JP2002515736A - A stable biocatalyst for ester hydrolysis - Google Patents

Abstract

  (57) [Summary] The present invention includes an isolated, stable esterase enzyme characterized by its ability to remain stable at a particular temperature, substrate specificity, and activity profile.

Description

DETAILED DESCRIPTION OF THE INVENTION A stable biocatalyst for ester hydrolysis   This application discloses a patent application 80 / _________________ filed on June 12, 1996; No. 80 / 009,704 filed on Jan. 10, 1995; and filed on Aug. 7, 1995 No. 80 / 001,995, all of which are incorporated by reference. The entirety of which is incorporated herein. State of government rights   The work disclosed in this application was made by ThermoGen Inc. . Approval number from NIH-SBIR for NCI1-R43-CA638 Partially supported by 76-01, the United States Government has It may have certain rights in the invention. Field of the invention   The present disclosure provides commercial pharmaceutical and chemically synthesized, recombinant ester hydrolysed proteins. A DNA vector, a host cell transformed with such a vector, And recombinant esters produced by such vectors and transformed cells Isolated stable biocatalyst components suitable for enzymatic use in hydrolyzed proteins Pointed to the field. Background of the Invention   Esterases and lipases. Esterases and lipases are listed below. As shown, hydrolysis of ester bond To produce alcohols and carboxylic acids.   Esterases and lipases have different substrate specificities, R group or chain length preferences, and And unique inhibitors (1, 2). Many estelar And lipases preferentially hydrolyze esters with long carbon chain R groups. From a range of hydrolases such as carboxylesterases, cholinesterers And acetylesterases that act on very specific substrates Across. In many cases, these hydrolases are fixed at the protein active site. It is also known to exhibit stereoselectivity and regioselectivity arising from its chirality. This preferential hydrolysis activity requires them to have different regioselectivities and stereoselectivities. Useful for required reactions or kinetic decomposition methods for racemic mixtures . For enzymes that exhibit stereoselectivity, when R * is a racemic mixture, the enzyme catalyst The hydrolysis product R1 is the most rapidly hydrolyzing stereoisomer, whereas Thus, the remaining esters, named R * ', are substituted for any remaining enantiomerically enriched R It was mixed with 1. The products are then chromatographed. To obtain pure R1. Esterer with various specificities The availability of a large pool of enzymes and lipases Useful for screening enzymes and developing optimal protocols for specific chemical synthesis . The convenience of this process scales up the production of many useful pharmaceutical products. make it easier.   In aqueous solvent systems, esterases and lipases react in their natural reaction: Hydrolyze the tell bond. In vitro, these enzymes Include cyclic and acyclic alcohols, mono- and di-esters, and lactams. Can be used to perform reactions on a wide range of substrates, including esters. 3). By conducting the reaction in an organic solvent from which water has been excluded (4, 5), The reaction of terases and lipases can be reversed. These enzymes are It is possible to form an ester bond by catalyzing an acylation or acylation reaction (3 , 6, 7). This process also involves transesterification of the ester and ring closure or opening. It can be used for ring reactions.   Optically pure chiral drug. Currently, most synthetic chiral drugs are racemic mixtures And sold. However, optically pure (single isomer) chiralization This situation is changing with advances in compound synthesis (7). Racemic drug Is often combined with one isomer that is therapeutically active and at best inactive and At worst include other enantiomers that are the main cause of potentially harmful side effects. Racemic drug This unusual isomer in objects is increasingly seen as a contaminant. In fact, The FDA's policy speech on new drug development is based on a pharmacokinetic overview of each of the isomers. After characterization in animals, the clinical pharmacokinetics obtained in the Phase I drug trials It is recommended to compare with a stoichiometric summary (8). Therefore, the pharmaceutical company A synthesis or separation route to produce each of the pure isomers of each new synthetic drug Need to develop.   Enzymatic synthesis of optically pure drugs and intermediates. Identified by classical chemical synthesis Optically pure chiral molecule Because it is often very difficult to generate a solution, new enzymatic biocatalysts Play a major role in this effort. In some cases, enzymes are dangerous chemicals It is possible to replace synthesis procedures with biological synthesis processes that are more environmentally friendly. You. Also, if the enzyme removes several chemical protection and deprotection steps simultaneously, Enzymatic production of pharmaceutical intermediates may be more cost effective (7). How many As described in several in-depth reviews (3, 7), the six major classes of enzymes (acid Reductase, transferase, hydrolase, lyase, isomerase, and ligation enzyme) Is useful in the synthesis of optically pure compounds. Hydrolase hydrolyze Large amounts of enzymes, information about them, their independence from cofactors, and Have proven to be the most useful group of enzymes due to the diversity of acceptable substrates. I have.   By examining the literature, the medium temperatures used in chemical synthesis or chiral decomposition Many examples of sex hydrolases, especially esterases and lipases, are given. these Esterases derived from pig (9, 10) and horse (3) liver and Spergillus species (11), Candida species (12-16), Pseudomonas species (17 -19), various lipases derived from Rhizopus species (20) and the like. Some Lipase is used in the treatment of propanolol, angina and hypertension -Used in the synthesis of adrenergic blockers (7). Ibuprof Non-steroidal anti-inflammatory drugs are It has been synthesized by the stereoselective hydrolysis of esters (7). These enzymes Despite beginning to show the usefulness of biocatalysts in chemical synthesis, various substrate specificities , Regioselectivity, and stereoselectivity There is still a great need for various esterases and lipases with Exist. In addition, these enzymes need to be used in large-scale industrial equipment, They have increased stability, higher heat resistance, and longer "shelf life" There is a need.   Thermostable enzyme. Thermophilic microorganisms catalyze reactions at high temperatures and are more stable It has already provided a rich source of some useful proteins (21,22). One example is , Derived from Thermus aquaticus DNA polymerase I and its use in the polymerase chain reaction (PCR) (23, 24). Thermophilic enzymes have long-term stability and ease of use. , Has become the most commercially successful enzyme in the industry. Most successful to date A successful enzyme, α-amylase, has been used in the processing of corn, Degree thermophile B. B. stearothermophile rus) (25). Another commercially successful industrial enzyme is subchi Lysine, a serine protease also found in various Bacillus strains, Widely used in rinsing detergents and other cleaning solutions.   The commercial success of these enzymes may result in their ease of use. Machine at high temperature In addition to functioning, thermostable enzymes generally have an increased shelf life, A specific range of articles used by methanogens for those untrained in biochemistry Significantly improve the handling conditions for working in the case. Enzymes are a large source of chemicals If they play a significant role in the It must be able to withstand various conditions. Thermostable enzymes are easy to handle And longer There is also a suitable fixed support that should be persistent and reusable for multiple uses. Have been done.   Finally, the hydrophobic and electrostatic forces that allow these enzymes to survive at elevated temperatures are Also allow them to function better in organic solvents in general (26- 31). While most enzymes lose most of their activity in organic solvents, To make thermostable enzymes more resistant to denaturing conditions in many organic solvents. Can be proven. Esterases and lipases that are highly thermostable There is a need for expanding the use of these biocatalysts in scale industrial reactions.   Thermostable esterases and lipases. To date, one esterase and Only a few lipases have been reported to have moderate thermostability. Tu Lin et al. (32) reported that Bacillus brevis (B) is stable at 70 ° C. for up to 10 minutes. (Bacillus stearothermo) cloned into B. acillus brevis) Filus esterase was reported. Sugihara et al. (33, 34) Microorganisms, Bacillus soil isolates and Pseudomonas cepacia Omonas cepacia) Isolation of a novel thermostable lipase from a soil isolate ing. The former lipase is stable at 65 ° C for up to 30 minutes, but above this temperature Rapidly inactivates. 75 ° C lipase from Pseudomonas cepacia Was stable when heated at pH 6.5 for 30 minutes, but assayed at this temperature. In that case it had only 10% of its activity. Thermo Alkaholic ( thermocalophilic) lipase (35) is Bacillus sp. MC7 And a half-life of 3 hours at 70 ° C. Had. Most Later, Sigurgisladottir et al. (6) reported that Olly was warmed to 80 ° C. One Therms strain and two Bacillus strains with lipase active against bu oil Reported isolation of strain, but no report on enzyme stability in this study .   These enzymes outline limited changes in substrate specificity and moderate thermostability Only provide. These are the need for different substrate specificities, economical commercialization It does not address the need to produce large quantities that can Stability is limited. In this application, we use a series of (position selectivity, Providing substrate specificity (including body selectivity), enhanced enzyme stability, and commercial Of a series of esterases and lipases that can be produced in large quantities for use are doing. Summary of the Invention   The present invention is characterized by esterase activity and is disclosed in FIGS. 1-4 and Table 1. Prepare for the isolation and characterization of the commercial grade enzyme preparation corresponding to the data. Like In a preferred embodiment, the present invention is characterized by an esterase activity and comprises Table 1 and FIG. Isolation and special isolation of a specially purified esterase corresponding to the data disclosed in JT-9 Prepare for signing. In a most preferred embodiment, the present invention has an esterase activity. Be prepared for proteins produced by recombinant DNA technology. The enzyme of the present disclosure From a variety of sources, including soil, water and waste landfills throughout the United States and elsewhere in the world From thermophilic microorganisms. These microorganisms are generally not at 45 ° C And grow in a temperature range of 90 ° C., whereby they are separated into moderate to extremely thermophilic bacteria. Be classified. Proteins isolated from this group of microorganisms Growing at a low temperature of 5 ° C to 37 ° C, but isolated from a species lacking thermal stability And functionally similar. The enzymes of the present disclosure can be used to produce carboxylic acids and alcohols. By thermophilic microorganisms, including esterase enzymes responsible for the hydrolysis of tell bonds. And proteins produced by the method. The protein of the present disclosure is a native mesophilic enzyme. Has a much longer active life than that found in Activity even after exposure to water and inert in the presence of organic co-solvents Resistant to chemical transformation. The proteins encompassed by the present disclosure are standard purification methods, particularly ionic It can be isolated by exchange chromatography. All enzymes of the present disclosure are Intracellular protein, which is recovered by cell disruption and Can be used. Purified esterases of the present disclosure are eluted with a NaCl gradient Fractions with a single activity are pooled and concentrated prior to lyophilization for storage I do. Specific activity is determined by the Pierce BCA method or by measuring UV absorption at 280 nm. And subsequent activity assay based on the initial hydrolysis rate of p-nitrophenyl propionate It is determined by measuring the total protein concentration by standard determination. Tamper of the present disclosure The proteins were grown at 55 ° C and 65 ° C in TT medium, the bacterial strain from which they were isolated. Length, as well as esterase hydrolysis activity. The present disclosure Protein has esterase activity in a screening microtiter plate assay Can be characterized by: In addition, the protein of the present disclosure is characterized by its protein Characterized by quality temperature summary, protein stability summary, and pH summary It is also possible. The proteins of the present disclosure can be used on native gradient PAGE gels Characterizing by apparent molecular weight corresponding to the enzyme activity staining Can be. The intrinsic molecular weight can be further determined by chromatography. Thus, the specific activity can be further determined under standard conditions. Table 10 shows the standard conditions Many of these features for selected proteins below are listed. This As described above, the proteins of the present invention have their amino acid protein sequences or their protein In addition to the nucleic acid sequence encoding the amino acid protein sequence of the protein, Can be characterized.   