EP0952847A1 - Biocatalyseurs stables pour l'hydrolyse d'esters - Google Patents

Biocatalyseurs stables pour l'hydrolyse d'esters

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Publication number
EP0952847A1
EP0952847A1 EP96929678A EP96929678A EP0952847A1 EP 0952847 A1 EP0952847 A1 EP 0952847A1 EP 96929678 A EP96929678 A EP 96929678A EP 96929678 A EP96929678 A EP 96929678A EP 0952847 A1 EP0952847 A1 EP 0952847A1
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EP
European Patent Office
Prior art keywords
activity
enzyme
protein
esterase
lambdatge
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP96929678A
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German (de)
English (en)
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EP0952847A4 (fr
Inventor
Larry Allen
John Aikens
David Demirjian
Veronika Vonstein
Michael Fonstein
Malcolm Casadaban
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Thermogen Inc USA
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Thermogen Inc USA
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Application filed by Thermogen Inc USA filed Critical Thermogen Inc USA
Publication of EP0952847A1 publication Critical patent/EP0952847A1/fr
Publication of EP0952847A4 publication Critical patent/EP0952847A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)

Definitions

  • the instant disclosure is directed to the field of isolated stable biocatalysts that are suitable for enzymatic application in commercial pharmaceutical and chemical synthesis, DNA vectors for the production of recombinant ester hydrolyzing proteins, host cells transformed by such vectors, and recombinant ester hydrolyzing proteins produced by such vectors and transformed cells.
  • Esterases and Lipases catalyze the hydrolysis of ester bonds to produce alcohols and carboxylic acids as shown below.
  • Esterases and lipases can be characterized by different substrate specificities, R group or chain length preference, and unique inhibitors (1, 2).
  • the many esterases and lipases range from hydrolases such as the broad carboxyl esterases which preferentially hydrolyze esters with long carbon chain R groups, to choline esterases, and to acetyl esterases which act on very specific substrates.
  • these hydrolases are also known to show stereo- and regio-selective preferences resulting from the chiral nature inherent in protein active sites. This preferential hydrolytic activity make them useful for reactions requiring different regioselectivity and stereoselectivity or for kinetic resolution methods on racemic mixtures.
  • R* is a racemic mixture
  • Ri t would be the most rapidly hydrolyzed stereoisomer while the remaining ester designated R*' would be the enriched antipode mixed with any remaining Rj .
  • the products can then be separated by chromatography to provide pure R ⁇ .
  • the availability of a large pool of esterases and lipases with varying specificities would be useful for screening the enzymes for specific reactions, and developing optimal protocols for specific chemical synthesis. The expedience of this process would facilitate the production scale- up of many useful pharmaceutical products.
  • esterases and lipases carry out their natural reactions: the hydrolysis of ester bonds.
  • these enzymes can be used to carry out reactions on a wide variety of substrates, including esters containing cyclic and acyclic alcohols, mono- and di-esters, and lactams (3).
  • organic solvents (4, 5) where water is excluded, the reactions of esterases and lipases can be reversed.
  • These enzymes can catalyze esterification or acylation reactions to form ester bonds (3, 6, 7). This process can also be used in the transesterification of esters and in ring closure or opening reactions.
  • Enzymatic synthesis of optically pure pharmaceuticals and intermediates Since it is often very difficult to generate optically pure solutions of certain chiral molecules by classical chemical synthesis, new enzymatic biocatalysts will play a major role in this endeavor. In some cases, enzymes may be able to replace hazardous chemical synthesis procedures with more environmentally-friendly biological synthesis processes. It can also be much more cost effective to produce a pharmaceutical intermediate enzymatically if an enzyme can eliminate several chemical protection and deprotection steps at once (7). All six major classes of enzymes (oxidoreductases, iransferases, hydrolases, lyases, isomerases, and ligases) have been useful in the synthesis of optically pure compounds as described in several detailed reviews (3, 7).
  • hydrolases have proven to be the most useful group of enzymes, due to the abundance of hydrolases, the information about them, their independence from cofactors, and the wide variety of substrates they can accept.
  • a survey of the literature shows many examples of mesophilic hydrolases particularly esterases and lipases used in chemical synthesis or chiral resolution. These include esterases from pig (9, 10) and horse (3) livers and a wide variety of lipases from Aspergillus sp, (11) Candida sp. (12-16), Pseudomonas sp., (17-19), Rhizopus sp. (20) and others.
