beta.-D-mannose
.alpha.-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
[0460]
20 TABLE 5 RELATIVE COMPOUND FLUORESCENCE A 60.6 B 73.6 C 100.0 D
84.3 E 29.1 F 5.4 G 7.1 H 0.9 I 0.0 M 9.4 N 0.5 O 0.5 P 4.0 Q 11.3
S 0.6 T 0.1 U 0.3 W 0.2
* * * * *