Thus, the present disclosure provides a selection of chiral pharmaceutical intermediates and other fine chemicals. Esterases isolated from thermophilic microbial stocks useful in dynamic preparation Libraries. This library contains at least 23 purified enzymes Which are available in various combinations as screening kits or powered For performing chiral reactions or the preparation of chiral products using chemical degradation techniques It can be used as a separate protein preparation. Under these conditions, The rate of hydrolysis of racemic esters catalyzed by these enzymes is It depends on whether the isomer best fits the structural parameters of the enzyme active site. Ki Products having one or more ral centers are carboxylic acids or alcohols. It can be a shift. In addition, many esterases described herein use a solvent Reaction proceeds in the direction of synthesis under transesterification conditions with limited water content Is used to prepare a chiral ester from a carboxylic acid and an alcohol. Can be   The present disclosure provides a recombinant ester hydrolyzing protein of the invention from a transformed cell host. Lambda phage expression vector having an insert that can be used for quality production Is included. The indium contained in this lambda phage expression vector The salt may be, for example, a phage-plasmid hybrid expression vector or other suitable vector. Expression vectors, such as, but not limited to, plasmids, Y It can be used in AC, cosmid, phagemid and the like. In a preferred embodiment The lambda expression vector is a vector named in Table 7, namely, A vector having an insert encoding a substantially similar recombinant protein One of them. The present disclosure is also capable of transforming host cells, And a vector encoding the recombinant ester hydrolysed protein, and its transformed host Main cells and recombinant ester hydrolyzed proteins are also provided. The right host cell Include, but are not limited to, Escherichia coli, Bacillus, Thermus species, etc. is not. Recombinant ester hydrolysis protein encoded by this vector The quality is 5-bromo-4-chloro-3-indolyl-acetate (X-acetate) Can be hydrolyzed. Recombinant ester water produced by this vector Degraded proteins have half-lives comparable to the corresponding proteins purified from the isolate Characterized by stability. Also, this recombinant ester hydrolyzed protein is Safe at temperatures comparable to or better than the corresponding protein from the debris It features the ability to remain constant. The recombinant vector encoded by this vector Stele hydrolyzed proteins are also characterized by certain substrate specificities discussed below, This is comparable to the corresponding purified protein from the isolate. Favorable condition In another embodiment, the vector is the vector named in Table 7 or 8, That is, a vector having an insert encoding a substantially similar recombinant protein. Is a doctor. In a preferred embodiment of the present invention, certain recombinant esters Vectors encoding the hydrolyzed proteins are named and listed in Table 8, American type under the terms and conditions of the Budapest Treaty on the Deposit of Microorganisms ・ Call to Culture Center (ATCC, Rockville, Maryland, USA) One of the vectors that have been deposited and have received a specific designation from the ATCC. Yes, when this number is notified, it will be added to the present specification by amendment. BRIEF DESCRIPTION OF THE FIGURES   FIG. Esterase screening plate. 50 microliter cell extraction The effluent was suspended in 0.7% agarose in 5-bromo-4-chloro-3-indo. Either ril acetate or butyrate (for esterase activity) 0 . Micro tie consisting of 1 mg / ml and 0.1 M Tris-HCl pH 8.0 Transfer to the wells of the tar plate. Control wells contain 20 U of porcine liver esterase ( PLE) or 20 U of porcine pancreatic lipase (PPL) buffer Consists of addition. Normally sufficient time to allow full color development in control wells, usually Incubate the plate at 37 ° C. for about 20 minutes. Blackish wells are active Indicates that the sex is positive. This photo shows the screenin of 65 candidate isolates. And the resulting positive.   FIG. Esterase activity staining of crude extracts from thermophiles. After electrophoresis, it These gels were equilibrated with Trizma buffer at pH 7.6, then Stain for activity with any of 0.15% X-acetate. Then this The gels are incubated at 55 ° C. for up to 30 minutes.   FIG. Molecular weight calibration curve. FIG. 3 shows the standard molecular weight calibration curve. Represent.   4A-T. Features of the enzyme. 4A-T provide a summary of activities that characterize the enzymes of the present disclosure. The point is. Graph 1 shows the relative esterase activity for each of the listed enzymes. 1 shows an overview of the temperature of the enzyme, plotting against temperature. Graph 2 is at 25 ° C. Listed, plotting relative residual activity at 40 ° C. and 65 ° C. against time 2 shows the residual esterase activity of the enzyme. Graph 3 shows relative esterase activity versus pH. 1 shows a summary of the pH of listed enzymes, plotted against.   Figure 5.8. Overview of E100 migration on 5.8% SDS-PAGE. Lane 1. DEAE And boiling E100 following Q Sepharose chromatography. Lane 2. Non-boiled Purified E100. Lane 3. Boiling E100. Lane 4. Molecular weight marker.   FIG. Kinetic analysis of E100. The enzymes are: a) Lineweaver-Burk (L ineweaver-Burke) and b) Edie-Hofstie (Ea normal Michaelis kinetics yielding linear data in both die-Hofstee analysis Km = 7.2 × 10 using p-NP as a substrate^-FiveM and Vmax = 1.8X10^-FiveMinute^-1Is obtained.   FIG. Summary of temperature and pH of E100. a) Summary of temperature of E100. p-nit Prop as a function of temperature for E100 catalyzed hydrolysis of rophenylpropionate G. Enzyme activity upon exposure to different temperatures was determined. Nitrophenylpropione The initial rate of hydrolysis of_Three0.25 mM substrate dissolved in CN, then 50 mM borate buffer pH 8.5 equilibrated to the desired temperature with added enzyme Was determined. Of the absorbance at 405 nm Determine the rate by observing the change and replace bovine serum albumin with the enzyme. Correction was made for spontaneous hydrolysis of the substrate used. b) Summary of pH of E100. E On the hydrolysis of p-nitrophenylpropionate catalyzed by H.100 PH effect. An overview of the pH of the enzyme can be obtained by using different buffers suitable for the desired pH. It was determined by preparing at a concentration of 0 mM. CH_ThreeSubstrate dissolved in CN (0. The reaction was performed by adding the enzyme to this buffer solution. . After incubating the reaction for 5 minutes, CH_Three0.1 mM P dissolved in CN The reaction was stopped by adding MSF. Add 0.1M Tris-HCl To adjust the pH of the mixture to 8.5. The absorbance at 405 nm Record and ε = 17 mM for product nitrophenol^-1cm^-1Concentration based on Was calculated. Product formation was corrected for spontaneous hydrolysis of the substrate.   FIG. Addition of p-nitrophenylpropionate, measured by relative velocity E100 resistance to the presence of organic co-solvents during water splitting. Various concentrations of CH_ThreeC By measuring the initial rate of enzyme-catalyzed hydrolysis of pNP in the presence of N Determine the residual activity of the enzyme in the presence of an organic solvent. Theory in determining activity As indicated, react at 37 ° C. in 50 mM Tris-HCl pH 8.5. I do. Changes in absorbance in spontaneous hydrolysis of substrates and in the presence of organic co-solvents Correct for changes in the extinction coefficient of the product.   FIG. Purification of E101. a) E10 indicated by 10% SDS-PAGE Step in the purification of 1. Lane 1. Molecular weight marker. Lane 2. (Included as standard Purified E100. Lane 3. NH_FourSO_FourDialysis after fractionation Protein. Lane 4. DEAE introduction / washing. Lane 5. SP Sepharose Introduction / cleaning. Lane 6. Purified E101. Eluted from S200 gel column. b) 10% SDS-PAGE of E101. Lane 1. Boiling E101. Lane 2. Non-boiled Boiling E101. Lane 3. Molecular weight marker.   FIG. Substrates used for stereoselectivity and regioselectivity screening. S Terases have substrate structures that fit into four types and can mediate stereoselectivity and regioselectivity. It is a flexible biocatalyst in the sense that The listed compounds are produced from chemical intermediates. Range of different structural features encountered in common substrates of potential Indicates the box. Some of these substrates are enantiomers or diastereomers Can be purchased in a pure form, and the quantitative Can be used in cleaning procedures. Hydrolysis to break down chiral centers Four classes of substrates most commonly associated with biocatalysts have been considered. A) Ta The substrate of Ip positions the desired product on the carboxylic acid side of the product, whereas In addition, the type II compound has the functionality required for alcohol. B) Type Substrates of type III and type IV can be considered a subset of types I and II. However, their unique characteristics dictate that they be classified separately. Type III molecules require that the enzyme distinguish between prochiral substrates, In contrast, type IV compounds have a meso structure. These last two substrate types are It is very unlikely that these types of compounds will be selectively activated by other chemical means. The importance of the biocatalyst-based decomposition method in the synthesis is shown.   FIG. A method for selecting a recombinant esterase. a) X-acetate gel overlay Strain isolate 28 (E Screening of phage library derived from 009). Departs esterase The blue color surrounds the single phage plaque that appears. b) 54 (E002) strains Purification of hybrid phage produced from. Hydrolyzed X-acetate color The amount of priming substrate surrounding each of the three phage stock phage plaques I have. c) and d) phage λTGE1.1; λTGE1.2; λTGE1.3. ΛTGE2.1; λTGE2.2; λTGE2.3; λTGE2.4; E2.8; λTGE3.2; λTGE3.3; λTGE3.4; λTGE4.1. ΛTGE4.2; λTGE4.3; λTGE11.1; λTGE11.3; TGE11.4; λTGE11.7; λTGE11.9; λTGE11.10; λTGE15.1; λTGE15.3; λTGE15.5; λTGE15.8; Studies on the hydrolytic activity of plasmid-bearing strains derived from λTGE15.9 Pot test. Higher activity detected by X-acetate leads to weaker growth Strongly related.   