JP2000511437A - A stable biocatalyst for ester hydrolysis - Google Patents

Abstract

  (57) [Summary] The present invention includes an isolated, stable esterase enzyme characterized by the ability to remain stable at a particular temperature, substrate specificity, and activity profile; an expression vector that expresses the protein; And a nucleic acid corresponding to the amino acid sequence of this protein.

Description

DETAILED DESCRIPTION OF THE INVENTION         A stable biocatalyst for ester hydrolysis   This application is based on the patent application Ser. No. 08 / 827,810 filed on Apr. 11, 1997. All of these applications are hereby incorporated by reference in their entirety. Be impregnated. State of government rights   The work disclosed in this application was made by ThermoGen Inc. Approval number from IH-SBIR: According to NCI1-R43-CA63876-01 Was partially supported by the U.S. Government, and May have certain rights. Field of the invention   The present disclosure relates to commercially available pharmaceutical and chemical synthesis, recombinant proteins that hydrolyze esters. DNA vector for producing a protein, a host transformed with such a vector Cells and esters produced by such vectors and transformed cells Isolated stable suitable for enzymatic use in recombinant proteins that hydrolyze It is directed to the field of biocatalysis. Background of the Invention   Esterases and lipases. Esterases and lipases are listed below. Catalyze the hydrolysis of ester bonds 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 resolution 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 And the remaining esters, named R * ', are any remaining R1 mixed ena Enriched in enantiomers. afterwards, Separation of these products by chromatography to give pure R1 it can. Utilizes a large pool of esterases and lipases with various specificities What we can do is screen enzymes for specific reactions and Useful for developing optimal protocols for synthesis. The convenience of this process is many useful Facilitates scale-up of the production of new pharmaceutical products.   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 a therapeutically active one The main types of isomers and the best, inert and, at worst, potentially harmful side effects And other enantiomers that are responsible for this. This unusual isomer in racemic drugs Is increasingly seen as a pollutant. In fact, the FDA has The policy speech was to characterize the pharmacokinetic profile of each of the isomers in animals Compared to clinical pharmacokinetic profiles obtained in Phase I drug trials (8). Therefore, the pharmaceutical company must There is a need to develop synthetic or separation routes to produce each of the smart isomers.   Enzymatic synthesis of optically pure drugs and intermediates. Identified by classical chemical synthesis It is often very difficult to produce optically pure solutions of chiral molecules of Therefore, novel enzymatic biocatalysts play a major role in this effort. some In some cases, enzymes may be used in biological synthesis processes to make dangerous chemical synthesis procedures more environmentally friendly. Can be replaced with Also, the enzyme has some chemical protection and deprotection Enzymatic production of a pharmaceutical intermediate is costly if steps are eliminated simultaneously. May be more effective (7). As described in some detailed reviews (3,7) , Six major classes of enzymes (oxidoreductases, transferases, hydrolases, lyases) , Isomerases and ligation enzymes) are useful in the synthesis of optically pure compounds. You. Hydrolases are large amounts of hydrolases and Information, their independence from cofactors, and the diversity of substrates they accept Has proved to be the most useful group of enzymes.   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 Against various esterases and lipases with sexual, regioselective, and stereoselective properties There is still a great need to do so. In addition, these enzymes are Because they need to be used in equipment, they have increased stability, higher thermal resistance, and It needs to have a longer "shelf life".   Thermostable enzyme. Thermophilic microorganisms catalyze reactions at high temperatures and are more stable A rich source of useful proteins Sources have already been obtained (21, 22). One example is Therms Aquatics DNA polymerase and polymerase chain from (Thermus aquaticus) Its use in reactions (PCR) (23, 24). Thermophilic enzymes have their length The most commercially successful enzymes in the industry due to the stability of the period and the ease of use I am plain. The most successful enzyme to date, α-amylase, is corn Medium thermophile B. Stearothermophilus (B. stearothermophilus) (25). Another commercially successful industrial The enzyme is subtilisin, a serine protease also found in various Bacillus strains And are widely used in laundry 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 Should be longer-lasting and reusable for multiple uses. A sharp fixed support is provided.   Finally, enable these enzymes to survive at elevated temperatures Hydrophobicity and electrostatic forces make them generally work better in organic solvents (26-31). Most enzymes do not show their activity in organic solvents. While most are lost, thermostable enzymes are more resistant to denaturing conditions in many organic solvents. Can prove more resistant. Estella is highly thermostable Enzymes and lipases are the key to the use of these biocatalysts in large-scale industrial reactions. Necessary for expansion.   Thermostable esterases and lipases. To date, one esterase and Only a few lipases have been reported to have moderate thermostability. Tuli n et al. (32) reported that Bacillus brevis (Bacillu s brevis) Bacillus stearothermophilus asteriser Ze reported. Sugihara et al. (33, 34) describe two microorganisms, Bacillus soil alone. New from isolated and Pseudomonas cepacia soil isolates A thermostable lipase has been isolated. The former lipase is stable at 65 ° C for up to 30 minutes , But rapidly inactivates above this temperature. Pseudomonas cepacia Is stable when heated at 75 ° C. and pH 6.5 for 30 minutes. However, it had only 10% of its activity when assayed at this temperature. Thermoalcalophilic lipase (35) is Bacillus Species identified as MC7 and isolated by continuous culture at 70 ° C. Had a half-life of 3 hours. Finally, Sigurgis1adottir et al. (6) One type of therms strain having a lipase active against warmed olive oil; and Although two Bacillus strains have been isolated, this study does not address the stability of the enzyme. There were no reports.   These enzymes have limited changes in substrate specificity and moderate thermostability Offers only the feel. These are the need for different substrate specificities, economical Does not address the need to produce large quantities that can be commercialized in Has limited overall stability. In this application, we use a series of Provide substrate specificity (including selectivity, stereoselectivity), enhanced enzyme stability and A series of esterases and lippers that can be produced in large quantities for industrial use Has been identified. Summary of the Invention   The invention is characterized by the esterase activity and the data disclosed in Table 1. Provides the isolation and characterization of commercial grade enzyme preparations is there. In a preferred embodiment, the invention is characterized by an esterase activity. Isolation of a specifically purified esterase corresponding to the data disclosed in Table 1 And characterization. In a most preferred embodiment, the present invention provides Provide protein produced by recombinant DNA technology having sterase activity I do. The invention is shaped A recombinant protein that hydrolyzes the ester of the present invention from a transformed cell host. Includes lambda phage expression vector containing insert used to produce I do. The insert contained in this lambda phage expression vector is, for example, Phage-plasmid hinilid expression vector or other suitable expression vector, e.g. Examples include, but are not limited to, plasmids, YACs, cosmids, It can be used in zimid and the like. In a preferred embodiment, lambda expression The vector is a vector named in Table 7, or a substantially similar set This is one of the vectors containing the insert encoding the replacement protein. Also, The present disclosure is capable of transforming host cells and hydrolyzing esters. Encoding recombinant proteins, transformed host cells, and Also provided are recombinant proteins that hydrolyze ter. Suitable host cells Include, but are not limited to, Escherichia coli, Bacillus, Thermus species, etc. Not. Recombinant to hydrolyze the ester encoded by this vector The protein was 5-bromo-4-chloro-3-indolyl-acetate (X-acetate). Tate) can be hydrolyzed. The ester produced by this vector is hydrolyzed Degraded recombinant protein is comparable to the corresponding protein purified from isolated cells. It is characterized by a comparable half-life stability. Also, a recombinant tag that hydrolyzes this ester The protein is the original isolated cells At a temperature comparable to or better than the corresponding protein from Characterized by the ability to remain stable. This vector Recombinant proteins that hydrolyze the ester being esterified are specific to the specific proteins discussed below. It is also characterized by its substrate specificity, which is the corresponding purified protein from the isolated cells. It is comparable to protein. In a preferred embodiment, the vector is Or the vector designated in 8 or a substantially similar recombinant protein. This is one of the vectors containing the insert encoding the protein. Departure In certain preferred embodiments, recombinant proteins that hydrolyze certain esters are The encoding vectors are those named and listed in Table 8.   The present invention provides a novel nucleic acid isolated from the expression vector of the present invention, and For the loaded protein. In particular, the present invention relates to the DN of said vector. The nucleic acid sequence of the A insert and the amino acid sequence of a protein that can be expressed therefrom Is for BRIEF DESCRIPTION OF THE FIGURES   FIG. Features of the enzyme. FIG. 1 provides an illustration of the profile of activity to be characterized and The following enzymes are shown. Graph 1 plots relative esterase activity versus temperature. 2 shows the temperature profile of the enzyme performed. Graph 2 shows 25 ° C., 40 ° C., and 65 ° C. The relative residual activity at Figure 4 shows the residual esterase activity of the listed enzymes as lots. Graph 3 shows the relative energy PH profile of listed enzymes plotting sterase activity against pH Is shown. The data for the enzymes are summarized in Tables 1, 2, and 10.   FIG. Kinetic analysis of E100. The enzymes are: a) Line Weber-Bourke (Li neweaver-Burke) and b) Eadie-Hofstee analysis Shows normal Michaelis kinetics producing linear data in both, p-NP Using as a substrate, Km = 7.2 × 10^-FiveM and Vmax = 1.8 × 10^-FiveMinute^-1 Is obtained.   FIG. Temperature and pH profile of E100. a) Temperature profile of E100. E of p-nitrophenylproprionate 100 catalyzed hydrolysis was plotted as a function of temperature. When exposed to different temperatures Enzyme activity was determined. The initial rate of hydrolysis of nitrophenylproprionate , CH_Three0.25 mM substrate dissolved in CN is added to the enzyme at the desired temperature. Determined in 50 mM borate buffer, pH 8.5, equilibrated in. 40 The rate was determined by observing the change in absorbance at 5 nm, and the Correction was made for spontaneous hydrolysis of the substrate using sera albumin. b) E10 PH profile of 0. P-Nitrophenylpro catalyzed by E100 Effect of pH on prionate hydrolysis. Enzyme pH profile of Determined by preparing different buffers suitable for pH at a concentration of 10 mM . CH_ThreeSubstrate (0.25M) dissolved in CN, followed by the enzyme in this buffer The reaction was performed by adding to the solution. After incubating the reaction for 5 minutes, CH_ThreeThe reaction was stopped by adding 0.1 mM PMSF dissolved in CN. I let you. 7. The pH of the mixture is increased by adding 0.1 M Tris-HCl. Adjusted to 5. Record the absorbance at 405 nm and confirm the product against nitrophenol. Ε = 17 mM^-1cm^-1The concentration was calculated based on. Product formation Corrected for spontaneous hydrolysis.   