  • lipases have been used in the synthesis of propranolol (7), a beta-adrenergic blocking agent used in the treatment of angina and hypertension.
  • Ibuprofen a nonstearoidal antiinflammatory agent has been synthesized via stereo selective hydrolysis of its methyl ester using carboxyesterase (7). While these enzymes have begun to demonsorate the utility of biocatalysts in chemical synthesis, there is still a profound need for a wider variety of esterases and lipases which have varying substrate specificities, regioselectivities, and steroselectivities. In addition, since these enzymes need to be employed in a large-scale industrial setting, there is a need for them to have increased stability, higher thermotolerance and a longer "shelf life".
  • Thermostable enzymes Thermophilic organisms have already provided a rich source of useful proteins that catalyze reactions at higher temperatures and are stable for much longer periods of time (21, 22).
  • One example is the DNA Polymerase I from Thermus aquatic us and its use in polymerase chain reaction (PCR) (23, 24).
  • Thermophilic enzymes have become the most commercially successful enzymes in industry because of their long-term stability and ease of use.
  • alpha- amylase is used in corn processing and comes from the moderate thermophile B. stearothermophilus (25).
  • subtilisin a serine protease also found in various strains of Bacillus, has been widely used in laundry detergents and other cleaning solutions.
  • thermostable enzymes In addition to functioning at high temperatures, thermostable enzymes generally posses an increased shelf life which markedly improves handling conditions, especially by those not trained in biochemistry to work with the specific range of conditions used for mesophilic enzymes. If enzymes are to play a significant role in large scale processing of chemicals, they must be able to endure the harsh conditions associated with these processes. Thermostable enzymes are easier to handle, last longer, and given the proper immobilization support should be reusable for multiple applications
  • thermostable enzymes While most enzymes lose a significant portion of their activity in organic solvents, thermostable enzymes may prove more tolerant to the denaturing conditions of many organic- solvents. Highly thermostable esterases and lipases are necessary to expand the application of these biocatalysts in large scale industrial reactions.
  • the lipase from Pseudomonas cepacia was stable when heated for 30 minutes at 75 * C and pH 6.5 but had only 10% of its activity when assayed at this temperature.
  • a thermoalcalophilic lipase (35) was identified from a Bacillus species MC7 isolated by continuous culture and had a half-life of 3 hours at 70 * C.
  • NASAgisladottir et al. (6) have reported the isolation of one Thermus and two Bacillus strains which posses lipases active on olive oil up to 80 * C, although there was no report on enzyme stability in this study.
  • the instant invention provides for the isolation and characterization of commercial grade enzyme preparations characterized by esterase activity, and corresponding to the data as disclosed in Figures 1-4 and Table 1.
  • the instant invention provides for the isolation, and characterization of specifically purified esterase which is characterized by esterase activity, and corresponding to the data as disclosed in Table 1 and Figures 5-9.
  • the instant invention provides for proteins generated by recombinant DNA technology which have esterase activity.
  • the enzymes of the instant disclosure can be isolated from thermophilic organisms from various sources including soil, water and refuse sites from across the United States and elsewhere in the world. These organisms generally grow in the temperature range of 45°C to 90°C which classifies them as moderate to extreme thermophiles.
  • Proteins isolated from this group of organisms are similar in function to those isolated from species that grow at lower temperatures 25°C to 37°C, but are lacking in thermostable characteristics.
  • the enzymes of the instant disclosure encompass proteins produced by thermophilic organisms including the esterase enzymes which are responsible for the hydrolysis of ester bonds to yield carboxylic acids and alcohols.
  • the proteins of the instant disclosure possess activity lifetimes considerably longer than found for unmodified mesophilic enzymes: retain activity even after exposure to elevated temperatures for extended periods of time, and resist inactivation in the presence of organic cosolvents.
  • the proteins encompassed by the instant disclosure can be isolated by standard purification methods, specifically, and by ion exchange chromatography.
  • the enzymes of the instant disclosure are all intracellular proteins that can be recovered by cell disruption and loaded on to DEAE cellulose.
  • Purified esterases of the instant disclosure are eluted by NaCl gradients; fractions containing single activities are pooled and concentrated prior to lyophilization for storage. Specific activity is determined by measuring the total concentration of protein either by the Pierce BCA method or by measuring the UV absorbance at 280 nm followed by an activity assay based on the initial hydrolysis rate of p-nitrophenylproprionate.