Figures 12a-r. Esterase activity of recombinant protein derived from phage lysate Examples of screening techniques using sex staining. Once an esterase positive candidate is identified Once done, the phage lysate was purified with a native 4-15% gradient BioRad (BioRa). d) Check for correct ester hydrolysis activity on ReadyGel. Clean. After electrophoresis, the gel was washed with Trizma buffer pH 7.6. By using a 0.15% X-acetate overlay. Stain for activity. The gel is then incubated at room temperature for up to 30 minutes . These figures identify proteins that have the same migration properties as the native protein. How to use this technology Here is a typical example. a) The strain isolate S1 to identify E001 Screening of positive clones from the pools made from them. Each lane is lambda TGE 1 isolate, 1, 2, 3, 4, 5, 8 and native control protein (C) are shown; b 3.) Positive clones from pools made from strain isolate 54 to identify E002 Screening. Each lane is a lambda TGE2 isolate, 1, 2, 3, 4, 6, 8 and native control protein (C); c) strain to identify E003 Screening of positive clones from pools made from isolate 50. Each lane is Shows lambda TGE3 isolate, 1, 2, 3, 4, and native control protein (C) D) positives from pools made from strain isolate GP1 to identify E004 Screening of clones. Each lane is a lambda TGE4 isolate, 1, 2, 3, 4 5, 6 and the native control protein (C) are shown; e) to identify E005 Of positive clones from stocks made from strain isolate C-1. each Lanes show lambda TGE5 isolate, 1, 2, 3, 4, 5, 6 and native control protein. F) generated from strain isolate 55 to identify E006 Screening for positive clones from stock. Each lane is a lambda TGE6 isolate, 1, 2, 3, 4, 5, 6 and the native control protein (C) are shown; g) E008 Of positive clones from stocks made from strain isolate 30 to identify Learning. Each lane shows lambda TGE8 isolate, 1, 2, 3, 4, 5, 6, and unchanged H) Sex control protein (C); h) strain isolate 28 to identify E009 Screening of positive clones from stocks made from. Each lane is lambda TG E9 isolate, 1,2,3,4,5,6,7 and native control protein (C) Shown; i) Positive from pool made from strain isolate 29 to identify E010 Sex clone screening. Each lane is a lambda TGE10 isolate, 1, 2, 3, , 4, 5, 6, 7, 8, 9, 10, 11, 12 and native control protein (C) J) from a pool made from strain isolate 31 to identify E011 Screening for positive clones. Each lane was lambda TGE11 alone on the first gel. Release, 1, 2, 3, 4, 7, 8 and native control protein (C) and second gel Lambda TGE11 isolate, 7, 8, 9, 10 and native control protein (C K) a pool made from strain isolate 26b to identify E0012 Screening of positive clones from. Each lane contains lambda TGE12 isolate, 1 2, 3, 4, 5, 6, and the native control protein (C) are shown; Screening of positive clones from stocks made from strain isolate 27 to identify Ning. Each lane shows lambda TGE13 isolate, 1, 2, 3, 4, 7, 8 and unchanged Shows sex control protein (C); m) strain isolate 34 to identify E014 Screening of positive clones from stocks made from. Each lane is lambda TG E14 isolate, 3, 5, 6, 8, 9, and native control protein (C) are shown; n 3.) Positive clones from pools made from strain isolate 62 to identify E015 Screening. Each lane is a lambda TGE15 isolate, 1, 2, 3, 4, 5 , 6, 7, 8 and the native control protein (C) are shown; o) Identify E016 Screening of positive clones from stocks made from strain isolate 47 to isolate. Each lane shows lambda TGE16 isolate, 1, 2, 3, 4, 5, 6, 7 and native pair. Indicating control protein (C); p) Strain isolation to identify E019 Screening of positive clones from pools made from body 4. Each lane is lambda TGE19 isolate, 1,2,3,4,5,6 and native control protein (C) Shown; q) Positive from pool made from strain isolate 7 to identify E020 Screening of clones. Each lane is a lambda TGE20 isolate, 3, 4, 6, and And native control protein (C); r) identifies E021 (E017b) Screening of positive clones from stocks made from strain isolate 32 for storage. Each lane contains lambda TGE21 isolate, 6, 8, 9 and native control protein (C ).   FIG. Effect of temperature on clone stability. Active esterase protein Recombinant strains having a plasmid with are often found on X-acetate plates. A phenotypic separation of the esterase activity was shown. This separation indicates that the esterase activity If toxic to cells, this may be due to loss of plasmid or insert. There was ability. To overcome this, cells should be cooled at low temperatures (probably due to cloning It was possible to grow (with reduced activity of the thermoesterase). these In the figure, TGE15.2 (15) and TGE15.9 (14) strains were transformed with X-acete. It is shown that both the coatings are applied at 28 ° C. (a) and 37 ° C. (b). Faster Yellow colonies of the growing isolate were visible at both temperatures, but reversed at 37 ° C. The choice is more powerful. The same phenomenon was observed for T grown at 28 ° C. and 37 ° C., respectively. GE2.1 (1); (for TGE2.2 (2) and TGE3.2 (3) strains) Found in c) and (d).   FIG. Example of esterase staining of a recombinant protein derived from a plasmid. Not yet Denatured microorganism (single column purification) And protein extracts from both the recombinant production strains. protein Run the extract on a 4-15% gradient Bio-Rad ready gel. Electric swimming After running, the gels were equilibrated with Trisma buffer at pH 7.6 and then 0.4% in X-acetate using X-acetate overlay Stain for activity. The gels are then incubated at room temperature for up to 30 minutes. To In these examples, native without any visible activity on this stained gel E007 derived from microorganism (E007N) and protein derived from CE007 strain Extract; recE00 from E008 and CE008 from native strain 8; recE00 derived from E009 and CE009 strains derived from native microorganisms 9; recE01 from E010 and CE010 strains derived from native microorganisms 0; recE01 from the E011 and CE011 strains derived from native microorganisms 1; recE01 from E014 and CE014 strains derived from native microorganisms 4; recE01 derived from E015 and CE015 strains derived from native microorganisms 5; recE from E017b and CE017b strains derived from native microorganisms 017b; rec from E019 and CE019 strains derived from native microorganisms E019: E021, derived from a native microorganism, r derived from CE009 (R) strain recE028 from ecE020 and CE028 strains-both of the same gene Isolated from reserve. recE028 has a low level of background on native protein preparations. N = native protein; R = recombinant protein Protein; R * = alternative recombinant protein with different migration pattern (in this case , E028 cloned from the same strain as E020).   Figure 15. Digestion pattern for the recombinant esterase of Figure 24. Listed in Table 8 The restriction endonuclease digestion patterns for a set of 24 plasmids are shown. Is done. (A) Plasmid 24 was replaced with EcoRI (1-24) and BamHI (25 -48), cut with HindIII (49-72) and EcoRV (73-96) I do. (B) Gel showing PstI digestion pattern for plasmids 1-18. ( c) PstI digestion pattern for plasmids 19-24 Gel showing the XbaI digestion pattern for 1. (D) Plasmids 12-24 And a gel showing an XbaI digestion pattern. Lanes 1-24 for all gels The following plasmids are referred to in the following order: pTGE1.1, pTGE2.1, pTGE GE2.2, pTGE3.2, pTGE4.6, pTGE5.3, pTGE6. 3, pTGE 7.1, pTGE 8.5, pTGE 9.4, pTGE 10.3, p TGE 11.10, pTGE 12.2, pTGE 13.2, pTGE 14.3, pTGE14.6, pTGE15.9, pTGE16.1, pTGE19.4, pTGE20.4, pTGE21.8, pTGE21.8x, pTGE20.3 , PTGE16.3. Plasmid pTGE21.8x is a variant of pTGE21.8 Which was isolated and had lost activity.   FIG. Nucleic acid sequence and amino acid sequence of translated protein. The enzyme of the present invention Isolation and cloning of the encoding gene generates a DNA segment, which is Open readies corresponding to the amino acid sequence of the translated protein A frame (ORF) can be found. Alternative start codons Recognized in the field, but few encoded proteins Both contain the nuclear protein ORF. FIG. 16A corresponds to the ORF of the E001 enzyme. Isolated nucleic acid sequence, and translated amino acid sequence, with alternative start codons Is underlined. FIG. 16B shows the cloned clone containing the E001 ORF. Isolated nucleic acid sequence. FIG. 16C shows the isolated corresponding to the ORF of the E009 enzyme. Nucleic acid sequence and translated amino acid sequence, with alternative start codons Lines are attached. FIG. 16D shows a cloned unit containing the ORF of E009. The isolated nucleic acid sequence. FIG. 16E shows the isolated nucleus corresponding to the ORF of the E011 enzyme. Acid sequence and translated amino acid sequence; alternative start codons are underlined Have been. FIG. 16F shows the cloned isolated nucleic acid containing the E011 ORF. Is an array. FIG. 16G shows the isolated nucleic acid sequence corresponding to the ORF of the E101 enzyme. , And the translated amino acid sequence, with alternative start codons underlined. I have. FIG. 16H shows the cloned isolated nucleic acid sequence containing the ORF of E101. is there. DETAILED DESCRIPTION OF THE INVENTION   The present invention has been selected for enzyme activity and has apparent molecular weight, pH, and temperature stability. Isolated and commercially available from thermostable bacteria characterized by To prepare a useful protein preparation. The isolated proteins of the present disclosure can be used to perform biocatalysis. Molecular weight markers to find similar enzymes besides functionally used enzymes Can be used as Commercial chemical synthesis of certain racemic products is often Often, the use of such isolated enzyme preparations is required.   The results of the characterization assay are based on the described esterase Indicates that the prime has a series of optimal parameters. For example, E100 and E10 1 has an optimal operating temperature above 70 ° C, consistent with enzymes isolated from extremely thermophilic bacteria. E001-E021 have been isolated from thermophilic microorganisms to a lesser extent It has an optimal commercial temperature in the range of 40-50 ° C., corresponding to the enzyme. However, Both groups are stable and functional compared to other known esterases from thermophiles Has been added. E001-E021 work in a non-extreme temperature range It provides an optimal temperature environment for chemists wishing to work, and works well even at room temperature. Also, These results indicate that the enzymes described do not show a clear preference under experimental conditions. Although it has various pH optimum conditions including near neutral or near neutral It is a tendency of most proteins to have an optimal pH slightly below Also shown.   The following examples are intended to be illustrative, as are certain aspects of the present invention. It is illustrated and is not intended to be limiting. Normal techniques in the art Have many equivalents to the invention beyond the scope of routine experimentation. You will understand what can be done. Embodiment 1 FIG. Isolation and propagation of thermophilic microorganisms   Strain-Therms T351 (ATCC 31677) is American Type Cal Available from the Char Center (ATCC). All isolates and cultures Grow in TT medium (36). This medium (per liter) is Puton (8 gm), Difco East Ixtrak (Difco Yeas) t Extract) (4 gm), and NaCl (2 gm). Screen Small cultures at 65 ° C. 250-300 rpm in 1 liter medium in 2 liter flask Lengthen. Larger cell production for enzyme purification requires a 17 liter fermentor (LH Fermentation, Model 20 Grow within the 00 series 1). These fermenters have an effective volume of 15 liters Cultures in TT broth at 250 rpm, 0.3-0.5 vv m (air volume / medium volume per minute) at 65 ° C. The temperature is 28 liters Circulate 56 ° C water from 65 ° C water tank through hollow baffle in stirring jar Maintain by doing. E. coli strains are grown as described in (37).   New and improved thermophile enrichment procedure-sediment and soil samples comprising organic matter Is used for isolation of a new strain. These samples are located on many lands in the midwest to southeast. Collect from. Samples (〜1 gm) were suspended in 2 ml of TT broth and TT agar plate containing 50-100 μl of the mixture twice the normal volume of agar (3%) Apply to. Agar is usually added to solid media to a final concentration of 1.5%. This This allows very motile microorganisms to crowd plates at the expense of other microorganisms It is escaped from being made. Plates are incubated at 55-65 ° C for 1-2 days. Bait and isolate, then place on a fresh plate to isolate a single colony Purify by re-streaking multiple times. Initial criteria for sorting are color, colony morphology , Microscopy, growth temperature, and lipase and esterase activity. First, Hundreds of strains were isolated. Sixty-five different microorganisms were selected for further study. Embodiment 2. FIG. Esterase identification and assay method   Esterase plate assay-microbes were cultured in liquid culture using TT medium Grow at either ° C or 65 ° C. Centrifuge cells (3,000 RPM at 2 0 min) and save the supernatant for testing. 2 volumes of pellets Of the cell pellet after washing three times with 10 mM Tris HCl pH 8.0. Is resuspended in fresh Tris buffer and disrupted by sonication. Cell debris Was removed by centrifugation and the crude extract was subjected to esterase activity as shown in FIG. Was tested. Both cell extracts and culture supernatants can be treated with Test for activity. Only cell extracts showed significant esterase activity.   Esterase liquid assay and determination of specific activity-bovine serum algal concentration is determined Determine protein concentration by Pierce BCA assay using Bumin as standard . The protein concentration was obtained from the calibrated absorbance at 562 nm of the sample solution, In milligrams. Esterase activity was equilibrated at 40 ° C and 346 Observed at nm (iso-absorption point of acid / carboxylate pair ε = 4800), 50 m P-Nitrophenylpropriate in M sodium phosphate buffer pH 7.0 Onoate (0.5 M from a 10 mM stock dissolved in CH3CN) It is routinely measured by determining the ratio. Specific activity was equivalent to milligrams of total protein Of p-nitrophenol produced in micrograms per minute You.   Identification of extremely stable esterases-native (non-denatured) 10% polyacylase Run the crude extract on a rilamide gel. Subsequently, these gels were treated with 5-bromo- 4 -Chloro-3-indolyl acetate (X-acetate), 5-bromo-4-octane Loro-3-indolyl butyrate (X-butyrate) or 5-bromo-4-chloro B-3 Esthetics containing any of indolyl caprylate (X-caprylate) It can be stained with an enzyme activity stain to produce an indigo precipitate. Therms The crude extract clearly showed two major bands in those lanes. colon A single small band of activity is seen in the bacterial control lane. Esterase is Therms T 351 and several new isolates. These microorganisms The activities found therefrom are summarized in Table 1.Embodiment 3 FIG. Procedure for purifying and homogenizing esterase activity   Protein Isolation--Generate large batch cell cultures by the method described in Example 1. Allow to grow and collect cell paste by centrifugation and store at -80 ° C. 100g The cell paste was prepared at pH 7.0 with 200 mM KCl and 0.1 mM EDTA. Thaw in 200 ml of stirring solution comprising 5 mM phosphate buffer of 5 . Once dissolved, the suspension was warmed to room temperature and then lysozyme (0.1 mg / ml ) For 2 hours. Next, the solution is sonicated to completely destroy the cells. 