FIG. P-Nitrophenylproprionate, as measured by relative velocity E100 resistance to the presence of organic co-solvents during hydrolysis. Various concentrations of CH_Three By measuring the initial rate of enzyme-catalyzed hydrolysis of pNP in the presence of CN, The residual activity of the enzyme in the presence of an organic solvent is determined. Explained in the determination of activity The reaction is performed at 37 ° C. in 50 mM Tris-HCl pH 8.5, as described. The change in absorbance is determined by spontaneous hydrolysis of the substrate and the product in the presence of an organic co-solvent. Correct for changes in extinction coefficient.   FIG. Substrates used for stereoselectivity and regioselectivity screening. Beauty treatment Lase may have four types of substrate structures that mediate stereoselectivity and regioselectivity In this sense, it is a flexible biocatalyst. Listed compounds Are different from those encountered in common substrates that are potentially important to the chemical intermediates industry Indicates the range of structural features. Some of these substrates are enantiomerically Can be purchased in diastereomerically pure form. Can be used in the qualitative screening technique described in Chiral center Classes of substrates most commonly associated with hydrolyzing biocatalysts for splitting Is taken into account. A) For Type I substrates, the desired product is the product carboxyl. On the acid side, whereas Type II compounds require alcohol It will include the functionality that is performed. B) Type III and Type IV substrates are Type I Can be considered as a subset of Will be classified separately. Type III molecules distinguish prochiral substrates by enzymes In contrast, type IV compounds have a meso structure. This The last two of these substrate types show that these types of compounds can be exposed to other chemical means. Therefore, it is very difficult to perform selective operation. This indicates the importance of the medium in the synthesis.   FIG. Nucleic acid sequence and amino acid sequence of translated protein. The enzyme of the present invention Isolation and cloning of the gene to be encoded yields a DNA segment, Open readies corresponding to the amino acid sequence of the translated protein To find a moving frame (ORF) Can be. Although alternative start codons are recognized in the art, The encoded protein contains at least the core protein ORF. Figure 6A is an isolated nucleic acid sequence corresponding to the ORF of the E001 enzyme, and Amino acid sequence, alternative start codons are underlined. FIG. 6B Isolated nucleic acid sequence corresponding to the ORF of E009 enzyme and translated amino acids Sequence, alternative start codons are underlined. FIG. 6C shows E011. An isolated nucleic acid sequence corresponding to the ORF of the enzyme, and a translated amino acid sequence. And the alternative start codon is underlined. FIG. 6D shows O101 of the E101 enzyme. An isolated nucleic acid sequence corresponding to RF, and a translated amino acid sequence; The start codon is underlined. FIG. 6E shows the ORF of the E019 enzyme. The corresponding isolated nucleic acid sequence, and the translated amino acid sequence, Codons are underlined. FIG. 6F shows a simple pattern corresponding to the ORF of the E005 enzyme. Isolated nucleic acid sequence, and translated amino acid sequence, with an alternative start codon Is underlined. FIG. 6G shows a cloned and isolated containing the E004 ORF. The alternative start codon is underlined. FIG. 6H , A cloned and isolated nucleic acid sequence containing the ORF of E006, The start codon is underlined. FIG. 61 shows a claw containing the ORF of E008. And isolated nucleic acid sequences Yes, and alternative start codons are underlined. FIG. 6J shows the OR of E010. F is a cloned and isolated nucleic acid sequence containing F, with alternative start codons Lines are attached. FIG. 6K shows a cloned and isolated clone containing the E013 ORF. And the alternative start codon is underlined. FIG. 155 is a cloned and isolated nucleic acid sequence containing the ORF of 015; Codons are underlined. FIG. 6M shows cloning containing the E016 ORF. And isolated nucleic acid sequences, with the alternative start codon underlined. FIG. 6N is a cloned and isolated nucleic acid sequence containing the ORF of E017, Alternative start codons are underlined. FIG. 60 includes the ORF of E020. Cloned and isolated nucleic acid sequence, with alternative start codons underlined Have been. FIG. 6P shows the cloned and isolated nucleic acid containing the E027 ORF. Sequence, alternative start codons are underlined. 6Q, 6R, 6S , 6T, and 6U are subsequences.   FIG. Substrate chain length specificity. FIG. 7A shows bis-p-nitrophenyl-carbonate. Figure 4 is a graph of data from an esterase colorimetric assay performed with the substrate as a substrate. FIG. 7B shows esterase performed using p-nitrophenyl-acetate as a substrate. It is data from colorimetric analysis. FIG. 7C shows bis-p-nitrophenyl-propio. Nate substrate. FIG. 7D shows bis-p-nitrophenyl-butylene. Substrate. FIG. 7E is a bis-p-nitrophenyl-caproate substrate. . FIG. 7F is a bis-p-nitrophenyl-caprylate substrate. FIG. 7G Bis-p-nitrophenyl-laurate substrate. B, C, D, and F Thus, E009 was diluted 80-fold compared to other enzymes.   FIG. Enantiomeric substrate specificity. FIG. 8A shows enantiomeric specificity. The results of the colorimetric esterase activity assay are summarized. FIG. 8B-D can not be detected. Colorimetric data expressed in time (minutes) required for the color change Reporting.   FIG. Enzyme activity on para-nitroanilide compounds. For various substrates The results of the enzyme activity analysis are shown in the table. Data is reported by standardized OD reading are doing. 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 provide a protein preparation for use. The isolated protein of the present disclosure has a biocatalytic effect. Molecular weight to find similar enzymes, as well as functionally use as enzymes to perform It can be used as a marker. Commercial chemical synthesis of specific racemic products Often such isolated enzyme preparations Need to be used.   The results of the characterization assay indicate that the esterase enzyme described is a series of optimal parameters. Data. For example, E100 and E101 are simply from extremely thermophilic bacteria. Having an optimal working temperature above 70 ° C. consistent with the enzyme to be released, 1 corresponds to an enzyme isolated from a less extreme thermophilic microorganism. It has an optimal commercial use temperature in the range of 50 ° C. However, both groups suffered from thermophilic bacteria. Additional stability and functionality compared to other known esterases from . E001-E021 is ideal for chemists wishing to work in a non-extreme temperature range. It provides a suitable temperature environment and works well at room temperature. In addition, these results Various enzymes, including those for which the described enzymes do not show a distinct advantage under experimental conditions, Although it has an optimal pH, most proteins are near neutral or It shows that it has an optimal pH slightly below.   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 (8gm), Difco Yeast Extract ) (4 gm), and NaCl (2 gm). Small rules for screening The mock cultures were grown at 65 ° C, 250-300 rpm, using 2 liters of 1 liter medium. Grow in a flask. Larger cell production for enzyme purification 17 liter fermenter (LH Fermentation, model Propagate in the Le 2000 series 1). These fermenters are 15 liters effective The cultures have a volume of 250 rpm, 0.3 to 0. Growing at 65 ° C., 5 vvm (air volume / medium volume per minute). The temperature is 28 lit. Circulating 56 ° C water from the 65 ° C water storage tank through a hollow baffle in a stirring jar To be maintained. E. coli strains are grown as described in (37) .   Enrichment procedure for freshly isolated thermophiles-multiple sediments and soil comprising organic matter The stream of the soil sample is used for the isolation of the new strain. These samples range from the Midwest to the Southeast. Gather from many lands. The sample (〜1 gm) was suspended in 2 ml of TT broth , 50-100μ of these samples 1 is spread on a TT agar plate containing twice the usual amount of agar (3%). Solid culture Agar is usually added to the ground to a final concentration of 1.5%. This makes it very lucky It prevents highly mobile microorganisms from crowding the plate at the expense of other microorganisms. Plates were isolated by incubation at 55 ° C. to 65 ° C. for 1-2 days. Multiple streaks on fresh plates to isolate single colonies To purify. Initial criteria for sorting are color, colony morphology, microscopy, growth temperature And lipase and esterase activity. First, several hundred 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 Is removed by centrifugation and the crude extract is placed on an esterase screening plate. Was tested for esterase activity. Briefly, 50 μl of cells The extract is taken up in 0.7% agarose and 0.1 M Tris-HCl pH 8.0 0.1 mg / ml suspended 5-bromo-4-chloro-3-indolylacetate Micros containing either phosphate or butyrate (for esterase activity) Transferred to wells of titer plate. Control wells are either buffer or 20 U pups. Add porcine liver esterase (PLE) or 20 U porcine pancreatic lipase (PPL) It has been added. Allow the plate to sit for enough time for the control wells to fully develop color. , Usually at 37 ° C for about 20 minutes. Dark wells are positive It shows activity.   Both cell extracts and culture supernatants were tested for esterase activity by this method. Test. Only cell extracts showed significant esterase activity.   Esterase liquid assay and determination of specific activity-bovine serum algal concentration is determined Protein concentration was determined by the Pierce BCA assay using Bumin as a standard. To determine. The protein concentration is obtained from the calibrated absorbance at 562 nm of the sample solution, Expressed as milligrams of protein. Esterase activity was equilibrated at 40 ° C. P-Nitrophenylpropane in 50 mM sodium phosphate buffer pH 7.0 Lionate (CH_Three0.5 M) hydrolysis from a 10 mM stock solution in CN Of 346 nm (iso-absorption point of acid / carboxylate pair ε = 480) Measurements were made routinely as determined by observation in 0). Specific activity is Microphone of p-nitrophenol produced every minute per milligram of protein Defined as romole.   Identification of extremely stable esterases-normal (non-denatured) 10% polyacylase Run the crude extract on a rilamide gel. After electrophoresis, the gel was washed with Tris pH 7.6. Equilibrate in Trizma buffer and then 0.15% X-acetate (May be shifted) for activity. The gel was then loaded at 55 ° C for 30 minutes. Cubate. These gels are available as 5-bromo-4-chloro-3-indolyl alcohols. Acetate (X-acetate), 5-bromo-4-chloro-3-indolylbutyle (X-butyrate) or 5-bromo-4-chloro-3-indolylcapryle Stain with an esterase activity stain containing any of the Ndigo precipitation can be produced. The Sams crude extract Two major bands were clearly visible in the Single activity in E. coli control lane A small band can be seen. Esterases have been identified as Therms T351 species and some new Identified from various isolates. The activities found from these microorganisms are summarized in Table 1. .Embodiment 3 FIG. Procedure for purifying and homogenizing esterase activity   Protein isolation-large batches of cell culture were prepared by the method described in Example 1. Grow and collect cell paste by centrifugation and store at -80 <0> C. 100g Cell paste at pH 7 containing 200 mM KCl and 0.1 mM EDTA. . Thaw in 200 ml of stirring solution composed of 5 mM phosphate buffer I do. 