  • the proteins of the instant disclosure can be characterized by the strain of bacteria from which they were isolated, the growth in TT media at 55 * C and 65 * C, and by esterase hydrolytic activity.
  • the proteins of the instant disclosure can be characterized by esterase activity in selection microtiter plate assay.
  • the proteins of the instant disclosure can also be characterized by the temperature profile, protein stability profile, and pH profile of the protein.
  • the proteins of the instant disclosure can be characterized by apparent molecular weight corresponding to esterase activity stain on native gradient PAGE gels. Specific molecular weight can be further characterized by chromatography, and specific activity can be further determined under standard conditions, where Table 10 contains a summary of many of these characteristics for selected proteins.
  • the proteins of the instant invention can be characterized by inherent prope ⁇ ies as well as by their amino acid protein sequence, or by a nucleic acid sequence which will encode for the amino acid protein sequence of the protein.
  • the instant disclosure encompasses a library of stable esterases isolated from a bank of thermophilic organisms, which are useful in the selective preparation of chiral pharmaceutical intermediates and other fine chemicals.
  • the library consists of at least 23 purified enzymes that can be used either in various combinations as a screening kit, or as individual protein preparations to carry out chemical reactions or prepare chiral products using kinetic resolution techniques. Under these conditions, racemic esters will have different rates of hydrolysis catalyzed by the enzymes depending on which stereoisomer best fits the structural parameters of the enzyme active site.
  • the products carrying the chiral center(s) may be on either the carboxylic acid or the alcohol.
  • many of the esterases described herein may be used to prepare chiral esters from carboxylic acids and alcohols if the reaction is run in the synthetic direction under transesterification conditions in which water is limited in solvent.
  • the instant disclosure encompasses lambda phage expression vectors which contain an insert that can be used for the production of recombinant ester hydrolyzing proteins of the instant invention, from a transformed cell host.
  • the insert contained on the lambda phage expression vector may be used in, for example, a phage-plasmid hybrid expression vector or other suitable expression vector such as, but not limited to, plasmids, YACs, cosmids, phagemids, etc.
  • a lambda expression vector is one of the vectors named in Table 7, or one which contains an insert which encodes for a substantially similar recombinant protein.
  • the instant disclosure also provides for vectors which are capable of transforming a host cell, and which encode for recombinant ester hydrolyzing proteins, the transformed host cells, and the recombinant ester hydrolyzing protein.
  • Appropriate host cells include but are not limited to: E. coli, Bacilli, Thermus sp., etc.
  • the recombinant ester hydrolyzing protein encoded by the. vector is capable of hydrolyzing 5-bromo-4-chloro-3- indolyl-acetate (X-acetate).
  • the recombinant ester hydrolyzing protein produced by the vector can be further characterized by a half-life stability comparable to that of a corresponding protein purified from the isolates.
  • the recombinant ester hydrolyzing protein is also characterized by the ability to remain stable at temperatures comparable to, or better than that of the corresponding protein from the original isolates.
  • Recombinant ester hydrolyzing protein encoded for by the vector can also be characterized by certain substrate specificities as discussed below, which are comparable to those of the corresponding purified protein from the isolates.
  • the vector is a vector named in Table 7 or 8, or one which contains an insert which encodes for a substantially similar recombinant protein.
  • a vector which encodes specific recombinant ester hydrolyzing protein is one of the vectors named and listed in Table 8, and deposited with the American Type Culture Collection (ATCC, Rockville, Maryland, USA) under the terms and conditions of the Budapest Treaty for the Deposit of Microorganisms, and given a specific designation number by the ATCC, to be amended to the specification upon receipt of such numbers.
  • ATCC American Type Culture Collection
  • FIG. 1 Esterase Screening plate. Fifty microliters of cell extract is transferred to a well on a microtiter plate consisting of 0.1 mg/ml of either 5-bromo-4-chloro-3-indolyl acetate or butyrate (for esterase activities) suspended in 0.7% agarose and 0.1M Tris-HCl pH 8.0. Control wells consist of addition of either buffer, 20 U of Pig Liver Esterase (PLE), or 20 U of Porcine Pancreatic Lipase (PPL). Plates are incubated for sufficient time to allow full color development in control wells, usually about twenty minutes at 37°C. Dark wells represent positive activity. This photograph demonstrates the screening of 65 candidate isolates, and the resulting positives.