375 watt Sonics & Materials Bibra with standard 1/4 "horn Cell (Sonics & Materials Vibra Cell) Ultrasound The setting used in the processor was an output setting of a destruction degree of 8 at a pulse ratio of 50% for 5 minutes. . Alternative methods of cell destruction include French press, Gaullen e Processing cells with a homogenizer, microfluidizer, or other homogenizer May be included. Cell debris is removed by centrifugation and NH to 60% saturation._FourSO_FourMinute The protein can be precipitated by the fractionation. Centrifuge the precipitated protein, Minimum volume of 50 mM phosphate containing 1 mM β-mercaptoethanol (BME) Resuspend at pH 6.5.   DEAE purification-The protein solution was suspended using a 10 Kd pore size dialysis tubing system. Dialyze three times against turbidity buffer. The obtained protein solution is 100 ml bed volume DEAE column diluted 2-fold and equilibrated with the same buffer To After washing the column with 200 ml of equilibration buffer, Elute with a linear gradient of 0.5 M NaCl.   Q resin purification-activity isolated by DEAE purification Pool the sexual fractions and exchange this buffer three times against the equilibration buffer. Dialysate, and the dialysate is equilibrated with the above buffer to a 50 ml bed volume It was applied to the resin Q resin. The column contains 0.1 M KCl and 1 mM BME. Wash with 100 ml of 50 mM phosphate pH 6.5, then add Elute with 150 ml of added 0.1 M to 0.6 M KCl gradient.   Ultrafiltration concentration-pool the active fractions and attach a 30 kd cut-off membrane Using the Amicon Ultrafiltration System Concentrate.   Separated SDS-PAGE-Concentrated protein solution is transferred to standard SDS introduction buffer Using a gel, guide the sample to a 10% SDS-PAGE gel for separation without boiling. Enter. After development, 0.1 M phosphate pH 7.5 and 0.1 mg / ml Gels were treated with 0.7% agarose containing lomo-4-chloro-indolyl acetate Manage. The resulting blue band was cut out of the gel, placed in a dialysis tubing, and treated with 0.05M Recover protein by electroelution for 1 hour in Tris buffer pH 8.5 . Native and SDS-PAGE stained with either Coomassie or silver stain At this stage, the protein is uniformly purified, as observed by both. The protein can be stored at 4 ° C. for future use.   Gel filtration-using a gel filtration column as a further or alternative purification step You can also. Embodiment 4. FIG. Commercial grade purification of isolated esterases   In many industrial applications, the cost A purified enzyme preparation is neither required nor desired. Meaningful d Tans of interest in isolated form to contain proteins with sterase activity A quick and inexpensive protocol for producing protein is desired. Of such semi-purified procedures One is described here. 50 g of cell paste is added to 0.1 M NaCl and 0.0 6. 100 ml of 50 mM Tris HCl buffer containing 1 mM EDTA pH7. Thaw in 5. Break cells by sonication and remove cell debris by centrifugation You. The crude cell lysate was diluted 3-fold with 50 mM Tris-HCl pH 7.5. A DEAE cellulose column (bed volume 6) equilibrated with this material with dilution buffer. 0 ml). The column is filled with 3 column volumes of dilution buffer, then 4 columns. Wash with a salt gradient of 0-0.5M NaCl over the whole volume. The active fraction is Elution from the on-exchange resin into a 0.25-0.35M salt gradient window. Fraction Were assayed for activity as described in the determination of specific activity and showed the highest activity. Pool and pool for ultrafiltration using a 10Kd molecular weight cut-off membrane More concentrated. Store the concentrated enzyme sample at 4 ° C. for future use. In case Thus, a substrate agarose overlay of proteins separated by native PAGE More than one ester hydrolytic activity is still detected under prolonged exposure to Which is not an obstacle for most industrial applications. Shows a small amount of secondary esterase activity. If necessary, further purification (eg, Ammonium sulfate precipitation, gel filtration or other methods described in Example 3) can do. This process can be scaled up or scaled up as desired. It is also possible to call-down. Embodiment 5 FIG. Method for determining the temperature profile   The outline of the optimal temperature of the esterase protein is 30 ° C, 35 ° C, and 45 ° C, respectively. 0.1M sodium phosphate buffer equilibrated for 5 minutes at 55 ° C, 55 ° C and 65 ° C It is achieved by measuring the activity of esterase diluted at pH 7.0. So After that, in Example 2 (modified at the various temperatures used in this example) Added to this equilibration solution under the reaction conditions described for the measurement of specific activity By measuring the rate of hydrolysis of the p-nitrophenyl proprionate Determine the temperature summary. Enables correction of temperature-dependent autohydrolysis of the substrate Use a control reaction with bovine serum albumin instead of the esterase enzyme. You. The data is then plotted as a relative activity against the temperature of the reaction. Embodiment 6 FIG. Methods for determining enzyme stability   The long-term stability of the esterase enzyme as an enzyme after exposure to various temperatures Evaluate by testing for residual activity. Enzyme stock solution at 0.1 M phosphorus Dilute with sodium acid buffer pH 7.0 to avoid concentration effects due to evaporation Equilibrated to 25 ° C, 40 ° C or 60 ° C, respectively, under sealed conditions Put in a temperature bath. Thereafter, aliquots are removed at regular intervals and By measuring the rate of hydrolysis of p-nitrophenyl proprionate, Determine the remaining activity. Results are plotted against time as relative activities . These results (see FIG. 4) are based on exposure to temperatures up to and including 40 ° C. Show that all enzymes retain most of their initial activity for at least 48 hours. Activity Properties are particularly relevant for enzymes isolated from microorganisms with optimal growth temperatures near 55 ° C. Decreases at 60 ° C. Embodiment 7 FIG. Method for determining pH overview   An overview of the esterase pH is determined as follows. Determination of specific activity in Example 2 Under reaction conditions similar to those described for Using a buffer with a broad useful pH window that overlaps the Determine the rate of hydrolysis of trophenylproprionate. Of these experiments Therefore, two kinds of buffers satisfying the above criteria, female (a useful range of 6-6.5) and Bis-trispropane (useful buffer range 6.5-9) was selected. All p The H test was based on experimental control using bovine serum albumin instead of esterase. The result was subtracted to correct for spontaneous autohydrolysis. This control data Processing becomes particularly important for pH above 7.5. Embodiment 8 FIG. Effect of solvent on esterase activity.   In industrial applications for biocatalysts, enzymes are often not It is required to function under severe conditions. Exposure to temperature rise and pH fluctuations Is a potential challenge to the activity of enzymes, but as a substrate target for biocatalysts Lack of water solubility of many compounds that can be useful is a major challenge for industrial organic chemists is there. Organic co-solvents are commonly used in reactions, and isolated enzymes Must be able to survive under the conditions of the co-solvent. Various organic solvents, For example, ethanol, acetonitrile, dimethylformamide, dioxane, toluene Ruene, hexane and SDS, Triton X100 and The experiment is performed in the presence of a detergent such as Tween 20. In addition, the isolated enzyme Activity was determined under near anhydrous solvent conditions and lyophilization of the isolated enzyme from buffer. Further experiments testing at a suitably active pH are also performed. Embodiment 9 FIG. Methods for protein characterization by migration on native PAGE   The number of esterase enzymes in each of the semi-purified samples was determined by 4-15% acrylamide gradient (Bio- Native gel PAGE using preformed gel purchased from Rad Laboratories) To decide from. The gel shows traces of contamination with other enzymes, which are Indolyl acetate not easily detectable by HPLC due to dilution effects The hydrolysis of the salt is possible. It is clear from this gel experiment that most samples Has a single major activity with migratory properties similar to those shown in FIG. That is. Embodiment 10 FIG. Determination of relative molecular weight by chromatography   The estimated native molecular weight of the protein of interest was determined by Hitachi HPLC 6200 (Pharmacia) Superdex (Supe) rdex) Determined by separation on a S200 FPLC column. protein With a 0.05 M sodium phosphate buffer, pH 7.0, containing 0.1 M NaCl. Separated by isocratic elution on The flow rate of the solvent is 0.5 ml / The protein was detected by UV at 280 nm. Esterase activity The fraction is first In the mo-3-chloro-3-indolyl acetate plate assay, the next most active The fraction was analyzed by a follow-up assay by hydrolysis of p-nitrophenyl proprionate. (Both methods are described in Example 2). The molecular weight is Protein: β-amylase 200Kd, alcohol dehydrogenase 150Kd, c Cysteine albumin 66Kd, carbonic anhydrase 29Kd, cytochrome c12.3K d (using a logarithm of the molecular weight versus time expressed in minutes). Estimated by comparison to a standard elution profile (lotted). Embodiment 11 FIG. Characterization of substrate specificity   Enzyme substrate priorities for hydrolysis activity on various esters are as follows: Can be determined. Each of the substrates is CH_ThreeDissolve in CN (final concentration 0.1 M), by mixing with 0.1 M phosphate buffer pH 7.5. Prepare a lattice of molecules on the interplate. The partially purified enzyme is then Add to these wells and incubate the reaction mixture for 30 minutes. Crude melting Demolition can also be tested in this way. Remove the plate after 10, 20 and 30 minutes Check to determine relative activity. For experiments with colorless substrates, dilute the reaction. Except for three extractions with chloromethane, except as described for colored substrates The reaction is performed in a test tube under the same conditions. Combine the organic layers and add MgSO_FourDry with Allow to concentrate to 0.1 ml in a stream of nitrogen. Next, this concentrate is applied to silica gel T Spot on LC plate, 80: 20: 0.01 Hexane: Ethyl A Tell: develop with a solvent mixture of acetic acid. UV and I TLC plates_TwoVisualize with You. Embodiment 12 FIG. Rapid screening assays for rapid substrate specificity characterization   Release of hydrogen ions during ester hydrolysis lowers the pH of the system. Rapid screening for esterase activity based on the mechanism of the medium hydrolysis reaction Therefore, a new method was developed. The flow of hydrogen ions in the reaction depends on the enzyme activity. Observed by using an indicator dye that has an enzyme-dependent color change within the desired pH range can do. The best indicators tested work best at slightly elevated pH Phenol red (starting point pH 8.5) for the more neutral enzyme For enzymes that work well under the conditions, bromothymol blue (starting point pH 7.2) ).   The response of the indicator is observed in one of two ways. Spectroscopic studies are different Phenol red or bromothymol dissolved at a concentration of 5 mM in a pH buffer By measuring the UV / visible maximum of any 0.001% solution of blue Do. Next, the substrate (final concentration 0.1 mM) was added to a 5 mM buffer solution (bromothymo Sodium phosphate pH 7.2, phenol red finger For the indicator, add sodium borate pH 8.5) and raise the temperature to 25 ° C for 5 minutes. Equilibrate and then start the reaction by adding 0.1 U of target enzyme. And a hydrolysis reaction is carried out.   