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. You. 375-watt Sonics & Materials B with standard 1/4 "horn The settings used for the Sonics & Materials Vibra Cell sonicator are: It was 5 minutes with an output setting of a destruction degree of 8 at a 50% pulse ratio. Alternative methods of cell destruction Includes a French press, Gaullen homogenizer, microfluidic Treatment of the cells with an idizer or other homogenizer may be included. Cell debris Removed by centrifugation and NH to 60% saturation_FourSO_FourPrecipitation of proteins by fractionation Can be The precipitated protein is centrifuged and 1 mM β-mercapto Resuspend in a minimum volume of 50 mM phosphate pH 6.5 containing ethanol (BME) Let   DEAE purification-The protein solution was resuspended using 10 Kd bore size dialysis tubing. Dialyze three times against turbidity buffer. The obtained protein solution is 100 ml bed volume of DEAE diluted 2-fold and equilibrated with the same buffer Apply to the column. After washing the column with 200 ml of equilibration buffer, Eluting with a linear gradient of to 0.5 M NaCl.   Active fractions isolated by Q resin purification-DEAE purification are pooled and equilibrated Dialyze the buffer against three changes of this buffer, and Apply 50 ml bed volume of Sepharose Q resin equilibrated with the above buffer. Was. The column was filled with 100 ml of 50 containing 0.1 M KCl and 1 mM BME. mM phosphate, pH 6.5, then 0.1M Elute with a 150 ml 0.6M KCl gradient.   Ultrafiltration concentration-pool the active fractions and attach a 30 kd cut-off membrane Enrich using the Amicon Ultrafiltration System You.   The preparative SDS-PAGE-concentrated protein solution is transferred to a standard SDS loading buffer. 10% SDS-PAGE for fractionation without boiling the sample using a buffer Get Hang on After developing, 0.1 M phosphate pH 7.5 and 0.1 mg / ml 0.7% agarose containing 5-bromo-4-chloro-indolyl acetate Treat the gel with. The resulting blue band was cut out of the gel, placed in a dialysis tube, and . Protein elution by electroelution for 1 hour in 05M Tris buffer pH 8.5 Collect. Native-stained with either Coomassie or silver stain , And SDS-PAGE, as observed by both The protein is uniformly purified. Store protein at 4 ° C for future use Can be   Gel filtration-using a gel filtration column as a further or separate purification step You can also. Embodiment 4. FIG. Commercial grade purification of isolated esterases   In many industrial applications, fully purified enzyme preparations are considered due to production costs. Neither needed nor desired. With significant esterase activity Fast and inexpensive to obtain the protein of interest in isolated form to contain the protein A costly protocol is desired. One such semi-purification procedure is described herein. 5 0 g of the cell paste was added to 1 containing 0.1 M NaCl and 0.01 mM EDTA. 00 ml of 50 mM Tris HCl buffer Thaw in pH 7.5. Crush cells by sonication and centrifuge for cell debris Is removed. The crude cell lysate was diluted with 50 mM Tris-HCl pH 7.5 for 3 hours. DEAE cellulose column diluted 2-fold and this material equilibrated with dilution buffer (Bed capacity 60 ml). Use this column with 3 column volumes of dilution buffer Then, wash with a salt gradient of 0-0.5 M NaCl over 4 column volumes. Activity Fractions were dissolved from this ion exchange resin in a 0.25-0.35 M salt gradient window. Issued. Fractions are assayed for activity as described in the determination of specific activity and Those showing a high activity are pooled and a 10Kd molecular weight cut-off membrane is used. And concentrated by ultrafiltration. Store concentrated enzyme samples at 4 ° C for future use I do. In some cases, substrate agarose of protein separated by native PAGE Two or more ester hydrolysis still under prolonged exposure to overlay Activity may be detected, which hinders most industrial applications Nevertheless shows only a small amount of second esterase activity. If necessary, additional Purification (eg, ammonium sulfate precipitation, gel filtration, or other as described in Example 3) Method) can be applied. This process can be scaled up if desired And scale it down. Embodiment 5 FIG. Method for determining the temperature profile   The optimal temperature profiles of the esterase protein are 30 ° C and 35 ° C, respectively. 0.1 M sodium phosphate equilibrated at 45 ° C, 45 ° C, 55 ° C and 65 ° C for 5 minutes By measuring the activity of the diluted esterase in buffer pH 7.0 went. Then the reaction conditions described in Example 2 for the measurement of the specific activity Add to solution equilibrated in (modified at various temperatures used in this example) Measuring the rate of hydrolysis of the added p-nitrophenylproprionate Thus, a temperature profile is determined. Compensate for temperature-dependent autohydrolysis of substrate Control using bovine serum albumin instead of esterase enzyme to enable Use the reaction. The data is then plotted as relative activity to the reaction temperature. To 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. The solution of the enzyme stock solution was Dilute with sodium acid buffer pH 7.0 to avoid concentration effects due to evaporation Temperatures equilibrated to 25 ° C, 40 ° C or 60 ° C, respectively, under sealed conditions for Put in the bath. Thereafter, aliquots were removed at regular intervals and p- By measuring the rate of hydrolysis of the nitrophenyl-proprionate, The activity to be retained You. The results are plotted as a relative activity versus time. These results are 4 When exposed to temperatures up to and including 0 ° C, all enzymes tested 48 hours indicates that most of the initial activity is retained. The activity is particularly near 55 ° C. For enzymes isolated from microorganisms with optimal growth temperature, does not decrease at 60 ° C . FIG. 4 is an example of typical data obtained. Tables 1 and 2 for data on enzymes And 10. Embodiment 7 FIG. Method for determining pH profile   The esterase pH profile is determined as follows. Specific activity in Example 2 Under reaction conditions similar to those described for determining sex, at least one Using a buffer with a broad and useful pH window that overlaps with two data points To determine the rate of hydrolysis of p-nitrophenyl proprionate. these For two experiments, two types of buffers satisfying the above criteria, female (6-6.5 useful Range) and bis-trispropane (useful buffers in the 6.5-9 range). Was. All pH tests were performed using bovine serum albumin instead of esterase. Correction for spontaneous autohydrolysis was obtained by subtracting the experimental results from the control. This Is especially 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 and serves as a substrate target for biocatalysts. Lack of water solubility of many viable compounds poses a major challenge for industrial organic chemists Become. 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 Washing such as Ruen, Hexane and SDS, Triton X100 and Tween 20 The experiment is performed in the presence of a cleaning agent. Also, the enzyme is lyophilized from the buffer. Between the activity of the isolated enzyme under almost anhydrous solvent conditions and the pH of the optimal activity. Additional experiments were also performed to test. Embodiment 9 FIG. For protein characterization by migration on native PAGE Method   The number of esterase enzymes in each of the semi-purified samples was determined by 4-15% acrylamide gradient (Bio- Native gel P using preformed gel purchased from Rad Laboratories Determine by AGE. This gel can hydrolyze indolyl acetate Trace enzyme contamination with other enzymes, which is Therefore, it cannot be easily detected by HPLC. This gel experiment It is clear that most samples have a single major activity buffer with similar migration characteristics. Has a zone or zone. 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 Superdex S200 attached to the car Determined by separation on an FPLC column. The protein was converted to 0.1 M (I) N Isolation with 0.05M sodium phosphate buffer at pH 7.0 containing aCl Separated by latic elution. The flow rate of the solvent was maintained at 0.5 ml / min. The quality was detected by UV at 280 nm. The esterase activity fraction was first The Lomo-3-chloro-3-indolyl-acetate plate assay was the second most active Follow-up assay by hydrolysis of the p-nitrophenylproprionate fraction (Both methods are described in Example 2). The molecular weight is Protein: β-amylase 200Kd, alcohol dehydrogenase 150 Kd, bovine serum albumin 66Kd, carbonic anhydrase 29Kd, cytochrome c Generated by using 12.3 Kd (expressed in minutes as logarithm of molecular weight Standard elution profile (plotted against time) Estimated by comparing to Lofil. Embodiment 11 FIG. Characterization of substrate specificity   The substrate advantage of esterases for their hydrolytic activity on various esters It can be determined as follows. Each of the substrates is CH_ThreeDissolve in CN (final Concentration 0.1M) and mix with 0.1M phosphate buffer pH 7.5 Thus, a molecular lattice is prepared on a microtiter plate. Then, partially The purified enzyme is added to these wells and the reaction mixture is incubated for 30 minutes. To Crude lysates can also be tested in this way. 10, 20, and 30 Check the plate after one minute to determine the relative activity. About experiments on colorless substrates For the colored substrate, except for extracting the reaction with dichloromethane three times, The reaction is carried out in a test tube under the same conditions as described above. Combine the organic layers and add MgS O_FourAnd concentrated to 0.1 ml in a stream of nitrogen. The concentrate is then Spot on a Ricagel TLC plate, 80: 20: 0.01 hexane: ethyl The mixture is developed with a mixed solvent of toluene and acetic acid. UV and I TLC plates_TwoVisible in Become Embodiment 12 FIG. Rapid screening assays for rapid substrate specificity characterization   The release of protons during the hydrolysis of the ester results in Rapid esterase activity based on the mechanism of pH-reducing enzyme-catalyzed hydrolysis A new method for screening was developed. Proton flow in reactions An indicator dye having a pH-dependent color change within the desired pH range of the enzyme activity It can be observed by using. The best indicators tested are slightly elevated Phenol red (starting point pH 8.5) ) And bromothymol blue for enzymes that work well in more neutral conditions. -(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 / Vis 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 (bromothiocyanate). Sodium phosphate pH 7.2 for Mohr Blue indicator and pheno Elured indicator, added to sodium borate pH 8.5) By equilibrating at a temperature of 5 ° C. for 5 minutes, followed by addition of 0.1 U of target enzyme. The hydrolysis reaction was performed by starting the reaction.   Alternative Methods for Monitoring Hydrolysis Reactions Are Applicable to Broad Screening Applications And useful. 0.001% indicator dye and less than 10% organic solvent composition Una C H_Three5 mM buffer containing CN, DMF or substrate dissolved in DMSO Add to agitated 24-well microtiter trays. 5 minutes, 25 ° C temperature After equilibration, the reaction was started by adding 0.1 U of esterase. You. The progress of the reaction is monitored by the color change of the solution. An aliquot is added to return the reaction color to the starting point. Extend the incubation The reaction is complete if no further color change is detected after Is determined. The product was determined by TLC analysis of the reaction acidified with 0.1 M HCl. Confirm formation, extract with ethyl acetate,_TwoSO_FourDry with N_TwoConcentrate under distribution You. For testing of substrates for which enzyme-based chiral resolution is being screened The product was resolved and isolated by chiral phase HPLC and the peak for each isomer The enantiomeric purity (enantiomeric purity) is determined by integrating the areas.   