  • PLE Pig Liver Esterase
  • PPL Porcine Pancreatic Lipase
  • Figure 2 Esterase activity stain of crude extracts from thermophiles. After electrophoresis, the gels are equilibrated in pH 7.6 Trizma buffer and then stained for activity in either 0.15% X-acetate. The gels are then incubated at 55°C for up to 30 minutes.
  • Figure 3. Molecular Weight calibration curve. Figure 3 depicts a standard molecular weight calibration curve.
  • Figures 4A-T Enzyme Characteristics.
  • Figures 4A-T depict the activity profiles which characterize enzymes of the instant disclosure. For each enzyme listed, Graph 1 depicts the Temperature Profile of the enzyme plotting relative esterase activity versus temperature. Graph 2 depicts the Residual Esterase Activity of the listed enzyme plotting relative remaining activity versus time in hours, at 25 * C, 40°C, and 65°C. Graph 3 depicts the pH profile for the listed enzyme plotting Relative Esterase Activity versus pH. Figure 5. Migration profile of E10 . 0 on 8% SDS-PAGE. Lane 1. Boiled E100 following DEAE and Q Sepharose chromatography. Lane 2. Non boiled purified El 00. Lane 3. Boiled El 00. Lane 4. Molecular weight markers.
  • FIG. 1 Kinetic analysis of E100.
  • Figure 7. Temperature and pH profiles of E100. a) Temperature profile of El 00. Plot of El 00 catalyzed hydrolysis of p-nitrophenyl proprionate as a function of temperature. Enzyme activity was determined upon exposure to different temperatures.
  • Figure 8 The tolerance of E100 to the presence of organic cosolvents on the hydrolysis of p-nitrophenyl proprionate as determined by relative rates. Residual activity of the enzyme is determined in the presence of organic solvent by measuring the initial rate of enzyme catalyzed hydrolysis of pNP in the presence of various concentrations of CH3CN. Reactions are run in 50 mM Tris-HCl pH 8.5 at 37°C as described in determination of activity. Changes in absorbance are corrected for spontaneous hydrolysis of the substrate and the changes in extinction coefficient of the product in the presence of organic cosolvent.
  • FIG. 9 Purification of E101. a) Steps in the purification of E101 as shown by 10% SDS-PAGE. Lane 1. Molecular weight markers. Lane 2. purified E100 (included as standard). Lane 3. dialyzed protein after NH4SO4 fractionation. Lane 4. DEAE load/wash.
  • FIG. 10 Substrates used to screen stereo- and regioselectivity. Esterases are versatile biocatalysts in the sense that stereo- and regio-selectivity can be mediated by substrate structure which fall into four types. The compounds listed represent a range of different structural features encountered in .common substrates with potential importance for the chemical intermediate industry. Several of the substrates are commercially available in entantio- or diastereomerically pure form and can be used in qualitative screening procedures described in the text. Four classes of substrates most commonly associated with hydrolytic biocatalysts for chiral centers resolution are considered. A) Type I substrates position the desired product on the carboxylic acid side of the product, while Type II compounds the alcohol contains the requisite functionality.
  • Type III and Type IV substrates can be considered subsets of Types I and II, but their unique properties dictate that they be classified separately.
  • Type III molecules require that the enzyme differentiates a prochiral substrate while Type IV compounds are meso structures.
  • These last two substrate types demonstrate the synthetic importance of biocatalyst based resolution methods as these types of compounds are very difficult to selectively operate upon by other chemical means.
  • Figure 11. Selection process for Recombinant Esterases. a). Screening of the phage library from strain isolate 28 (E009) using an X-Acetate gel overlay. Blue halos surround single phage plaques expressing esterase. b) Purification of hybrid phages produced from the 54 (E002) strain.
  • Figures 12a-r Examples of screening technique using esterase activity stain of recombinant protein from phage lysates. Once esterase-positive candidiates are identified, phage lysates are screened for the correct ester hydrolysis activity on a native 4- 15% gradient BioRad ReadyGel. After electrophoresis, the gels are equilibrated in pH 7.6 Trizma buffer and then stained for activity by using a 0.15% X-acetate overlay. The gels are then incubated at room temperature for up to 30 minutes.
  • the figures shows a typical examples of how the tequnique is used to identify proteins with the same mobility characteristics as the native protein, a) Screening positive clones from a bank made from strain isolate SI to identify E001.