Alternative methods for monitoring hydrolysis reactions are useful in a wide variety of screening applications is there. CH such that 0.001% indicator dye and less than 10% organic solvent composition_Three Stir 5 mM buffer containing substrate dissolved in CN, DMF or DMSO 2 Add to 4-well microtiter tray I can. After equilibrating the temperature at 25 ° C. for 5 minutes, 0.1 U of esterase is added. To initiate the reaction. Monitor the progress of the reaction by changing the color of the solution. When changes occur, an aliquot of NaOH is added to return the color of the reaction to the starting point. If no further color change is detected after prolonged incubation The reaction is determined to be complete. TL of reaction acidified with 0.1 M HCl The product formation was confirmed by C analysis and extracted with ethyl acetate to_TwoSO_FourDried in Let N_TwoConcentrate under stream. Enzyme-based chiral degradation has been screened For testing of different substrates, the products are separated and isolated by chiral phase HPLC, The enantiomeric purity is determined by integrating the peak areas of the isomers.   A rapid assay for various hydrolytic activities, in this case esterases, is Determined in experiments in microtiter plates using different enzymes and substrates You. Hydrolysis of total protein and the normal substrate p-nitrophenylproprionate By using the same specific activity for each enzyme determined from the initial solution ratio, Accurate comparison of enzymes sold can be guaranteed. The data is for a given indicator dye. Is recorded as the time required to visualize the pH-dependent color change. BSA In the control experiment using as a protein source, the color of the indicator did not change, It is established that the change in H is the result of enzyme-catalyzed hydrolysis. Enzymes and indicators A control test of the reaction solution with and without substrate shows a It was established that this was not the result of the salt or enzyme alone.   Is the format of the microtiter plate acceptable for small-scale preparative chemistry? The research performed to determine whether this is done is as follows. Racemic phene Chilua The reaction was carried out using acetate and porcine liver esterase and 0.1N NaOH Keep the original color of the solution until titration with aliquots no longer causes a color change When the color change no longer occurs, the reaction is stopped. The product is isolated by TLC And confirm the extent of the reaction by comparing the total amount of base added. Flash mosquito By column chromatography and then by chiral phase HPLC, The tyl alcohol is separated from the starting acetyl ester. Water content from peak integration The enantiomeric excess of the digest was determined and performed in the absence of the indicator dye Compare with the same reaction. The results from these experiments indicate that the It has no effect on the stereospecificity of esterase-catalyzed degradation of phenethyl acetate And suggest.   To test for utility in broad-based enzyme screening methods, Seven types of microorganisms isolated from various sources in the environment were transformed into groups of structurally diverse compounds. They were tested for their ability to produce enzymes that catalyze hydrolysis. Table 2 shows this The results of these studies are shown.Embodiment 13 FIG. Further characterization of substrate specificity   Shown in FIG. 10 are the substrates that can be tested for the activity of each enzyme. It is an example. Because of their importance as intermediates, these molecules are Specially selected in the literature with the potential for industrial use . Experiments were performed on the crude lysate, or its activity could include substrate preference and stereochemistry. If it is known to quickly determine the reaction chemistry, Can be carried out using the extracted protein. All structure activity tests were performed on pig liver Compare with a standard mesophilic biocatalyst such as esterase. Reaction is analyzed by TLC Observe and purchase the product from commercial sources or use chemical means (eg, (Base-catalyzed hydrolysis of ter).   The study of stereochemical priority by each esterase is one of two approaches. Therefore, it can be evaluated. In the first method, commercially available enantiomerically pure The standard single stereoisomer of a novel substrate ester is hydrolyzed with each enzyme and the enantiomer is added. The relative ratio of water splitting is used as a diagnostic qualitative determinant of potential chiral selectivity. In the second method, molecules that cannot be purchased as a single stereoisomer Using a kinetic cracking method (generally performing the reaction to less than 50% completion) Hydrolyze as a mixture. The reaction product was isolated and purified by chiral phase HPLC (de Diacel Chiralcel OD or OB ) Or the product is a Mosher derivative (alcohol product) or Prepared by derivatization to a menthyl derivative (carboxylate product) Of their corresponding diastereomers by 1H NMR their enantiomeric excess (ee) About And analyze. The ratio of diastereomers determined from the NMR spectra corresponds to Based on the integral of the peak, Compare with any of the standards obtained from commercial sources using the FT reagent. Then raw The optical rotation and absolute configuration of the product are determined by optical rotation analysis and the value or quotient found in the literature. Compare with values determined from standards obtained from industrial suppliers. Embodiment 14 FIG. Characterization of protein E001-E021 / 17b   Strains from the identified sources listed in Table 1 were identified as TT as described in Example 1. Isolated by growth at 65 ° C. in media (ie, Sl etc). Esterase hydrolysis specific activity was identified by the method described in Example 2, Assign identifiers to the isolated esterase proteins as listed in Table 1. (Ie, E001 etc.). To prepare the enzyme, a 15 liter culture of the isolate The nutrients are grown and the cells are spun down and collected as described in Example 1. The cells were lysed and the isolated preparation was prepared according to the procedure outlined in Example 4. Purified. This protein was used to determine the temperature profile in Example 5, Example 6 to determine the stability of the protein, and to determine the pH overview Characterized using the method described in Example 7. The results are shown in FIG. . This protein was used for migration on native gradient PAGE as described in Example 9. Therefore characterized. The data is shown in FIG. As described in Example 2 The specific activity was determined and analyzed by chromatography as described in Example 10. The amount of offspring was determined. They are shown in Table 1. Substrate specificity of some proteins The properties are shown and are shown in Table 2. Thus, identified and characterized Esterases are useful and have unique activities in commercially useful purity It is shown to be. The definitive results are summarized in Table 10. Embodiment 15 FIG. Characterization of E100   Purification of E100-E100 was described in Example 3 from Therms species T351. A series of four steps: DEAE purification, Q resin purification, ultraconcentration, and separation SDS P Purify 300 times by AGE. Because the second esterase activity was present (E101), the specific activity could not be measured in the crude lysate. this The second activity is completely removed from the target esterase during the first chromatography step. It was possible to remove it. In this chromatography step, the second step The sterase passed through the DEAE column without binding. Various technical grays In order to purify E100 of DEAD, DEAE purification alone is required from all other contaminating activities. Sufficient to obtain substantially purified E100. Q resin purification and ultrafiltration Enables the production of purer products according to the requirements of a particular application I do. The final SDS PAGE purification step involves the use of proteins to characterize the molecule. It allows the quality to be uniformly purified.   Protein characterization-separation of active bands electroelution on SDS-PAGE gel And return to 10% SDS-PAGE under denaturing conditions. As a result, A single band with a relative molecular weight of 45 Kd is shown (FIG. 5). Unboiled sample By running on the same SDS-PAGE gel, the approximate proposed monomer fraction Child A plurality of bands are shown at the increased positions. In addition, this non-boiled sample But can be stained, but are compatible with multimeric forms of the enzyme, starting with dimeric species. Only relevant bands are found to retain activity. Purified protein ratio The activity was determined using 4-methyl-umbelliferyl-butyrate (MUB) as a substrate. About 3.2X10^-6M minutes^-1mg^-1It is.   Measurement of enzyme activity of E100-p-nitrophenylpropionate (pNP) , Or, in some cases, the hydrolysis of MUB Measure activity. Each substrate is dissolved in acetonitrile and the temperature-dependent pH change To a reaction mixture containing 50 mM Tris HCl pH 8.5 adjusted for kinetics. (Final concentration 100 μM). After thermally equilibrating the reaction at 37 ° C. for 5 minutes, The reaction was started by adding 10 μL of enzyme sample, whereas the control was In the reaction, an equivalent amount of BSA was used instead. This reaction was performed for 405 nPNP. m ε = 17 mM^-1cm^-1For MUB, 360 nm ε = 7.9 mM^-1 cm^-1And observe spectrophotometrically.   The rate of enzyme-catalyzed hydrolysis is corrected for spontaneous hydrolysis of the substrate. Protein The concentration of the substance was determined by either the absorbance at 280 nm or the Lowry test. Determined by Approximate activity is 0.7% agar on a microtiter plate. Of 5-bromo-4-chloro-3-indolyl ester suspended in a matrix Determined by a colorimetric assay based on hydrolysis. 0.1mg of this indolyl derivative / Ml solution in a minimum volume of acetonitrile and 0.1M phosphate buffer p Add to a warm solution of 0.7% agar containing H7.5. 10 μL of this solution is cooled Can be used with as much as 100 μL of enzyme sample if rejected Dispense into titer plates and incubate at ambient or> 65 ° C You.   E100 is effectively inhibited when exposed to tosyl fluoride; It is not affected by the presence of either a stimulating agent or a reducing molecule (Table 3). ).   The reaction conditions were the same as in the general experiment above, except that the specified components were added. Is explained. The relative ratio is based on the ratio of spontaneous hydrolysis of non-catalytic reactions. Has been corrected.   Substrate specificity of E100-Substrate specificity was tested as outlined by Example 11. And the results from this structure activity experiment for E100 are summarized in Table 4. E100 contains many nitrophenyls, cumaryls and Shows a broad substrate specificity that catalyzes the hydrolysis of thiolester. This enzyme is Shows hydrolytic activity towards both the straight and aromatic moieties on the carboxylate side, ,> Also include a carboxylate R group or a naphthyl leaving group of a long alkyl chain of C8. Is not a substrate. This enzyme is assayed by the zona pellucida on the agar plate. As such, it does not show significant activity toward either casein or milk.  Substrate activity assay for esterase E100 partially purified from the species of Therms. (++) (a) color formation in less than 10 minutes or (b) significant product formation on TLC Best activity determined by. The measurement of the remaining activity was +> +/−> −> −− Continue in order. Structure abbreviations are as follows: I, chloro-bromo-indolyl, N, a-naphthyl, U, methylumbelliferyl, pN, p-nitrophenyl, oN, o-nitrophenyl, PA, phenylacetate.   Determination of kinetic characteristics-The kinetic characteristics are based on the enzyme nitrophenylproprionate. Determined by measuring the concentration dependent initial ratio of elemental catalyzed hydrolysis. The reaction is p H8.5 in 50 mM Tris-HCl buffer equilibrated to 37 ° C. It is started by adding nitrogen. 40 due to the formation of product nitrophenol The ratio is determined from the change in absorbance at 5 nm. The ratio of the spontaneous Correct for mechanical hydrolysis. Double reciprocal plots and concentration Analyzed by both Hanes Wolff plots, K Determine m, Vmax and Vmax / Km. Plot of initial ratio of hydrolysis reaction The kinetic characteristics of E100 determined from are shown in FIG.   Summary of the temperature of E100 and determination of the optimum pH-a summary of the temperature of this enzyme is given in FIG. Decide as shown. The activity of the enzyme increases with increasing temperature of the reaction. C (background signal due to substrate auto-hydrolysis becomes very large (It can no longer be corrected). This suggests that the lower limit of the optimal activity of E100 is above 70 ° C. E100 is It was observed that the lower limit of optimal activity, the limit of substrate stability at 37 ° C., was about 9.0. An overview of the basic pH is shown (FIG. 7b).   Determination of enzyme stability in the presence of organic solvents-polar aprotic co-solvent E100 is resistant to organic solvent compositions using acetonitrile as a reserve system Test for The enzyme is present in a solvent mixture of 20% by volume organic co-solvent. Retained 50% of the activity (FIG. 8).   