The rapid assay for different hydrolysis activities, in this case esterases, is Determined in experiments in microtiter plates using different enzymes and substrates You. An accurate comparison of the commercially available enzymes is based on the total protein versus the normal substrate p-nitrophenyl. Identical for each enzyme determined from the initial rate of hydrolysis of propionate Is guaranteed by using the specific activity of Data is for a given indicator dye Is reported as the time required to visualize the pH-dependent color change of Protein BSA as a quality source In the control experiment used, the color of the indicator did not change, and the change in It is established that it is the result of water splitting. Contains enzymes and indicators, no substrates A control test of the reaction solution indicates that the color change of the solution It was established that the result was not.   The format of the mitaro titer plate is easy to adapt to small-scale prepared chemicals The research performed to determine whether or not this is done is as follows. Race The reaction was carried out using mycotic phenethyl acetate and porcine liver esterase. Titrate with an aliquot of 1N NaOH and dissolve until no more color change occurs. The original color of the solution was maintained, and the reaction was stopped when no color change occurred. Product Is isolated and tested by TLC to determine the extent of the reaction by comparing the total amount of base added. You. Phenethyl alcohol as starting material by flash column chromatography And separated by chiral phase HPLC. Determine the enantiomeric excess of the hydrolysis product from the integrated value of the peaks, Compared to the same reaction performed in the absence of charge. The conclusions from these experiments The result is an esterase-catalyzed splitting of phenethyl acetate that can include an indicator dye. Does not affect the stereospecificity of   To test for utility in broad-based enzyme screening methods, Seven types of microorganisms isolated from various sources in the surrounding environment were Group of things Were tested for their ability to produce enzymes that catalyze the hydrolysis of. Table 2 The results of these studies are shown.  The results are reported as the amount of time required to change the color of the indicator. This These data indicate different substrate specificities among the isolated cells in different ambient environments. Special Note that the relative rate of hydrolysis of the substrate to the enantiomer is important. It is an indication of the stereoselectivity to be determined. The control reaction is a substrate using the above commercially available enzyme. Similar to that described in specificity studies. 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 on its activity including substrate dominance and If it is known to quickly determine the reaction chemistry to be obtained, simply This can be done using the isolated protein. All structure-activity tests were performed on pig liver Compare with standard mesophilic biocatalysts such as esterases. TLC analysis of the reaction The product was purchased from commercial sources or was chemically Compare to standards prepared by means (eg, base-catalyzed hydrolysis of esters) .   The study of stereochemical dominance by each esterase is one of two approaches. Can be evaluated more. In the first method, commercially available enantiomers (Mirror image Hydrolysis of the standard single stereoisomer of the (sterically) pure substrate ester with each enzyme Diagnostic diagnostic qualities of potential chiral selectivity, relative rates of hydrolysis of each enantiomer Use as a determinant. In the second method, purchased as a single stereoisomer Kinetic resolution methods (typically less than 50% complete) As a racemic mixture. Isolate the reaction product And chiral phase HPLC (Diacel Chiralcel) OD Or OB) or the product as a Mosher derivative (alcohol product) or Is prepared by derivatization to a menthyl derivative (carboxylate product) Of the corresponding diastereomer¹1 H NMR shows their enantiomeric excess. The remainder (ee) is analyzed. Diastereomers determined from NMR spectra The ratio of the mer is based on the integrated value of the corresponding peak, which is Any of the standards obtained from commercial sources using chiral shift reagents if necessary Compare with The optical rotation and absolute configuration of the product are then determined by optical rotation analysis, Compare with values found in the literature or determined from standards obtained from commercial suppliers I do. Embodiment 14 FIG. Characterization of protein E001-E021 / 17b   Perform strains from identified sources listed in Table 1. Isolated by growing at 65 ° C. in TT medium as described in Example 1. (Ie, S1 from soil). Esterase hydrolysis specific activity is described in Example 2. Esterase starch identified by the methods described and isolated as listed in Table 1. An identifier was assigned to the protein (ie, E001, etc.). To prepare enzymes Example 1 A 15 liter culture of the isolated cells was grown and the cells were spun down. Collect as described in. Lyse these cells and perform an isolated preparation Purified according to the procedure outlined in Example 4. This protein is Example 5 to determine protein stability and Example to determine protein stability The method described in Example 6 and Example 7 for determining the pH profile Was characterized using The results are shown in FIG. Example of this protein Characterized by migration on a native gradient PAGE as described in 9 . The data is shown in FIG. Determine specific activity as described in Example 2 The molecular weight was determined by chromatography as described in Example 10. Specified. They are shown in Table 1. Substrate specificity for some proteins And they are shown in Table 2. Thus, identified and characterized Esterase is useful and has unique activity in commercially useful purity Is shown. 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 preparative SDS Purify 300 times or more by PAGE. A second esterase activity is present Therefore, the specific activity could not be measured in the crude lysate (E101). . This second activity is released from the target esterase during the first chromatography step. It was possible to completely remove it. In this taromatography process, Two esterases passed through the DEAE column without binding. Various technical DEAE purification alone is sufficient to purify grade EOOO, Substantially purified E100 was obtained from any contaminating activity. Q cash register Purification and ultrafiltration produce purer products depending on the requirements of a particular application Make it possible. Final SDS PAGE purification step determines molecular characteristics To purify the protein homogeneously.   Protein characterization-active bands were electrophoresed on preparative SDS-PAGE gels Collect by evacuation and run again on 10% SDS-PAGE under denaturing conditions. This , A single band with a relative molecular weight of about ４５45 Kd is shown. Unboiled sample Run on SDS-PAGE gel As a result, multiple bumps can be placed at positions that have been increased by approximately the molecular weight of the proposed monomer. Is shown. In addition, this unboiled sample can be stained for activity. However, only the band corresponding to the multimeric form of the enzyme starting from the dimeric species retains its activity. Is found to have. The specific activity of the purified protein is 4-methyl-umbe. Using referyl-butyrate (MUB) as a substrate, about 3.2 × 10^-6M minutes^-1 mg^-1It is.   Measurement of enzyme activity of E100-p-nitrophenylproprionate (pNP) Or, in some cases, the esterase activity by observing the hydrolysis of MUB. Measure gender. Dissolve each substrate in acetonitrile, temperature-dependent pH fluctuation To a reaction mixture containing 50 mM Tris HCl pH 8.5 adjusted for (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 Concentration was determined by either absorbance at 280 nm or the Lowry assay. Decide You. Approximate activity was determined using a 0.7% agar matrix on a microtiter plate. Hydrolysis of 5-bromo-4-chloro-3-indolyl ester suspended in Determined by colorimetry. 0.1mg / ml solution of this indolyl derivative In a minimum volume of acetonitrile and 0.1 M phosphate buffer pH7. 5 to a warm solution of 0.7% agar. 10 μL of this solution is cooled Microtiter plates that can be used with 100 μL of enzyme samples Aliquot into rates and incubate at ambient temperature to a temperature of> 65 ° C.   E100 is effectively inhibited when exposed to tosyl fluoride, Are not affected by the presence of any chelating agents or reducing molecules. (Table 3).   The reaction conditions were the same as those described above except for the addition of the specified components. It is explained in the experiment. Relative rate is the spontaneous hydrolysis of non-catalytic reactions Is corrected for speed.   Substrate specificity of E100-Substrate specificity was tested as outlined by Example 11. The results from this structure activity study of E100 are summarized in Table 4. E100 is used to hydrolyze many nitrophenyl, cumaryl and alkyl esters Shows a broad substrate specificity that catalyzes This enzyme is located on the carboxylate side of the substrate. Shows hydrolytic activity for both linear and aromatic moieties, but> C8 long chain alkyl Those containing a carboxylate R group or a naphthyl leaving group on the ruthenium chain are not substrates. This Enzyme is assayed by casei as assayed by the zona pellucida on the agar plate. It does not show significant activity on either milk or milk.   Substrate activity assay for esterase E100 partially purified from the species of Therms. (++) indicates (a) color formation in less than 10 minutes or (b) significant production on TLC The highest activity determined by the formation of the product. The measurement of the remaining activity is +> +/−> −> −−. Structure abbreviations are as follows: I, chloro-bromo-i N, a-naphthyl, U, methylumbelliferyl, pN, p-nitro Phenyl, oN, o-nitrophenyl, PA, phenylacetate.   Determination of the kinetic characteristics-The kinetic characteristics are based on the enzyme nitrophenylproprionate. Determined by measuring the concentration dependent initial rate of elemental catalyzed hydrolysis. The reaction is Performed in 50 mM Tris-HCl buffer equilibrated to 37 ° C. at pH 8.5 The reaction is started by adding the enzyme. For the formation of product nitrophenol The rate is determined from the change in absorbance at 405 nm. The rate is determined by the Correct for spontaneous hydrolysis of quality. Double reciprocal plot of concentration versus velocity data And Km, analyzed by both the and Harness Wolff plots. , Vmax and Vmax / Km. A plot of the initial rate of the hydrolysis reaction? The kinetic characteristics of E100 determined from this are shown in FIG.   E100 temperature profile and determination of optimal pH-temperature profile of this enzyme The feel is determined as shown in FIG. 7a. The activity of the enzyme depends on the temperature of the reaction. As the temperature rises, it exceeds 70 ° C (background signal due to autohydrolysis of the substrate) Signal becomes very large and can no longer be corrected) Steady increase to the limit of analysis Which indicates that the lower limit of the optimal activity of E100 is above 70 ° C. Suggest. E100 is a base with a lower limit of optimal activity observed around 9.0. PH profile, 9.0 is also the limit for substrate stability at 37 ° C. (FIG. 7b).   Determination of enzyme stability in the presence of organic solvents-polar nonprototype Acetonitrile, an acidic co-solvent, is used to evaluate the resistance to organic solvent compositions. To test E100. This enzyme is prepared in a solvent mixture of 20% by volume of organic co-solvent. Retained 50% of its activity (FIG. 8).   