  • Lanes indicate lambdaTGEl isolates 1, 2, 3, 4, 5, 8 and native control protein (C); b) Screening positive clones from a bank made from strain isolate 54 to identify E002. Lanes indicate lambdaTGE2 isolates 1, 2, 3, 4, 6, 8 and native control protein (C); c) Screening positive clones from a bank made from strain isolate 50 to identify E003. Lanes indicate lambdaTGE3 isolates 1, 2, 3, 4 and native control protein (C); d) Screening positive clones from a bank made from strain isolate GP1 to identify E004. Lanes indicate lambdaTGE4 isolates 1 , 2, 3, 4, 5, 6 and native control protein (C); e) Screening positive clones from a bank made from strain isolate C-1 to identify E005.
  • Lanes indicate lambdaTGE5 isolates 1, 2, 3, 4, 5, 6 and native control protein (C); f) Screening positive clones from a-bank made from strain isolate 55 to identify E006. Lanes indicate lambdaTGE ⁇ isolates 1 , 2, 3, 4, 5, 6 and native control protein (C); g) Screening positive clones from a bank made from strain isolate 30 to identify E008. Lanes indicate lambdaTGE ⁇ isolates 1, 2, 3, 4, 5, 6 and native control protein (C); h) Screening positive clones from a bank made from strain isolate 28 to identify E009. Lanes indicate lambdaTGE9 isolates 1, 2, 3, 4, 5, 6, 7 and native control protein (C); i) Screening positive clones from a bank made from strain isolate 29 to identify E010.
  • Lanes indicate lambdaTGElO isolates 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 and native control protein (C); j) Screening positive clones from a bank made from strain isolate 31 to identify E01 1. Lanes indicate lambdaTGEl 1 isolates 1, 2, 3, 4, 7, 8 and native control protein (C) on the first gel and lambda TGE11 isolates 7, 8, 9, 10 and native control protein (C) on the second gel ; k) Screening positive clones from a bank made from strain isolate 26b to identify E012. Lanes indicate lambdaTGE12 isolates 1, 2, 3, 4, 5, 6 and native control protein (C); 1) Screening positive clones from a bank made from strain isolate 27 to identify E013.
  • Lanes indicate lambdaTGE13 isolates 1, 2, 3, 4, 7, 8 and native control protein (C); m) Screening positive clones from a bank made from strain isolate 34 to identify E014. Lanes indicate lambdaTGE14 isolates 3, 5, 6, 8, 9 and native control protein (C); n) Screening positive clones from a bank made from strain isolate 62 to identify E015. Lanes indicate lambdaTGEl 5 isolates 1, 2, 3, 4, 5, 6, 7, 8 and native control protein (C); o) Screening positive clones from a bank made from strain isolate 47 to identify E016. Lanes indicate lambdaTGEl 6 isolates 1, 2, 3, 4, 5, 6, 7 and native control protein (C); p) Screening positive clones from a bank made from strain isolate 4 to identify E019.
  • Lanes indicate lambdaTGE19 isolates 1, 2, 3, 4, 5, 6 and native control protein (C); q) Screening positive clones from a bank made from strain isolate 7 to identify E020. Lanes indicate lambdaTGE20 isolates 3, 4, 6, and native control protein (C); r) Screening positive clones from a bank made from strain isolate 32 to identify E021 (E017b). Lanes indicate lambdaTGE21 isolates 6, 8, 9 and native control protein (C);
  • Figures 14 Examples of esterase stain of recombinant protein from plasmids. Protein extracts from both the native organism (single column purified) and a recombinant production strain are compared. Protein extracts are run on a 4-15% Gradient BioRad Ready Gel. After electrophoresis, the gels are equilibrated in pH 7.6 Trizma buffer and then stained for activity in either 0.4% X-acetate using an X-Acetate overlay. The gels are then incubated at room temperature for up to 30 minutes.
  • FIG. 15 Digestion patterns for 24 recombinant esterases.
  • the restriction endonuclease digestion patterns for the set of 24 plasmid listed in Table 8 is shown, (a) The 24 plasmids are cut by EcoRI (1-24), BamHI (25-48), Hindlll (49-72) and £coRV(73-96). (b). A gel showing the Pstl digestion pattern for plasmids 1-18. (c). A gel showing the Pstl digestion patterns for plasmids 19-24 and the Xbal digestion patterns for plasmids 1-11. (d). A gel showing the Xbal digestion patterns for plasmids 12-24.