N-terminal sequence of E100-purified protein on 10% SDS-PAGE gel After pouring into an electric blotte Transfer to PVDF membrane by ringing. Several times of double distilled water from the membrane Replace and wash to remove residual SDS or other contaminants, then remove -Stain with blue. Next, the membrane was washed with 50:40:10 MeOH: H_TwoO: Destain using several changes of AcOH, then wash once with 10% MeOH . Thereafter, the membrane is air dried and then subjected to sequencing. N-terminal of E100 The sequence was determined at the University of Illinois at Urbana Plain Genetic Engineering Facility.   The N-terminus of E100 was purified by 10% SDS-PAGE and applied to a PVDF support. The transferred polypeptide was determined by automated sequencing. The sequence obtained is: MKLL EWLK? EV, where each letter refers to the standard amino acid one-letter code, "?" Indicates an undefined amino acid. Thus, E100 is commercially viable in pure purity. It has been shown to be a useful esterase with its own activity. Embodiment 16 FIG. Characterization of E101   E101 is one of the two esterase activities isolated from Thermus T351. It is one of. E101 is the second esterase, E10, in the early purification step. 0 can be purified.   Purification of E101-Therms T35 prepared as described in Examples 1 and 2 One supernatant was fractionated with NH4OH and the precipitated protein was collected at 20-60% saturation. Gather. The pellet is washed with 30 ml of buffer (50 mM Tris-HCl pH8. 0, 1 mM BME) and the same buffer using a 30 Kd cut-off dialysis tubing. Dialyze the buffer. The dialysate was applied to a 100 ml bed equilibrated with the above buffer. Volume of DEAE resin introduced into the column Is washed with 150 l of equilibration buffer. Active protein introduced and washed fraction Amicon concentrator with pooled YM30 membrane Concentrate using. Then, the concentrated protein is equilibrated with the above buffer. Directly into a 25 ml bed volume of Sepharose SP resin. Active fraction introduced and As it appears in the wash fraction, it is pooled and concentrated as described above. Then the concentrate To a Sephacryl HR200 gel filtration column (1 × 40 cm) and collect 0.5 ml fractions at a flow rate of 2 ml / h. Active fraction Collect and analyze by SDS-PAGE. To perform N-terminal sequencing, Concentrate fractions that are considered to be submitted to the Protein Sequencing Service Center . Store this enzyme at 4 ° C. for future use.   E101 can be purified more than 35-fold by these methods. Has a dramatically different characteristic from E100, another esterase isolated from a strain of I do. Attempts to use ion exchange chromatography resulted in negative purification. this Is because there was no fact that the protein was retained. The tested resin had a pH It varies from 6.0 to 9.0 and ranges from phosphates including Tris and Hepes to borates. , Q Sepharose, CM cellulose, SP under buffer conditions Sepharose and hydroxyapatite are included. After two ion exchange steps The protein is purified to homogeneity by gel filtration chromatography. However However, because the retention is much longer than suggested by the molecular weight, this protein The quality appears to be interacting with the column. Native and denatured SDS respectively -As determined from PAGE, the molecular weight of this protein is about 135 Kd, ~ 3 5Kd It appears to have monomer mass (Figure 9).   Characteristics of E101-Specific activity of this enzyme is 4-methyl-umbelliferyl butyrate Plate (MUB) was used as a substrate and was 1% higher than that observed for E100. 0 times higher. E101 is inhibited by PMSF; Insensitive to chelating agents. The specific activity of the purified protein is 3.2 × 10^-FiveMo Le minutes^-1mg^-1Which is a methyl umbelliferyl butyrate It was determined from the initial ratio of hydrolysis using the rate as a substrate. Table 5 shows E100 1 provides a summary of the inhibitory effects of various substrates on activity.   The reaction conditions were as described in the general experiment above, except that the specified compounds were added. It is explained in. Relative ratios are based on the spontaneous rate of noncatalytic hydrolysis. Has been corrected.   Substrate specificity of E101-The substrate specificity of E101 is as described in Example 11. Was decided. The results from the structure activity experiments for this E101 are shown in Table 6. I have. The hydrolysis activity of this enzyme is similar to that observed for E100, There is no observable protease activity towards luc or casein.   Substrate activity assay for esterase E101 partially purified from the species of Therms. (++) (a) color formation in less than 10 minutes or (b) significant product formation on TLC Best activity determined by. The measurement of the remaining activity was +> +/−> −> −− Continue in order. Structure abbreviations are as follows: I, chloro-bromo-indolyl, N, a-naphthyl, U, methylumbelliferyl, pN, p-nitrophenyl, oN, o-nitrophenyl, PA, phenylacetate.   Thus, E101 is a useful ester having unique activity with commercially useful purity. It has been shown to be a Terase. Embodiment 17 FIG. Esterase cloning   General cloning strategy-Stratagene ™ ) Was used to construct a library and assay for esterase activity. Can be used for exit. Similar results can be obtained with other cloning systems. Wear. The usual efficiency of cloning in λ vectors is equivalent to mg of cloned DNA. Rari hybrid clone 10^FiveOr 10⁷Individual, which is the normal amount of size Sufficient to generate a representative gene library from selected chromosomal DNA fragments It is. We have determined that esterase activity in phage plaques Contrary to Ronnie, it is more efficient because the substrate is more accessible to the enzyme. Heading. Phages generally respond to the toxic effects of the cloned protein. It is less sensitive and can survive at temperatures up to 70 ° C. Temperature rise & The ability of the cloning system to withstand the potential toxicity of the cloned protein is described herein. Is required to detect the activity of thermophilic proteins such as esterases described in .   Isolation of DNA to construct a gene bank-as described in Example 1 Genomic DNA is prepared from a culture of the appropriate strain containing the esterase of interest. The cells of the different lines were mixed with 100 ml of TT broth (polypeptone per liter (B BL11910) 8g, yeast extract 4g, NaCl 2g) at 55 ° C or Shaking at 65 RPM overnight at 250 RPM until late log phase Let it grow. The cells are collected by centrifugation and the pellet is dissolved in 5 ml of lysis buffer. (10 mM Tris-HCl, pH 7.0, 1 mM EDTA, and 10 mM N aCl). Lysozyme is added to a final concentration of 2 mg / ml. After incubating the cells at 37 ° C. for 15 minutes, SDS is added to 1%. So Of phenol / chloroform / isoamyl alcohol (25/24/1 ) And gently extract three times, and overlay the aqueous phase with 95% ethanol. Wind up the DNA.   Those of ordinary skill in the art will appreciate other methods for preparing DNA known in the art. (37). For example, fresh strains containing esterases of interest Colonies were inoculated into 50 ml of TT medium in a 250 ml Erlenmeyer flask and Lanswick Environmentalist Shaker (New Brunswi ck Environmental Shaker) at 55 ° C for 24 hours. Incubate at 200 rpm for hours. Centrifuge at 3000g for 15 minutes And collect the cells with 5 ml of GTE buffer (50 mM glucose, 25 mM M Tris-HCl pH 8, 10 mM EDTA) and 2 mg / ml With lysozyme at 37 ° C. for 10 minutes. Lysozyme-producing spheropla The lysate was dissolved by adding 1% SDS and 100 μg / ml protein was added. The protein is partially deproteinized by adding Kosease at 24 ° C. for 10 minutes. H The chromosomal DNA was further purified by three extractions with phenol / chloroform. Precipitated with 2.5 volumes of ethanol and washed with 20 ml of 75% ethanol Then resuspend in 1 ml TE (10 mM Tris pH 8.0; 1 mM EDTA) You. The extracted fractions are larger than 50Kb Consisting of DNA fragments at a concentration of 10 V / c using a 0.7% agarose gel. of about 0.5 ng / μl as detected by gel electrophoresis running at 4 m for 4 hours.   Construction of gene library-Genomic DNA is partially digested with restriction enzyme Sau3A Lambda ZAP Express (Lambda ZAP E) xpress) (Stratagene Cloning System) . The product of the ligation reaction was purchased from Promega using λ Pack in vitro with the packaging extract. This vector is length Contains up to 12 kb of DNA, expresses T3 and T7 promoters and Clone identification by both probe hybridization . This library is retained and screened for esterase activity.   Five samples of 10 μg of each chromosomal DNA of the strain prepared as described above were Different concentrations of Sau3A restriction endonuclease (New England Bio Manufacturers' instructions using New England BioLabs For 30 minutes at 37 ° C. in a volume of 50 μl each. Sau3A concentration Varies from 0.1 u to 0.002 u / μg of digested DNA in separate tubes . The reaction was stopped by inactivating the endonuclease at 70 ° C for 10 minutes. Stop and allow gel electrophoresis on a 0.7% agarose gel running at 10 V / cm for 4 hours. Analyze more (a typical digestion pattern is obtained, data not shown). 5 kb The fraction with the average fragment size is selected for cloning. E001, E00 2, E003, E006, E007, E008, E009, E010, E012 , Including E016, E020 For the strain, stain stained with Sau3A at a concentration of about 0.02 u / μg DNA There are a second five samples of body DNA. For the remaining strains, 0.1 u / DN Adequate partial digestion in a first tube containing μg Sau3A of A To achieve. 50 ng of the chromosomal DNA fragment was ligated with an equimolar amount of lambda ZAP phage vector. Using the dephosphorylated BamHI-arm of the Ligate in 5 μl containing gauze (New England Biolabs) You. The ligation reaction is performed at 18 ° C for 8 hours, and then heat-inactivated at 70 ° C for 10 minutes. And stop it. This ligation containing about 10 ng of DNA insert 1 μl of the reaction was transferred to a 10 μl lambda prohead (Promega) Used for in vitro packing. Perform a packing reaction at 28 ° C for 90 minutes. Combine with 100 μl of the XL1 blue overnight culture and add 2 ml of 0.7 per plate. The LB medium was collected using the upper layer agar (0.8% NaCl, 10 mM MgSO4). Apply to 5 90 mm Petri dishes containing. This is used to determine cloning efficiency. Make a serial dilution of the packing mixture. Cloning efficiency is generally about 1.0. X10⁷Individual hybrid phage / μg of cloned DNA. Of individual strains The cloning efficiency of each varies, and the size of the resulting library ranges from 0.5 to 2.5X10^FiveWas within the range. From this, 2 to 12 positive clones were separated. (Data not shown). Plate to collect amplified library Hybrid phages were collected from one and 3 ml of L containing 25% glycerol. Store in medium B. Connect the other four primary plates to the 5-bub as described below. Lomo-4-chloro-3-indolyl-acetate (X-acetate) The hybrid plaque containing the esterase gene was treated with the indicated agar containing Find out.   Screening of gene banks for esterase activity-above packing The products of the reaction are infected into E. coli XL1 Blue MRF (Stratagene). Non Primary plaques of the amplified gene library were placed on the plates using X-acetate. Cover with an upper layer of agar containing 65 ° C for 30 minutes at Clean. The concentration of the substrate in this indicator overlay may be ethanol or N From a 4% stock in N, dimethylformamide, generally 0.1% in the final solution. Dilute to 1% (usually about 0.4% is used). Add another suitable substrate. Can be used instead, including 5-bromo-4- Chloro-3-indolyl-butyrate (X-butyrate), 5-bromo-4-c Rollo-3-indolyl-proprionate (X-proprionate), 5-bromo Mo-4-chloro-3-indolyl-stearate (X-stearate), Chillumbelliferyl-acetate (MUA), 4-methylumbelliferyl- Butyrate (MUB), 4-methylumbelliferyl-propionate (MU P) or synthetically purchased from commercial sources such as Sigma Chemical Co. 5-bromo-4-chloro-3-indolyl- or 4-meth Includes, but is not limited to, tilumbelliferyl-ester . For inactivating background exogenous esterase activity from Escherichia coli Preheat plate at 65 ° C. for 20 minutes. Hybrids that survived this procedure Pick up the phage and rescreen three times. Next, the extract is Same mobility as denatured protein Will be analyzed for the presence of a protein band with The lambda ZAP cloning system cuts out smaller plasmid vectors and imports them. Allows simplification of the characterization of the sart. About esterase activity Gene bank screening, ie, protein isolation, purification, and N- Terminal sequencing; generation of modified nucleotide probes from N-terminal sequences; Other methods can be used to screen gene banks using lobes. However, this method is efficient for the isolation of promising clones, Not suitable.   