N-terminal sequence of E100-purified protein on 10% SDS-PAGE gel And transfer to PVDF membrane by electroblotting. Membrane Washing with several changes of double distilled water to remove residual SDS or other pollutants And then stained with Coomassie blue. Next, put the membrane at 50:40 : 10: MeOH: H_TwoDestain using O: AcOH with several changes, then Washed once with 0% MeOH. The membrane is then air-dried and then sequenced. Process. The N-terminal sequence of E100 is from the University of Illinois at Urbana-Champine Gene Engineering University (University if Illinois Urbana Champaign genetic engineering faci lity).   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 resulting sequence is: MKLLEWLK? EV, where each letter refers to the standard amino acid one letter code And “?” Indicates an undefined amino acid. Thus, E100 is a useful ester having unique activity with commercially useful purity. It has been shown to be a Terase. 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 kind of supernatant is NH_TwoSO_FourAnd the precipitated protein is subjected to 20-60% saturation. To collect. The pellet is washed with 30 ml of buffer (50 mM Tris-HCl p H8.0, 1 mM BME) and reconstituted using a 30 Kd cut-off dialysis tube. Dialyze against the same buffer. The dialysate was 100m equilibrated with the above buffer. 1 bed volume of DEAE resin and the column is loaded with a 150 ml equilibration buffer. Wash with fur. Active protein is observed in the loaded and washed fractions, which are pooled and Concentrate using an Amicon concentrator fitted with a membrane. Then the concentrated tan The protein was washed with a 25 ml bed volume of Sepharose S equilibrated with the above buffer. Introduced directly into P resin. The active fraction appears in the loading and washing fractions. Above Concentrate as described. Then, concentrate the concentrate to Sephaacryl HR200 gel It is introduced into a filtration column (1 × 40 cm) and 0.5 ml fractions are collected at a flow rate of 2 ml / h. collect. Active fractions are collected and analyzed by SDS-PAGE. N-terminal sequencing Fractions that are considered to be homogeneous Submit to the center. Store this enzyme at 4 ° C. for future use.   E101 can be purified 35 times or more by these methods. Has dramatically different characteristics from E100, another esterase isolated from the strain You. Attempts to use ion exchange chromatography resulted in negative purification. this is This is because there was no fact that the protein was retained. The tested resin had a pH of 6 . Phosphate varying from 0 to 9.0, including Tris and Hepes Under conditions with buffer from salt to borate, DEAE, Q Sepharose, C M cellulose, SP Sepharose and hydroxyapatite. Two After the ion exchange step, the protein is purified by gel filtration chromatography. Homogenized, but retention is much longer than suggested by molecular weight Thus, it appears that this protein is interacting with the column. Each This protein was determined from native (native) and denatured SDS-PAGE. Having a molecular weight of about 135 Kd and a monomer mass of ~ 35 Kd I think that the.   Characteristics of E101-Specific activity of this enzyme is 4-methyl-umbelliferyl butyrate Plate (MUB) as the substrate, which is 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 Determined from the initial rate of hydrolysis using the rate as a substrate. Table 5 shows E100 1 provides a summary of the inhibitory effects of various substrates on the activity of s.  The reaction conditions were as described in the general experiment above, except that the specified compounds were added. It is explained in. Relative rates are relative to the spontaneous rate of uncatalyzed hydrolysis. It 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, It has no observable protease activity on luc or casein.  Substrate activity assay for esterase E101 partially purified from the species of Therms. (++) indicates (a) color formation in less than 10 minutes or (b) significant product on TLC The highest activity determined by the formation of. The measurement of the remaining activity is +> +/->-> -Followed in order. Structure abbreviations are as follows: I, chloro-bromo-indo Ryl, N, a-naphthyl, U, methylumbelliferyl, pN, p-nitrofe Nil, oN, o-nitrophenyl, PA, phenylacetate.   Thus, E101 is a useful ester having unique activity with commercially useful purity. Shown to be Terase I have. Embodiment 17 FIG. Esterase cloning   General cloning strategy-Stratagene^TM(Stratagene^TMΛZA from) P cloning system for library construction and detection of esterase activity Can be. Similar results can be obtained with other cloning systems. it can. The usual efficiency of cloning in a lambda vector is 10 per g^FiveFrom 10⁷Up to a single hybrid clone, a convenient amount Sufficient to obtain a gene library representing the size-selected chromosomal DNA fragments of It is something. We found that the detection of esterase activity in phage plaques More efficient because the substrate is more accessible to the enzyme as opposed to bacterial colonies Is found. Phages generally contain the toxicity of the cloned protein. It is less sensitive to action and can survive at temperatures up to 70 ° C. Warm A cloning system that withstands increased levels and the potential toxicity of the cloned protein The ability of the protein is dependent on the activity of thermophilic proteins, such as the esterases described herein. Necessary for gender detection.   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. Different Cells of 100 ml of TT broth (polypeptone per liter (BB) L11910) 8 g, yeast extract 4 g, NaCl 2 g) at 55 ° C. or Grow to late log phase at 65 ° C. overnight with shaking at 250 RPM. cell Is collected by centrifugation and the pellet is washed with 5 ml of lysis buffer (10 mM -HCl, pH 7.0, 1 mM EDTA, and 10 mM NaCl). Cloudy. Lysozyme is added to a final concentration of 2 mg / ml. Cells at 37 ° C After incubation for 15 minutes, SDS is added to 1%. Dissolve the melt Gently with ethanol / chloroform / isoamyl alcohol (25/24/1) The DNA is wound by extracting once and overlaying the aqueous phase with 95% ethanol. take.   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 The colonies were inoculated into 50 ml of TT medium in a 250 ml Erlenmeyer flask, and New Brunswick Enviro Incubate at 200 rpm for 24 hours at 55 ° C in the nmental Shakcr). To The cells were collected by centrifugation at 3000 g for 15 minutes and 5 ml of GT E buffer (50 mM glucose, 25 mM Tris-HCl pH 8, 1 0 mM EDTA) and resuspend at 2 mg / ml. Treat with zozyme at 37 ° C. for 10 minutes. Lysozyme-producing spheroplast Was dissolved by adding 1% SDS, and 100 μg / ml of proteinase was dissolved. Add K and partially deproteinize at 24 ° C. for 10 minutes. Phenol / k The chromosomal DNA was further purified by three extractions with loroform, 2.5 volumes Of ethanol, washed with 20 ml of 75% ethanol, and then washed with 1 ml of ethanol. Resuspend in TE (10 mM Tris pH 8.0; 1 mM EDTA). Lottery The eluted fractions consist of DNA fragments larger than 50 kb and have a concentration of 0.7% Detection by gel electrophoresis at 10 V / cm for 4 hours using a garose gel resulted in approx. 5 ng / μl.   Construction of gene library-Genomic DNA is partially digested with restriction enzyme Sau3A Lambda ZAP Express (digested) Licensed to the Stratagene Cloning System Gate. The product of the ligation reaction was purchased from Promega. In vitro packaging using the obtained λ packaging extract. this The vector contains DNA up to 12 kb in length and contains T3 and T7 promoters. Clones by both expression and probe hybridization to plaques Enables the identification of Keep this library and scan for esterase activity. Clean. genome Other methods for obtaining DNA libraries are known in the art.   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 Labs, New England BioLabs) according to the manufacturer's instructions, 50 μl each. For 30 minutes at 37 ° C. The concentration of Sau3A was 0.1 ml in a separate tube. from 0.002 u / μg of DNA to be digested. Endonuclea The reaction was stopped by heating and inactivating the enzyme at 70 ° C. for 10 minutes. Analyze by gel electrophoresis on a 0.7% agarose gel for 4 hours at 100 cm. A typical digestion pattern is obtained, data not shown). Average fragment size of 5 kb Are selected for cloning. E001, E002, E003, E006, E007, E008, E009, E010, E012, E016, E For the native strain containing 020, a concentration of about 0.02 u / μg DNA Sa It was the second of five samples of chromosomal DNA digested with u3A. Remaining For the strain, the first in vitro tube with 0.1 u / μg Sau3A To achieve an appropriate degree of partial digestion. 5 ng of 50 ng chromosomal DNA fragment 1 unit of ligase (New England Biolabs) in equimolar Amount of Lambda ZAP Phage Vector (Stratagene) Dephosphorylated BamHI − To the right. The ligation reaction was performed at 18 ° C. for 8 hours and at 70 ° C. for 10 hours. Stop by heat inactivation for 1 minute. Contains about 10 ng of DNA insert Of ligation reaction was added to 10 μl of lambda prohead (Promega ) For in vitro packaging. Packaging reaction 2 Perform at 90 ° C for 90 minutes, combine with 100 μl of an overnight culture of E. coli XL1 blue, 2 ml per rate of 0.7% top agar (0.8% NaCl, 10 mM MgS O_Four) Is applied to five 90 mm Petri dishes containing LB medium. Claw A serial dilution of this packaging mixture is performed to determine the packaging efficiency. Claw The conversion efficiency is generally about 1.0 × 10⁷Single hybrid phage / cloning DNA μg. Cloning efficiency for individual strains fluctuates and the live Rally size 0.5 ~ 2.5 × 10^FiveWas within the range. 2 from here 12 positive clones were analyzed (data not shown). Amplified library Collect hybrid phage from one of the plates to collect Store in 3 ml LB medium containing glycerol. The other four primary plates, 5-Bromo-4-chloro-3-indolyl-acetate as described below (X-acetate), and treated with indicator agar containing esterase gene. Find bridging plaque.   Gene bank screening for esterase activity-above package Infection product into E. coli XL1 blue MRF (Stratagene) . Primary plaques of the unamplified gene library were placed on the plates using X-acetate. Enzyme activity was measured by covering with agar-containing upper agar at 65 ° C for 30 minutes. Screening. Substrate concentration in this indicator overlay may be ethanol or From a stock solution of 4% in N, N-dimethylformamide to a Dilute between 0.1 and 1% (typically about 0.4% is used). Other suitable substrates can be used instead in this procedure, including , 5-bromo-4-chloro-3-indolyl-butyrate (X-butyrate), 5-bromo-4-chloro-3-indolyl-proprionate (X-proprio ), 5-bromo-4-chloro-3-indolyl-stearate (X-stelate) Allate), 4-methylumbelliferyl-acetate (MUA), 4-methyl Umbelliferyl-butyrate (MUB), 4-methylumbelliferyl-pro Prionate (MUP) or a synthetic product such as Sigma Chemical 5-Promo-4-chloro-3-indoris, which may be purchased from a commercial source Or 4-methylumbelliferyl esters, including but not limited to It is not something to be done. Background exogenous esterase activity from Escherichia coli To inactivate Preheat the rate at 65 ° C. for 20 minutes. The hybrid fan that survived this procedure Pick up and rescreen three times. Next, the extract is The presence of a protein band with the same mobility as the Analyze as described. Lambda ZAP cloning system is smaller Cut out plasmid vectors to simplify insert characterization To Screening of gene banks for esterase activity, ie , Protein isolation, purification, and N-terminal sequencing; degradation from N-terminal sequences Preparation of nucleotide probe; Screening of gene bank using degradation probe Although other methods can be used for cloning, isolation of promising clones This method is efficient and uniquely 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 allates, and substrates based on 4-methylumbelliferyl 0.7% top agar (0.8% NaCl, 10 mM) MgSO_Four) Overlay about 2 ml on each plate. Of cloned esterase Expression is based on the blue halo around the phage colony (or Fluorescence halo in the case of 4-methylumbelliferyl substrate) Can be. A typical result of this process is that 1 : 3000 positive colonies.   