  • lanes 1-24 refer to the following plasmids in the following order: pTGEl . l , pTGE2.1 , pTGE2.2, pTGE3.2, pTGE4.6, pTGE5.3, pTGE6.3, pTGE7.1 , pTGE8.5, pTGE9.4, pTGE10.3, pTGEl l.lO, pTGE12.2, pTGE13.2, pTGE14.3, pTGE14.6, pTGE15.9, pTGE l ⁇ .
  • Plasmid pTGE21.8x is a variant of pTGE21.8 which was isolated that had a loss in activity.
  • Figure 16 Nucleic acid sequence and translated protein amino acid sequence. The isolation and cloning of the genes encoding for the enzymes of the instant invention will result in DNA segments in which an open reading frame (ORF) may be found which corresponds to the translated protein amino acid sequence. Alternative start codons are recognized in the art, however the encoded protein will comprise at minimum a core protein ORF.
  • Figure 16A is an isolated nucleic acid sequence, and translated amino acid sequence which correspond to E001 enzyme ORF, alternative start codons are underlined.
  • Figure 16B is the cloned isolated nucleic acid sequence which contains the E001 ORF.
  • Figure 16C is an isolated nucleic acid sequence, and translated amino acid sequence which correspond to E009 enzyme ORF, alternative start codons are underlined.
  • Figure 16D is the cloned isolated nucleic acid sequence which contains the E009 ORF.
  • Figure 16E is an isolated nucleic acid sequence, and translated amino acid sequence which correspond to E01 1 enzyme ORF, alternative start codons are underlined.
  • Figure 16F is the cloned isolated nucleic acid sequence which contains the E01 1 ORF.
  • Figure 16G is an isolated nucleic acid sequence, and translated amino acid sequence which correspond to ElOl enzyme ORF, alternative start codons are underlined.
  • Figure 16H is the cloned isolated nucleic acid sequence which contains the ElOl ORF.
  • the instant invention provides for isolated commercially useful protein preparations from themostable bacteria which are selected for enzymatic activity, and characterized by apparent molecular weight, pH, and temperature stability.
  • the isolated protein of the instant disclosure can be used as molecular weight markers for finding similar enzymes, as well as functionally as enzymes for carrying out biocatalysis. Commercial chemical synthesis of specific racemic products often require the use of such isolated enzyme preparations.
  • the results of characterization assays demonstrate that the esterase enzymes described have a range of optimal parameters. For instance, ElOO and ElOl have optimal operating temperatures above 70°C as would be consistent with enzymes isolated from an extreme thermophile, and E001-E021 have optimal commercial temperatures in the range of 40-50°C as would be consistent with enzymes isolated from the more moderate thermophilic organisms. Both groups, however, provide added stability and functionality as compared to other known esterases from thermophilic bacteria. E001-E021 provide an optimal temperature environment for chemists who wish to work in less extreme temperature ranges, and also function well at room temperature. The results also demonstrate that the enzymes described posses a variety of pH optima including some with no apparent preference under the conditions of the experiment, however the trend for most of the proteins is to have pH optima near or slightly below neutral.
  • thermophilic organisms Strains - Thermus sp. T351 (ATCC 31674) is available from the American Type Culture Collection (ATCC). All isolated strains and cultures are grown on TT medium (36). This medium consists of (per liter): BBL Polypeptone (8 gm), Difco Yeast Extract (4 gm), and NaCl (2 gm). Small scale cultures for screening are grown at 65°C at 250-300 rpm with 1 liter of medium in a 2 liter flask. Larger scale production of cells for enzyme purification are grown in 17 liter fermentors (LH Fermentation, Model 2000 series 1).
  • the fermentors have a working volume of 15 liters and cultures were grown in TT broth, 250 rpm, 0.3 to 0.5 vvm (volumes air/volume media per minute) at 65°C. Temperature is maintained by circulating 65°C water from a 28 liter 65°C water reservoir through hollow baffles withm the stirred jars E coli strains are grown as descnbed in (37)
  • Enri c hm en t P ro cedures fo r Ne wly Mud i ⁇ p leetf s ⁇ rh B ⁇ m op h i le sediments, composting organic matenals, and soil samples are used to isolate new strains These samples are collected from numerous geographic sites ranging from the Midwest to the Southeast.