In particular, four plates with phage colonies generated during the cloning described above Rate to inactivate some potential E. coli esterase activity. Incubate at 5 ° C for 30 minutes. About 1 mg / ml of colorimetric esterase substrate X -Acetate or other substrates (X-butyrate, X-proprionate, X-stelate) Including but not limited to allate and 4-methylumbelliferyl based substrates 0.7% top agar (0.8% NaCl, 10 mM) MgSO_Four) Approximately 2 ml is placed on each plate. Generation of cloned esterase Currently the blue halo around the phage colony (or 4-methylumbelliferyl) In the case of a substrate, it can be detected by a tendency halo. For example, strain isolation Expression pattern observed for a gene library derived from body 28 (E009) The pattern is shown in FIG. 11a. A typical result of this process is that the hybrid A 1: 3000 ratio of positive colonies to phage can occur.   Generally, 2 to 12 primary positive phage plaques are picked from each set of plates. And resuspend in 50 μL of LB medium Use a sterile piece of paper to streak and streak over E. coli XL1 Blue. You. These purified phage plaques were then transformed with X-acetate as described above. Covered with the indicated agar and positive plaques were selected as in the primary screening experiment. This 11b is shown in FIG. 11b. In general, perform three such purifications Is sufficient to generate pure hybrid clones that express esterase activity. is there. All of these clone candidates were used in the X-acetate plate assay. It shows the desired esterase activity. Each single primary phage plasmid from each strain Some candidate clones that represent the progeny of the worm are selected for further analysis.   Overview of the proteins produced by phage clones-these phage clones The production and analysis of protein from the protein is performed as follows, but alternatives are possible. You. A single plaque from each clone (10 mM MgSO4)_FourThe presence of Resuspend in 20 μl of an overnight culture of E. coli XL1 blue (grown in LB medium underneath). Turbid and at 24 ° C. in one of the wells of a 96-well microtiter plate Incubate for 20 minutes to adsorb, 15 ml test tube containing 2 ml LB New Brunswick Environmental Alamey set at about 300 rpm Grow overnight at 37 ° C. in a dental shaking incubator. Fine Cell residue can be removed by centrifugation at 12,000 g for 10 minutes. Next, clones were cloned for comparison with the native protein purified from the original microorganism. These phage lysates were subjected to 4-15% gradient native polyacrylamide gel electrophoresis ( PAGE). Pre-formed gradient gel is used for Bio-Rad Laboratories (Catalo) 161-0902), Generation of the gels shown in FIGS. 12a-m according to the manufacturer's instructions for native gels Used for Original strain purified to a single protein band by HPLC Esterase preparation from is used as a control on the same gel. Or even Unpurified but purified to a single esterase activity Can be used as a control. Has esterase activity The protein band is applied for 5-20 minutes at room temperature with X-acetate overlay applied. It can be detected by incubating for minutes. Clone candidate relative Mobility can be compared to native esterase protein.   FIGS. 12a-z show the ES detected in lambda clones as compared to the host strain. Figure 4 shows the results of a typical comparison of the activity of the Terase. 107 highs from 20 strains The data obtained for the candidate brid clones are summarized in Table 7. School Each of the cleaned gene libraries contains proteins purified from the original strain At least a candidate clone expressing an esterase protein having a mobility of There is one. Some λ clone candidates are esterases purified from the original strain. Expresses an esterase activity having a mobility different from that of the main component of the sample. Beauty treatment A band of similar size with the activity of the enzyme is associated with minor components in native microorganisms. (Data not shown). Hydrolysis activity of these cloned esters The sexes are given the names shown in Table 7.   Excision of plasmid vector from phage-Lambda ZAP vector The large clone is conveniently converted to a plasmid vector and the DNA insert Good physical characterization and regulated expression of cloned genes Make reality possible. M1 by co-infection with helper phage 3-Multiple copies with cloned insert by introducing specific replication Excision of the plasmid occurs. (About 10⁷Has colony forming units (CFU) 10 μl of phage stock of lambda hybrid and Exascist (Exas sis) M13 helper phage (about 10^TenCFU) 1 μl in LB 20 μl of an overnight culture of E. coli XL1 blue grown in. At 24 ° C After 20 minutes, transfer the cell suspension to one of the wells of a 96-well microtiter plate. Transferred to a 15 ml culture tube, diluted with 2 ml of LB, and diluted at 37 ° C. and 300 rpm. Grow overnight, heat at 65 ° C for 10 minutes, and centrifuge at 3000 g for 20 minutes To clarify. The excised plasmid packed in M13 particles was Transduce lambda resistant strain XLOLR that does not allow the generation of 13 helper phage You. 10 μl of the lysed phage lysate was transferred to a 96-microtiter plate. Mix in one of the wells with 30 μl of an overnight culture of E. coli XLOLR strain, Incubate at 20 ° C. for 20 minutes to absorb, dilute with 100 μl LB, and Incubate at 7 ° C for 40 minutes to obtain a kanamycin (Km) resistant marker of the plasmid. Ker (neo) is expressed. Cells were harvested using two plates supplemented with 40 mg / ml Km. Apply to LB plate. One of these plates contains 50 μl of 4% X -Also include the acetate storage solution.   Preliminary experiments were performed by growing the plates at 37 ° C. Significant Phenotypic Segregation Occurs in Transfectant Escherichia coli Colonies Expressing Esterase To indicate In the extreme case of the CE020 strain, Estella is very active. Primary transformant expressing the protease activity Very few colonies from Ronnie do not express any esterase activity Could not re-strike. This isolation and identification of the plasmid containing the active clone Due to the apparent instability, a protocol to manipulate most esterase clones Col provides a standard protocol for plasmid excision as recommended by Stratagene. Modifications were needed compared to Kor. This instability is due to intracellularly expressed May be due to the function of the The sterase is derived from a thermophilic microorganism and may not be active at low temperatures. Ability to overcome this separation problem by lowering the growth temperature Was assumed.   Thus, overcoming the instability problem due to the activity of the esterase-containing plasmid To culture E. coli cells with thermophilic esterase at 28 ° C and 30 ° C Doing so results in less effective segregation of phenotypes. Thus, the cloning thermophile The activity of the sex esterase is lethal or partially lethal to the host cell. In some cases, the growth temperature of the strain should be reduced to 30 ° C., or even room temperature. is there. This is shown in FIG. Temperature increases clone phenotypic stability After deciding to make a difference, apply all plasmid-based clones. Further experiments are performed at 26 ° C., generally for 48 hours. Including X-acetate Escherichia coli cells are spread on a medium containing Detect the expression and degree of segregation within or between primary colonies. this As such, growing transformed cells at a temperature that reduces the activity of the cloned esterase Length is important for effective isolation of the resulting plasmid.   In certain cases, each phage clone Picking up 8 bacterial colonies to be led from a plate without X-acetate To 100 ml LB supplemented with 40 mg / ml Km in 96 well plate Transfer and grow overnight. The progeny of these colonies were transformed with X-acetate-containing agar. Analyze by the spot test used. Light blue halo and minimal amount of Estella Derived from each phage by picking up what produces a solid Some of the resulting plasmid clones are selected for further study.   Selection of stable plasmid variants-plasmid based with esterase gene Because these vectors have often been determined to be unstable, A stable variant of the mid is isolated. One method for such isolation is as follows. It is something. E. coli cells with the excised plasmid were Was purified using an LB plate supplemented with a small amount of X-acetate, Any potential negative growth impact from the production of deadly indole products Reduce. Colonies (generally giving a moderate growth rate and a strong blue color) Selected by their phenotype and in a 15 ml test in 2 ml LB with Km 48 hours, about 0.1 OD₆₀₀Growing until reaching 12,000g And centrifuged for 1 minute. Transfer cell pellet to 500 ml 0.1 M Resuspended in acid buffer pH 7.0, Sonics & Materials Vibra Se Sonicate at 4 ° C. using a 375 watt sonicator. Ultrasonic treatment This is performed using a microtip, a maximum capacity of 40%, and a pulse of 50% for 45 seconds. Dissolution Centrifuge the lysate at 12,000 g for 5 minutes and test for its relative esterase activity. I do. Variants with highest activity for next growth And select for analysis. The application and then the growth and activity assay in liquid medium were performed in 3 To confirm the stability of the clone.   This procedure allows specific esterase activity between variants from the same plasmid line. Can be reduced from a factor of more than 100 to a factor of 3. Active Stabilization generally occurs at a level corresponding to the highest activity value detected at the beginning of stabilization. I will. This does not merely suppress the activity of the cloned toxic gene, Mutations in the E. coli host that allow for higher resistance of Could be shown.   Physical characterization of plasmid clones-standard alkaline lysis method or Extract plasmid DNA from E. coli by other procedures known in the field (37). I do. This DNA is transformed into a series of restriction endonucleases such as EcoRI, Ba digested with mHI, HindIII, PstI, EcoRV, and XbaI, Establish the digestion pattern of the clone and determine the size of the cloned DNA fragment You. The physical map pattern of the 24 selected product clones is shown in FIG. You. From this data, the size of the insert for each clone was calculated. It is summarized in.   Generation of tag sequences for PCR identification of esterases containing inserts   The DNA sequence at the end of the insert fragment carrying the esterase gene was cloned. Using standard T7 and S6 primers to generate unique tags for It can be determined by sequencing the ends of the insert. Sequence analysis Independently amplifies DNA inserts from both clones and host microorganisms It can be done to design possible PCR primers. These tags are To generate this DNA fragment from the chromosome of the microorganism to be studied using RPC technology Can be used potentially.   Screening of cosmid libraries using oligonucleotide probes -Other methods are used to clone enzymes that cannot be cloned due to activity. It is. Prepare a denatured probe for the N-terminus of the protein and Hybridize with the plaque from the ink. Alternatively, the protein content of the enzyme N-terminal sequence of the internal protein fragment obtained after digestive digestion, -A modified PCR amplification probe can be prepared using the sequence obtained from the terminal. Wear. Using these sequences, the N-terminal and internal fragment or two internal fragment sequences Probe from the amplification region between Carrying clones can be identified. Embodiment 18 FIG. Esterase overproduction and overexpression   Production of Recombinant Esterase-The producer strains used are listed in Table 8. clone The enzyme is produced from E. coli strain XLOLR. Alternatively, any suitable E. coli Inn The main can be used, including HB101, C600, TG1 and X L1-blue, but is not limited to these.   Several types of media can be used for the production of cloned esterases. LB ( 10 gm / 1 tryptone, 5 gm / l yeast extract and 10 gm / l NaCl) And Terrific broth (after autoclaving. 17M KH_TwoPO_Four, 0.72M K_TwoHPO_Four100 ml of sterile solution of 12 gm / l tryptone, 24 gm / l yeast extract and 4 gm / l glycero Have been tested and the results from the optimal growth conditions for the producing strains are listed in Table 9 below. Have been. Each medium is supplemented with 10-50 μg / ml kanamycin.   The optimal production medium depends on many factors, including the cost of the medium and the specific activity of the purified protein. Depends on. TB medium is a richer medium and is therefore more expensive. For example, in the case of CE009, a single fermentation operation produces more total units However, it does not produce enough to justify the higher cost of this medium. In addition Thus, the specific activity is higher for the preparation of LB medium.   