Generally, 2 to 12 primary positive phage plaques are picked from each set of plates. And resuspended in 50 μL of LB medium. Streaks on the loan. Next, these purified phage plaques were transformed as described above with X -Cover with indicator agar containing acetate and screen positive plaques for primary screening experiments Selected similarly. In general, such purification by re-streaking is performed three times to obtain It is sufficient to obtain a pure hybrid clone expressing the sterase activity. All of these clone candidates were significant in the X-acetate plate test. Shows sterase activity. Each represents the progeny of a single primary phage plaque Several clone candidates from each strain are selected for further analysis.   Examination of the protein profile produced by the phage clone-these files The production and analysis of proteins from clones is performed as follows, but alternatives are also available. It is possible. A single plaque from each clone (10 mM MgSO4)_Four E. coli XL1 blue overnight culture (grown in LB medium in the presence of and resuspend in wells of a 96-well microtiter plate. Incubate for 20 minutes at 24 ° C in one and adsorb, containing 2 ml of LB Transfer to a 15 ml test tube and set to about 300 rpm. At 37 ° C in a Qu Environmental Shaking Incubator Grow overnight. Cell debris is removed by centrifugation at 12,000 g for 10 minutes can do. Next, compare with native protein purified from the original microorganism. For comparison, a phage lysate from the clone was used on a 4-15% gradient of native port. Apply to acrylamide gel electrophoresis (PAGE). Bio molded gradient gel Purchased from Rad Laboratories (Catalog No. 161-0902) Used according to manufacturer's instructions for Ivgel. A single tag by HPLC An esterase preparation from the original strain, purified to a protein band, Serve as a control on the device. Alternatively, a single ester that is not homogeneously purified but Using native protein preparations that have been purified down to the enzyme activity Can be used. The protein band having esterase activity was identified as X-acetate. Apply a Tate overlay and incubate at room temperature for 5-20 minutes. More detectable. Clone candidate relative mobility Protein.   Data obtained for 107 hybrid clone candidates from 20 strains Data are summarized in Table 7, Typical of esterase activity detected in lambda clones compared to host strain The results of a typical comparison are shown. Each of the screened gene libraries An esterase protein having the mobility of a protein purified from the original strain Is present in at least one candidate clone. Some λ clone candidates are Has a different mobility than the main components of the esterase sample purified from the original strain Expresses esterase activity. A similar size band with esterase activity Is observed as a minor component in native microorganisms (data not shown). The hydrolytic activity of these cloned esters is given the name shown in Table 7. ing.   Excision of plasmid vector from phage-Lambda ZAP vector The large clone is conveniently converted to a plasmid vector and the DNA insert Physical characterization and controlled expression of cloned genes To be. M13-specific by co-infection with helper phage By introducing replication, multiple copy plasmids with cloned inserts can be Cutting out of the sumid occurs. Stock solution of phage of lambda hybrid (about 10⁷Ko 10 μl (with Ronnie forming units (CFU)) and Exassist M1 3 helper phages (about 10^Ten(CFU) and grown in LB with 1 μl Escherichia coli XL1 Blue 20 μl of the overnight culture were infected. After 20 minutes at 24 ° C., the cell suspension is Transfer from one of the wells of the microtiter plate to a 15 ml culture tube and add 2 ml Dilute in LB, grow overnight at 37 ° C. and 300 rpm, heat at 65 ° C. for 10 minutes And clarified by centrifugation at 3000 g for 20 minutes. Packed in M13 particles The excised plasmid thus obtained was not allowed to grow M13 helper phage. Transfect a lambda-resistant strain XLOLR. Lysate of excised phage 10 μl in one of the wells of a 96 microtiter plate in E. coli XLOLR. Mix with 30 μl of overnight culture of the strain and incubate at 37 ° C. for 20 minutes to absorb. Diluted with 100 μl of LB and incubated for 40 minutes at 37 ° C. To express the kanamycin (Km) resistance marker (neo). Cells to 40 Apply to two LB plates supplemented with mg / ml Km. These plates One of the plates also contains 50 μl of a 4% X-acetate stock solution.   Preliminary experiments were performed by growing the plates at 37 ° C. Significant Phenotype Separation in Escherichia coli Colonies of Transductants Expressing Sexual Esterase Indicates that it will occur. In the extreme case of CE020 strain, what Estella Colonies that do not express any protease activity are primary forms that actively express esterase activity. It was almost impossible to re-streak from transgenic colonies. this Due to the apparent instability of the plasmid containing the isolation and active clones, most Protocol for working with Terase clones recommended by Stratagene Modification required compared to standard protocol for plasmid excision . This instability is due to the function of the cloned protein expressed in the cell. Therefore, these esterases may be released by thermophilic microorganisms. Growth temperature, since it may come in and may not be active at low temperatures. It was hypothesized that reducing this would overcome this separation problem.   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 As a result, effective segregation of phenotypes was reduced. In this way, The activity of the loaned thermophilic esterase is lethal to the host cell, or If partially lethal, raise the growth temperature of the strain to 30 ° C, or even room temperature. Should be lowered. Contains plasmid containing active esterase protein The recombinant strain showed a phenotypic separation of esterase activity on X-acetate. This separation is useful if the esterase activity is toxic to the cells. This is due to the loss of the lathmid or insert. To avoid this If the cells are cooled to lower temperatures (probably the activity of the cloned thermophilic esterase (Reduce). like this The strains are spread with X-acetate at 28 ° C and 37 ° C. Proliferate faster Yellow colonies of the isolates were observed at both temperatures, but were counteracted at 37 ° C. It is much more intense in sorting. Temperature greatly differs in phenotypic stability of clones After deciding to let it go, apply all the plasmid-based clones. Further experiments are performed at 26 ° C., generally for 48 hours. Medium containing X-acetate Escherichia coli cells are spread on the ground, and the cloned esterase is expressed by the plasmid. And the degree of separation between these or primary colonies. in this way, Growth of transformed cells at temperatures that reduce the activity of cloned esterases produces Is important for the effective isolation of a specific plasmid.   In a particular case, eight bacterial colonies derived from each phage clone were Pick up knee from plate without X-acetate and place in 96-well plate Transfer to 100 ml LB supplemented with 40 mg / ml Km and grow overnight. The progeny of these colonies were spot-tested using X-acetate-containing agar. To analyze. Light blue halo and minimal amount of esterase-yields segregants By picking up the plasmid clones derived from each phage Some of the options are selected for further study.   Selection of stable plasmid variants-esterase inheritance It has been determined that vectors based on plasmids with offspring are often unstable. Therefore, stable variants of these plasmids are isolated. Such isolation One of the methods is as follows. Large with excised plasmid Enterococci cells were used on LB plates supplemented with Km and a limited amount of X-acetate. To produce the indole product, which is somewhat lethal for the colorimetric reaction. Reduces any potential negative growth impact. Colonies (generally gentle Selected by their phenotype, which contains Km. OD of about 0.1 for 48 hours in a 15 ml test in 2 ml LB₆₀₀Reached Grow to reach and collect by centrifugation at 12,000 g for 1 minute. Fine The cell pellet was resuspended in 500 ml of 0.1 M phosphate buffer pH 7.0. SONICS & MATERIALS VIBRA CELL 4 using a 375 watt sonicator Sonicate at ° C. Ultrasonic treatment, microchip, maximum capacity 40%, 45 seconds This is done with a 50% time pulse between. The lysate is centrifuged at 12,000 g for 5 minutes, Test for its relative esterase activity. The next variant with the highest activity Select for growth and analysis. Application, then growth in liquid medium and activity assay Is performed three times to confirm the stability of the clone.   This procedure allows specific esterase activity between variants from the same plasmid line. Factor of more than 100 To a factor of three. Activity stabilization is generally the first It occurs at a level corresponding to the highest activity value detected at any one time. This is a cloning toxicity Rather than simply silencing the activity of the gene, May indicate that a mutation in the E. coli host that allows for greater resistance has been selected. there were.   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 Digest with mHI, HindIII, PstI, EcoRV, and XbaI. Establish the digestion pattern of the loan and determine the size of the cloned DNA fragment . The physical map pattern for the resulting clone was determined. From this data The size of the insert for each clone was calculated and summarized in Table 8.   Generation of tag sequence for PCR identification of esterase containing insert   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 amplifying DNA inserts from both, clone and host microorganisms Can be performed by designing a PCR primer capable of These tags are for research To generate this DNA fragment from the chromosome of the target microorganism using RPC technology. Can be used locally.   Screening of cosmid libraries using oligonucleotide probes -Other methods may be used to clone enzymes that cannot be cloned due to activity. Can be. A degradation probe for the N-terminus of the protein was prepared and Hybridize with plaque from Dibank. Alternatively, the N-terminal sequence Alternatively, the N-terminal of the internal protein fragment obtained after proteolytic digestion of the enzyme Degraded PCR amplification probes can be made using sequences obtained from the ends . Using these sequences, between this N-terminus and the internal fragment, or two internal fragments A probe is made from the amplified region between the sequence and the D coding for the enzyme of interest. A clone carrying NA Can be identified. Embodiment 18 FIG. Overproduction and overexpression of esterases   Production of Recombinant Esterase-The producing strains used are listed in Table 8. clone The enzyme is produced from E. coli strain XLOLR. Alternatively, any suitable E. coli Hosts can be used, including HB101, C600, TG1 and XL1-Blue, but is not limited to these.   Several types of media can be used for the production of cloned esterases. LB ( 10 gm / l tryptone, 5 gm / l yeast extract and 10 gm / l NaC l) and Terrific broth (0.1 after autoclaving) 7M KH_TwoPO_Four, 0.72M K_TwoHPO_Four100 ml of a sterile solution of 12 gm / l tryptone, 24 gm / l yeast extract and 4 gm / l glyce Rolls) have been tested and the results from the optimal growth conditions for the producing strains are listed in Table 9 below. It is listed. Each medium is supplemented with 10-50 μg / ml kanamycin.   Optimal production media are many, including the cost of the media and the specific activity of the resulting protein. Depends on factors. TB medium is a richer medium and therefore more expensive. You. 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, The sex is higher in 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 0.5 vvm airflow. The seeding steps are as follows To establish. Production in 250 ml Erlenmeyer flasks using a full cup of frozen product culture Inoculate 50 ml of medium. The flask was introduced at 30 ° C. for 2 days (250 RPM). Cuvate and then inoculate a 1 liter flask containing 250 ml of production medium I do. The flask is incubated at 30 ° C. and 250 RPM for 1 day. this The fermenter is inoculated using a 1 liter flask.   