Fermentation production is performed at 30 ° C. in a 17 L fermenter (15 L effective volume / LH fermentation). , 600 RPM, and an airflow of 0.5 vvm. The seed procedure is as follows Establish. Production culture in a 250 ml Erlenmeyer flask using a full cup of frozen production culture Inoculate 50 ml of ground. Incubate the flask at 30 ° C. for 2 days (250 RPM). After incubating, inoculate a 1 liter flask containing 250 ml of production medium. Used. The flask is incubated at 30 ° C. and 250 RPM for 1 day. This 1 liter frus Inoculate the fermenter using a coconut.   Production of a substantially purified preparation from cell paste of a recombinant enzyme producing strain The synthesis was performed according to the method described in Example 4 and the native tamper in Examples 14-34. This is done in the same way as the specific protocol described for quality.  Optimization of Esterase Production-Further optimization of esterase production is achieved by shaking Study the medium in the flask, then on a 1-20 liter scale This is done by further optimization. Depending on the enzyme, the final fermentation conditions may be intermittent feed (fed- batch) or a continuous fermentation process. Estella to analyze Because the activity of the enzyme is intracellular, it must be defined or defined as in TT medium. It is necessary to use a fresh medium. Grow the microorganisms of interest and Collect by centrifugation. The pellets were broken by sonication, and Examples 3 and 4 Using standard techniques for ion exchange and gel permeation chromatography described in Element can be purified. Growth conditions, including medium composition, pH, and temperature, are Keeps the highest enzyme levels on scale (eg, shake flasks, and 1 liter fermenters) While optimizing for the highest cell density.   Isolation of high producing mutants-using some simple mutagenesis schemes, High production mutants with different activities are addressed and isolated. These include U Mutagenesis with V light and ethylmethane sulfonate (EMS) or N-methyl-N ' Includes chemical mutagenesis such as -nitro-N-nitrosoguanidine (MNNG) It is. Following the method described in Miller (38), cells were mutagenized at various concentrations. Treat with the original (or various exposure times to UV light). Different mutations in different microorganisms The optimum concentration of the stock varies. Generally, 80% survival for EMS, MNNG About 50% survival for UV light or 10-50% for UV light Is desirable. The mutagenized culture is then grown and the mutagen washed Rinse and apply to solid medium.   Mutants by applying esterase plate screening to cells Identify. For examples relating to esterase screening, see 5-bromo- 4-chloro-3-indolyl acetate or 4-methylumbelliferyl acetate An agar overlay containing a colorimetric or fluorogenic substrate such as a plate is applied. Base Colonies that show a significant increase in activity due to quality hydrolysis are identified.   Next, the candidate variants are analyzed by native polyacrylamide gel electrophoresis, Compare with parent strain. Next, the standard assay method described in Example 2 or Example 12 Apply the described rapid esterase / lipase screen to determine enzyme activity Can be identified. If the increase in generation level is large, After massy staining, the gels are either buffered on native or SDS polyacrylamide gels. May increase. Strains with multiple activities are also distinguished in this way, One can also confirm that the increase is in the enzyme of interest. Next Confirm that the variants have the same kinetic and substrate properties as the parent enzyme . Most of the mutants identified by this approach involve promoter regions, A gene that can be isolated either in the presser molecule, or in other regulatory elements. It is the current mutant. Most mutations in enzyme structural genes inactivate the enzyme However, enhanced activity can also be isolated. Mutation is desired Increase the activity of the protein band in the Coomassie stained gel If it is clear that the strength does not increase, the variant is re-characterized and Determine if it is a more efficient biocatalyst. Embodiment 19 FIG. Esterase screening kit   Packaging a large subset of enzymes for easy analysis of many enzymes at once An easy-to-use screening kit can be obtained. These kits can Configured to eliminate as many potential errors as possible, each enzyme If so, it is provided in a lyophilized form close to its optimal buffer and reaction conditions.   Various sizes of mys composed of different plastic materials from glass vials Many different kit formats are possible, up to crotiter plates. handling Microtiter plates are preferred because of their ease of operation. Most microphones Rotator plates are made of polystyrene, which is compatible with most organic solvents. Not resistant to. For experiments with aqueous solvents, polystyrene is no problem. No. Utilizing other more durable plastics like polypropylene Yes, it is ideal for this kit. Can test 3ml samples (This allows enough sample for multiplex TLC or HPLC analysis) A 24-well microtiter plate has been developed. Other formats are also different May be useful for applications.   Stir bar, buffer (0.1 M Na phosphate pH 7.0) and 1 U of each enzyme For each well of a 24-well polypropylene tray (Tomtec) Each kit is prepared by adding. Enzymes have equivalent activity provided for comparison. Aliquots into each well or vial in a set volume to ensure that I do. Thereafter, the entire kit is freeze-dried, sealed with a heat-sealed foil (3M), and sealed. Attach the bell. In separate experiments, earlier experiments comparing glass to plastic Suggested by experiment Even if the protein is freeze-dried in the tray of this kit, the enzyme activity is large. It has been found that it is not compromised. In addition to enzymes, each of the kits contains 6-9 4 control wells comprising a buffer of pH. This is the Some qualities have a tendency to become unstable in buffered solutions, Positive reactions and autohydrolysis were found to be confusing. You. Instructions, datasheets, sample preparation vials, glassware An eye device and a plastic eye drop device. This kit uses solvent and substrate for each well. It is configured so that it only needs to be added to the well. Described in Example 12. A rapid screening indicator dye method can be included in each well or vial. is there. This makes the preliminary qualitative determination of the effectiveness of the enzyme simple and fast. Embodiment 20 FIG. Cloning and characterization of recombinant proteins   Derived from the strain isolate producing the native isolated protein (listed in Table 1) Cloning and characterization of the recombinant protein was performed as described in Example 37. Was. The lambda expression vector was isolated as described above (the specific isolate named Are shown in Table 7). Escherichia coli containing the excised phage-plasmid Guided loan to be summarized in Table 7 and finalized by subsequent screening After three rounds of stabilization procedure, selectively for esterase activity Activity units per mg of total cell protein were estimated. Positive phage library -Estella used to screen candidates for recombinant proteins Fig. 12 Which also allowed the identification of alternative recombinant proteins. Recombinant The production of protein was performed using TB in the culture medium, and in Example 4, the native (non-recombinant) ) Purification of the enzyme as described for the protein and described in Example 38. To do so. Embodiment 21 FIG. Sequencing of recombinant proteins   By isolating and cloning the gene encoding the enzyme of the present invention, DNA A segment is obtained, and the translated protein amino acid is added to the DNA segment. Find open reading frames (ORFs) corresponding to sequences . Arrangement of a DNA insert having a corresponding nucleic acid sequence encoding the protein enzyme; Column determination can be done by hand or by the usual method using automated equipment. it can.   Once obtained, the presence of alternative start codons can be determined by analyzing the nucleic acid sequence. (This is a recognized phenomenon in the art) However, the encoded protein contains at least the nucleoprotein ORF. FIG. 16A shows the isolated nucleic acid sequence corresponding to the ORF of the E001 enzyme and the translated The alternative amino acid sequence is shown with the alternative start codon underlined. FIG. 16B , Is the isolated cloned amino acid sequence containing the ORF of E001. FIG. C is the isolated nucleic acid sequence corresponding to the ORF of the E009 enzyme, and the translated The amino acid sequence is shown, with alternative start codons underlined. FIG. 16D shows E0 9 is an isolated cloned amino acid sequence containing the 09 ORF. FIG. 16E An isolated nucleic acid sequence corresponding to the ORF of the E011 enzyme and translated amino acids Shows the sequence, with alternative start codons Underlined. FIG. 16F shows an isolated clone containing the E011 ORF. This is an amino acid sequence. FIG. 16G shows the isolated corresponding to the ORF of the E101 enzyme. Nucleic acid sequence and translated amino acid sequence, with the alternative start codons underlined Is attached. FIG. 16H shows an isolated cloned clone containing the E101 ORF. Amino acid sequence.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 9/18 C12Q 1/44 9/20 C12N 15/00 ZNAA C12Q 1/44 5/00 A (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), AL, AM, AT, AU, AZ , BB, BG, BR, BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, J, TM, T R, TT, UA, UG, US, UZ, VN (72) inventor Eikinzu, John United States 60,526 Illinois La Grange Park NV. Spring Street 833 (72) Inventor Demiljan, David United States 60610 Shikago, IL. Kingsbury Number 311 550 (72) Inventor Fonstein, Veronica United States 60615 Shikago S., Illinois. Cornell Avenue 4926-H. (72) Inventor Fontstein, Michael United States 60615 Chicago, Illinois. Cornell Avenue 4926-H (72) Inventor Casadavan, Malcolm United States 60615 Shikago S., Illinois. Kimberk 5227

Claims (1)

[Claims] 1. A commercial grade enzyme preparation isolated from a thermophilic microorganism, comprising E001 , E002, E003, E004, E005, E006, E007, E008, E009, E010, E011, E012, E013, E014, E015, E 016, E017b, E018, E019, E020, E021, E022, E 023, E024, E025, E026, E027, E028, E029, E0 30, E100, E101, E102, E103, E104, E105, and E Esterase activity, temperature profile, protein selected from the group consisting of 106 An enzyme preparation characterized by a summary of stability and a summary of pH. 2. A substantially pure enzyme preparation comprising E001, E002, E003, E0 04, E005, E006, E007, E008, E009, E010, E01 1, E012, E013, E014, E015, E016, E017b, E01 8, E019, E020, E021, E022, E023, E024, E025 , E026, E027, E028, E029, E030, E100, E101, Selected from the group consisting of E102, E103, E104, E105, and E106. Summary of esterase activity, temperature, protein stability, pH An enzyme preparation characterized by its apparent molecular weight. 3. E001, E002, E003, E004, E005, E006, E007 , E008, E009, E010, E011, E012, E013, E014, E015, E016, E017b, E018, E019, E020, E021, E022, E023, E024, E025, E026, E027, E028, E 029, E0 30, E100, E101, E102, E103, E104, E105, and E 106, characterized by an esterase activity selected from the group consisting of Enzymes produced by the expression of recombinant DNA in plants. 4. E001, E002, E003, E004, E005, E006, E007 , E008, E009, E010, E011, E012, E013, E014, E015, E016, E017b, E018, E019, E020, E021, E022, E023, E024, E025, E026, E027, E028, E 029, E030, E100, E101, E102, E103, E104, E1 05, and a cell that produces a protein selected from the group consisting of E106. 5. E001, E002, E003, E004, E005, E006, E007 , E008, E009, E010, E011, E012, E013, E014, E015, E016, E017b, E018, E019, E020, E021, E022, E023, E024, E025, E026, E027, E028, E 029, E030, E100, E101, E102, E103, E104, E1 05, and a nucleic acid vector encoding a protein selected from the group consisting of E106 Structure. 6. Rapid screening for enzymatic activity on test substrates in bacterial isolates A pH-dependent color change of an enzyme-containing sample derived from a bacterial isolate. Contact with the test substrate in the presence of an indicator dye having a color and observe the color change Detecting a change in pH in the solution containing the indicator dye, A method wherein the H-dependent color change indicates the degree of enzyme activity. 7. Keys to quickly identify enzymatic activity on specific substrates A panel of enzymes disposed in a reaction vessel, wherein the reaction vessel is The reaction vessel contains a buffered enzyme powder or solution, optionally an indicator solution; A material that is ropylene or otherwise resistant to organic solvents, Magnetic stirring; whereby the enzyme substrate to be tested and An appropriate solvent is added to the reaction vessel and incubated at the same time; A kit that detects whether it is reacting with element. 8. The optional indicator solution detects a color change in the solution in the reaction vessel. And the color change indicates enzyme activity. The kit according to claim 7, which is a kit.