Production of a substantially purified preparation from the cell paste of a recombinant enzyme producing strain Raw is the method described in Example 4 and native in Examples 14-34. Perform similar to the specific protocol described for proteins.  Optimization of Esterase Production-Further optimization of esterase production can be achieved by shaking Studies of media in Lasco, then on a 1 to 20 liter scale This is done by further optimization. Depending on the enzyme, the final fermentation conditions are fed batch (fe d-batch) or a continuous fermentation process. Esterase to be analyzed Because the activity is intracellular, a well-defined or defined, such as TT medium It is necessary to use a medium. Grow the microorganism of interest and remove the cell pellet. Collect by centrifugation. The pellet was broken by sonication and described in Examples 3 and 4. Enzymes can be purified using standard ion exchange and gel permeation chromatography techniques. It can be purified. Growth conditions, including medium composition, pH, and temperature, are (Eg, shake flasks and 1 liter fermenters) to maintain the highest enzyme loading While optimizing for the highest cell density.   Isolation of high producing mutants-using some simple mutagenesis schemes, Work on and isolate mutants with different activities and high productivity. These include U Mutagenesis with V light and ethyl methanesulfanoate (EMS) or N-methyl Chemical mutagenesis such as -N'-nitro-N-nitrosoguanidine (MNNG) Is included. Following the method described by Miller (38), cells were mutated at various concentrations. Varying the exposure time to the original (or UV light) ). The optimal concentration of different mutagens in different microorganisms will vary. General 80% survived for EMS, about 50% survived for MNNG, For UV light, a kill concentration that allows 10-50% survival is desirable. Next Then, the mutagenized culture is grown, and the mutagen is washed away and applied to a 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, candidate variants were separated by native polyacrylamide gel electrophoresis. Analyze and compare with parent strain. Then, the standard assay method described in Example 2 or Example By the rapid esterase / lipase screening method described in Differences in the amount of activity can be identified. If the increase in production level is large, Native or SDS polyacrylamide gel after Coomassie staining In some cases, an increase in bands may be seen. This method can also be used for strains with multiple activities. To confirm that the increase is in the enzyme of interest. it can. Second, those variants have the same kinetic and substrate properties as the parent enzyme Make sure that This approach Most of the mutants identified by H Or an expression variant that can be isolated in any of the other regulatory elements . Most mutations in an enzyme structural gene appear to inactivate the enzyme. Alternatively, enhanced activity can be isolated. The mutation in the desired protein Band activity on the Coomassie stained gel If not, re-characterize the variant and make it more efficient To determine if it is a biocatalyst. Embodiment 19 FIG. Esterase screening kit   Package large subsets of enzymes for easy analysis of many enzymes at once. To make an easy-to-use screening kit. These kits 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 composed of different plastic materials from a series of glass vials Many different kit formats are possible, up to a microtiter plate. Taking Microtiter plates are preferred because of their ease of handling and operation. Most The microtiter plate is made of polystyrene, which is Not resistant to solvents. For experiments with aqueous solvents, police There is no problem with styrene. Other more resistant plastics such as polypropylene It is ideal for this kit. Detect 3 ml sample (This provides enough sample for multiplex TLC or HPLC analysis) Large size 24-well microtiter plates have been developed You. Other formats may be useful for different applications.   Stir bar, buffer (0.1 M Na phosphate pH 7.0) and 1 U of each enzyme To each well of a 24-well polypropylene tray (Tomtec) Each kit is prepared by adding. Enzymes provide equal activity for comparison Aliquots into each well or vial in a set amount to ensure that It is. 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 Lyophilize the protein in the tray of this kit as suggested by It was found that the enzyme activity was not significantly impaired. In addition to enzymes, Each of the kits contains four control wells comprising a buffer at a pH of 6-9 . This tends to be unstable in buffered solutions for some of the substrates tested That may confuse a positive reaction with autohydrolysis. Is found. Instruction manual, data sheet, sample preparation Vials, glass eye drops and Plus It is an eye drop device made by Chick. This kit allows the addition of solvent and substrate to each well. And only be configured as needed. Rapid screenini described in Example 12. It is also possible to include the indicator dye method in each well or vial. This Preliminary qualitative determination of enzyme efficacy is simple and fast. Embodiment 20 FIG. Cloning and characterization of recombinant proteins   Due to strain isolates producing native isolated proteins (listed in Table 1) Cloning and characterization of the resulting recombinant protein is described in Example 37. Went like so. The lambda expression vector was isolated as described above (the named specific Are listed in Table 7). Excised hybrid phage-pla E. coli clones containing the Smid were induced as summarized in Table 7 and Final selection of esterase activity by cleaning, 3 stabilizations After the procedure, the activity units per mg of the total cell protein obtained were estimated. Screen positive phage library candidates for recombinant proteins The esterase activity stained gel used to obtain the same Also made possible. Production of recombinant protein was performed using TB in the medium and 4. Enzymes as described for native (non-recombinant) proteins Purify Then, as described above. Embodiment 21 FIG. Sequencing of recombinant proteins   By isolating and cloning the gene encoding the enzyme of the present invention, DN An A segment is obtained, and the translated protein amino acid is added to the DNA segment. Open reading frame (ORF) corresponding to acid sequence can be found You. Of the DNA insert containing the corresponding nucleic acid sequence encoding this protein enzyme Sequencing can be performed either manually or using automated equipment. It can be performed by a usual method.   Once obtained, the presence of alternative start codons can be determined by analyzing the nucleic acid sequence. At present, this is a recognized phenomenon in the art, but its existence is not clear. And the encoded protein enzyme is at least Contains ORF. FIG. 6A shows the isolated nucleic acid sequence and the ORF of the E001 enzyme. And the translated start amino acid sequence is underlined. ing. FIG. 6B shows the isolated nucleic acid sequence and the translation corresponding to the ORF of the E009 enzyme. The translated amino acid sequence is shown, with alternative start codons underlined. FIG. C shows the nucleic acid sequence containing the cloned and isolated E011 ORF, The start codon is underlined. FIG. 6D shows the cloned and isolated E1. 1 shows a nucleic acid sequence containing the ORF of 01, with alternative start codons Underlined. FIG. 6E shows the cloned and isolated E019 ORF. 4 shows the nucleic acid sequence comprising the alternative start codons are underlined. FIG. 3 shows the nucleic acid sequence containing the isolated and isolated E005 ORF, and alternative start codons. Is underlined. FIG. 6G shows the OR of E004 cloned and isolated. Figure 4 shows the nucleic acid sequence containing F, with alternative start codons underlined. FIG. 6H FIG. 3 shows the nucleic acid sequence containing the cloned and isolated E006 ORF, Don is underlined. FIG. 61 shows the cloned and isolated E008 Figure 3 shows the nucleic acid sequence containing the ORF, with alternative start codons underlined. FIG. 6J Indicates the nucleic acid sequence containing the cloned and isolated E010 ORF, The start codon is underlined. FIG. 6K shows the cloned and isolated E01. 3 shows a nucleic acid sequence containing 3 ORFs, with alternative start codons underlined. Figure 6L indicates the nucleic acid sequence containing the ORF of E015 cloned and isolated, The replacement start codon is underlined. FIG. 6M shows the cloned and isolated E Figure 16 shows nucleic acid sequence containing ORF of 016, with alternative start codons underlined . FIG. 6N shows the cloned and isolated nucleic acid sequence containing the E017 ORF. , The alternative start codon is underlined. FIG. 60 shows the cloned and isolated Nucleic acid sequence containing the ORF of E020, with the alternative start codons underlined I have. FIG. 6P shows the cloned 3 shows the isolated and isolated nucleic acid sequence containing the ORF of E027, with the alternative start codon underlined Is attached. FIG. 6Q shows the nucleic acid sequence at the 5 ′ end of the insert containing E003. Figure 6R contains the nucleic acid sequence at the 3 'end of the insert comprising E003. I have. FIG. 6S shows the nucleic acid sequence at the 5 'end of the insert containing the ORF of E004. FIG. 6T shows the nucleic acid sequence at the 3 'end of the insert containing the OFR of E004. Contains. Fig. 6U shows the nucleic acid at the 3 'end of the insert containing the ORF of E014. Contains an array. These nucleic acid sequences can be prepared by those skilled in the art using routine methods. Characterization of the full-length nucleic acid sequence of the insert, hybridization Detecting clones through, creating amplification primers for detection, It allows amplification and production of full-length homologous genes.Embodiment 22 FIG. Characterization of ester chain length specificity   The enzymes of the present invention can be tested for enzyme specificity for ester substrates of different chain lengths. And further characterized by: Such assays are based on the methods described above. The method can be carried out by using a method and selecting an appropriate substrate. FIG. The results of an enzyme colorimetric esterase activity assay are shown. Reaction conditions are 500 μg / ml Using the substrate, graphed data estimated to be about 0.1 U / 1 ml reaction was obtained. Was done. Here, 1 unit (U) means esterase for each enzyme stock solution. Calculated in relation to activity, one unit is about 1 μmol p-nitroper minute It is the amount of enzyme required to hydrolyze phenylproprionate. Data is , Approximate maximum OD during incubation_410nmAs reported.   FIG. 7A shows data using bis-p-nitrophenyl-carbonate as a substrate. It is a rough one. Maximum activity was found with enzyme E019, which was 4 hours OD of 0.30 after incubation_410nmshowed that. FIG. 7B shows p-nitto FIG. 3 illustrates data using rophenyl-acetate as a substrate. The greatest activity Sex was found with the enzyme E020, which was 3 hours after 400 seconds incubation. . 571 OD_410nmshowed that. FIG. 7C shows bis-p-nitrophenyl-propio. FIG. 3 is a diagram showing data using nate as a substrate. Maximum activity is enzyme E003 In the 600 second incubation. Later, 1.4 OD_410nmshowed that. FIG. 7D shows bis-p-nitrophenyl-butyle. 2 is a diagram illustrating data using a sheet as a substrate. Maximum activity is with enzyme E020 Which was found to be 1.19 OD after 160 seconds incubation._410nm showed that. FIG. 7E shows the data using bis-p-nitrophenyl-caproate as a substrate. FIG. The greatest activity is found with the enzyme EOO9, which is 5 OD of 0.37 after 60 seconds incubation_410nmshowed that. FIG. FIG. 4 shows data using sp-nitrophenyl-caprylate as a substrate. is there. Maximum activity was found with enzyme E003, which was incubated for 360 seconds. OD of 0.07 after the_410nmshowed that. FIG. 7G shows bis-p-nitrophenyl. 2 is a diagram illustrating data using lu-laurate as a substrate. Maximum activity is enzyme E016, which is 0.11 after 480 seconds of incubation. OD_410nmshowed that. Embodiment 23 FIG. PH-dependent assay for enantiomeric esterase specificity   The enzyme of the present invention is a substrate for specific enantiomers having different chiral structures. It can be further characterized by testing the enzyme specificity for tell. Such assays involve selecting the appropriate substrate using the methods described above. It is performed by FIG. 8 shows the results of the screening. FIG. 8A shows enantiomers. ー About specificity It summarizes the results of a colorimetric esterase activity assay. FIG. 8B shows the pH Quantitative colorimetric analysis in minutes of the time required for a detectable color change indicating a change Shows the data. Values are reported in minutes after enzyme addition. You. NH showing no hydrolysis was detected after 3 days, and o / n indicates that no hydrolysis occurs after overnight incubation (about 6-15 hours). It indicates that it was. Substrates 1, 2, 4, 6, 8, and 9 have ~ 0.005 % Bromothymol blue in 25 mM KPi buffer, pH = 7.4 At a concentration of 40 mM. Substrates 3, 5, and 7 have 〜0.00 5m with 5% promothymol blue and up to 10% MeCN as co-solvent M KPi buffer, dissolved at a concentration of 10 mM in pH = 7.4 . The esterases tested were determined by hydrolysis of PNP-propionate. As before, 1 U was added per well. Control reactions do not add enzyme It was a substrate solution. Embodiment 24 FIG. Characterization of enzyme activity on p-nitroanilide compounds   The enzymes of the present invention test enzyme specificity for another substrate similar to an ester This can be further characterized. Such a test is described above. By selecting the appropriate substrate using it can. The enzyme of the present invention is applicable to the anilide and ester listed below. The results are shown in FIG. The test was performed according to the general formula:  Test reactions were performed in microtiter plates using each reaction in a total volume of 100 μl. went. Each reaction contained approximately 75 μl of phosphate buffer, 5 μl of 5 mM substrate, And 50 U / ml (where 1 U is 1 μM p-nitrophenyl-pro Enzyme 20 prepared in an approximate amount necessary to hydrolyze the pionate) μl. The final reaction mixture contains 1 U enzyme and 0 U per well. . 25 mM substrate. Incubate the reaction for about 2.5 hours at 37 ° C. I did it. Control reactions lacking the enzyme were performed in adjacent wells. Control without substrate Was also performed on each plate. After the incubation, the plate is Read at 405 nm on a Model 3350 microtiter reader. Control value Was subtracted from the values in the tested wells to determine the true activity.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12N 9/18 C12N 5/00 A // (C12N 15/09 ZNA C12R 1:01) (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, BY) , KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, D K, EE, ES, FI, GB, GE, GH, GM, GW, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU , LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventor Fonstein, Michael United States 60615 Chicago, Illinois. Cornell Avenue 4926-H. (72) Inventor Von Stein, Veronica United States 60615 Chicago, Illinois. Cornell Avenue 4926-H. (72) Inventor Demiljan, David United States 60610 Shikago, IL. Kingsbury Drive Number 311 550 (72) Inventor Casadavan, Malcolm United States 60615 Shikago S., Illinois. Kinberg Avenue 5227

Claims (1)

[Claims] 1. An isolated nucleic acid having the nucleic acid sequence of FIG. 6E (E019). 2. An open reading frame encoded by the nucleic acid according to claim 1. A nucleic acid segment having the nucleic acid sequence of 3. 3. The nucleic acid according to claim 2, wherein the nucleic acid is operably linked and expressible. An expression vector nucleic acid construct having a target body. 4. A host cell transformed with the expression vector construct of claim 3. 5. A protein expressed by the expression vector construct according to claim 3. 6. An isolated nucleic acid having the nucleic acid sequence of FIG. 6F (E005). 7. An open reading frame encoded by the nucleic acid according to claim 6. A nucleic acid segment having the nucleic acid sequence of 8. 8. The nucleic acid according to claim 7, which is operably linked and expressible. An expression vector nucleic acid construct having a target body. 9. A host cell transformed with the expression vector construct of claim 8. 10. Expression by the expression vector construct according to claim 8. Protein. 11. An isolated nucleic acid having the nucleic acid sequence of FIG. 6G (E004). 12. An open reading frame encoded by the nucleic acid according to claim 11. A nucleic acid segment having the nucleic acid sequence of a genome. 13. 13. The nucleic acid according to claim 12, wherein said nucleic acid is operably linked and expressible. An expression vector nucleic acid construct having an insert body. 14． A host cell transformed with the expression vector construct of claim 13. 15. A protein expressed by the expression vector construct according to claim 13. 16. An isolated nucleic acid having the nucleic acid sequence of FIG. 6H (E006). 17． An open reading frame encoded by the nucleic acid according to claim 16. A nucleic acid segment having the nucleic acid sequence of a genome. 18. 18. The nucleic acid of claim 17, wherein said nucleic acid is operably linked and expressable. An expression vector nucleic acid construct having an insert body. 19. A host cell transformed with the expression vector construct of claim 18. 20. A protein expressed by the expression vector construct of claim 18. 21. An isolated nucleic acid having the nucleic acid sequence of FIG. 6I (E008). 22. An open reading frame encoded by the nucleic acid according to claim 21. A nucleic acid segment having the nucleic acid sequence of a genome. 23. 23. The nucleic acid of claim 22, operably linked to and capable of being expressed. An expression vector nucleic acid construct having an insert body. 24. A host cell transformed with the expression vector construct of claim 23. 25. A protein expressed by the expression vector construct according to claim 23. 26. An isolated nucleic acid having the nucleic acid sequence of FIG. 6J (E010). 27. An open reading frame encoded by the nucleic acid according to claim 26. A nucleic acid segment having the nucleic acid sequence of a genome. 28. 28. A nucleic acid according to claim 27, operably linked and expressible. An expression vector nucleic acid construct having an insert body. 29. A host cell transformed with the expression vector construct of claim 28. 30. A protein expressed by the expression vector construct according to claim 28. 31. 6K (E013). Nucleic acid. 32. An open reading frame encoded by the nucleic acid according to claim 31. A nucleic acid segment having the nucleic acid sequence of a genome. 33. 33. The nucleic acid of claim 32, wherein said nucleic acid is operably linked to and can be expressed. An expression vector nucleic acid construct having an insert body. 34. A host cell transformed with the expression vector construct of claim 33. 35. A protein expressed by the expression vector construct of claim 33. 36. An isolated nucleic acid having the nucleic acid sequence of FIG. 6L (E015). 37. An open reading frame encoded by the nucleic acid according to claim 36. A nucleic acid segment having the nucleic acid sequence of a genome. 38. 38. The nucleic acid according to claim 37, wherein the nucleic acid is operably linked and expressible. An expression vector nucleic acid construct having an insert body. 39. A host cell transformed with the expression vector construct of claim 38. 40. A protein expressed by the expression vector construct of claim 38. 41. An isolated nucleic acid having the nucleic acid sequence of FIG. 6M (E016). 42. An open reading frame encoded by the nucleic acid according to claim 41. A nucleic acid segment having the nucleic acid sequence of a genome. 43. 43. The nucleic acid of claim 42, wherein said nucleic acid is operably linked and expressable. An expression vector nucleic acid construct having an insert body. 44. A host cell transformed with the expression vector construct of claim 43. 45. A protein expressed by the expression vector construct of claim 43. 46. An isolated nucleic acid having the nucleic acid sequence of FIG. 6N (E017). 47. An open reading frame encoded by the nucleic acid according to claim 46. A nucleic acid segment having the nucleic acid sequence of a genome. 48. 48. The nucleic acid according to claim 47, wherein the nucleic acid is operably linked and expressible. An expression vector nucleic acid construct having an insert body. 49. A host cell transformed with the expression vector construct of claim 48. 50. A protein expressed by the expression vector construct of claim 48. 51. An isolated nucleic acid having the nucleic acid sequence of FIG. 60 (E020). 52. An o-encoded by the nucleic acid of claim 51. A nucleic acid segment having a nucleic acid sequence of an open reading frame. 53. 53. The nucleic acid of claim 52, wherein said nucleic acid is operably linked to and can be expressed. An expression vector nucleic acid construct having an insert body. 54. A host cell transformed with the expression vector construct of claim 53. 55. 54. A protein expressed by the expression vector construct of claim 53. 56. An isolated nucleic acid having the nucleic acid sequence of FIG. 6P (E027). 57. An open reading frame encoded by the nucleic acid according to claim 56. A nucleic acid segment having the nucleic acid sequence of a genome. 58. 58. The nucleic acid of claim 57, wherein said nucleic acid is operably linked to and expressable. An expression vector nucleic acid construct having an insert body. 59. A host cell transformed with the expression vector construct of claim 58. 60. A protein expressed by the expression vector construct of claim 58. 61. Figure 6Q (E003-5 ') at the 5' end of a nucleic acid molecule that is seamlessly adjacent And at the 3 'end the nucleic acid sequence of Figure 6R (E003-3'). Isolated by an intervening nucleic acid, Nucleic acid. 62. 62. An open reading frame encoded by the nucleic acid of claim 61. A nucleic acid segment having the nucleic acid sequence of a genome. 63. 63. The nucleic acid of claim 62, wherein the nucleic acid is operably linked to and can be expressed. An expression vector nucleic acid construct having an insert body. 64. A host cell transformed with the expression vector construct of claim 63. 65. A protein expressed by the expression vector construct according to claim 63. 66. Figure 6S (E004-5 ') at the 5' end of a nucleic acid molecule that is seamlessly adjacent And at the 3 'end the nucleic acid sequence of FIG. 6T (E004-3'). Isolated nucleic acid, which is interrupted by an intervening nucleic acid. 67. 67. An open reading frame encoded by the nucleic acid of claim 66. A nucleic acid segment having the nucleic acid sequence of a genome. 68. 78. The nucleic acid of claim 67, wherein said nucleic acid is operably linked and expressable. An expression vector nucleic acid construct having an insert body. 69. A host cell transformed with the expression vector construct of claim 68. 70. 69. The expression vector construct of claim 68. The revealed protein. 71. Isolated with the nucleic acid sequence of Figure 6U (E014-3 ') at the 3' end of the molecule Nucleic acid. 72. 72. An open reading frame encoded by the nucleic acid of claim 71. A nucleic acid segment having the nucleic acid sequence of a genome. 73. 73. The nucleic acid according to claim 72, wherein the nucleic acid is operably linked and expressible. An expression vector nucleic acid construct having an insert body. 74. A host cell transformed with the expression vector construct of claim 73. 75. A protein expressed by the expression vector construct of claim 73.