  • Esterase Plate assay - Organisms are grown in liquid cultures on TT media at either 55°C or
  • Isolates GP1, 27,28,29,30,31,32,34,62 appear to be thermophilic Acttnomyces.
  • ElOl. ⁇ ⁇ Specific activity is the amount of p-mtrophenol produced in micromoles per minute per milligram of total protein at 40°C after purification to homogeneity (for ElOO and ElOl) or semi-purification (for E001 -
  • E021) as descnbed in the Examples. 4 E021 is also referred to as E017b.
  • Protein Isolation A large batch cell culture is grown according to the methods described in Example 1 and the cell paste is collected by centrifugation and stored at -80°C. lOOg of cell paste is thawed in 200 ml of a stirred solution composed of 50 mM phosphate buffer at pH 7.5 containing 200 M KCI and 0.1 mM EDTA. Once dissolved, the suspension is allowed to warm to room temperature and then treated with lysozyme (0.1 mg/ml) for 2 hours. The solution is then sonicated to completely disrupt the cells. Settings used on a 375 watt Sonics & Materials Vibra Cell sonicator with a standard 1/4" horn were 5 minutes of power setting 8 disruption with a 50% pulse rate.
  • Alternative methods for cell disruption can include processing the cells through a device such as a french press, Gaullen homogenizer, microfluidizer or other homogenizer.
  • Cell debris is removed by centrifugation and proteins can be precipitated by NH 4 SO4 fractionation to 60% saturation.
  • Precipitated protein is centrifuged and resuspended in minimal volume of 50 mM phosphate pH 6.5 containing 1 mM ⁇ -mercaptoethanol (BME).
  • DEAE Purification The protein solution is dialyzed against the resuspension buffer 3 times using 10 Kd pore size dialysis tubing. The resulting protein solution is diluted two fold in the buffer and applied to a 100 ml bed volume DEAE column equilibrated in the same buffer.
  • the column is washed with 200 ml equilibration buffer and then eluted with a linear gradient from 0 to 0.5 M NaCl.
  • Q Resin purification - Active fractions isolated from DEAE purification are pooled and dialyzed against three changes of equilibration buffer and dialysate was applied to a 50 ml bed volume of sepharose Q resin equilibrated with the buffer above.
  • the column is washed with 100 ml of 50 mM phosphate pH 6.5 containing 0.1M KCI and 1 M BME and then eluted with 150 ml of a KCI gradient from 0.1 M to 0.6M added to the above buffer.
  • Ultrafiltration Concentration - Active fractions are pooled and concentrated using an
  • the crude cell lysate is diluted by three fold with 50 mM Tris-HCl pH 7.5 and the material is loaded to a DEAE cellulose column (bed volume 60 ml) equilibrated with the dilution buffer.
  • the column is washed with three column volumes of dilution buffer followed by a salt gradient of 0-0.5M NaCl over 4 column volumes.
  • Active fractions eluted from the ion exchange resin in the salt gradient window of 0.25-0.35 M. Fractions were assayed for activity as described under determination of specific activity and those showing the highest activity were pooled and concentrated by ultrafiltration with 10 Kd molecular weight cut off membrane. Concentrated enzyme samples are stored at 4°C for further use.
  • ester hydrolysis activity may still be detected under long term exposure to substrate agarose overlays of proteins separated on native PAGE, indicating very small quantities of a second esterase activity which should not interfere with most industrial applications.
  • a further purification (such as an Ammonium sulfate salt precipitation, gel filtration, or other methods as described in Example 3) can be applied if necessary. The process can be scaled up or down as desired.
  • Example 5 Method for determination of temperature profile.
  • Optimal temperature profiles for an esterase protein is performed by measuring the activity of the esterase diluted into 0.1M sodium phosphate buffer pH 7.0 equilibrated at 30°C, 35°C, 45°C, 55°C and 65°C respectively for five minutes. The temperature profile is then determined by measuring the rate of hydrolysis of p-nitrophenylproprionate added to the equilibrated solution under reaction conditions described for determination of specific activity in Example 2 (modified by the various temperatures used in this experiment). Control reactions that substitute bovine serum albumin for esterase enzymes are used to allow correction for temperature dependent autohydrolysis of the substrate. The data is then plotted as relative activity versus the temperature of the reaction.
  • Example 6 Method for determination of enzyme stability.
  • the long term catalytic stability the esterase enzyme is evaluated by testing the activity remaining after exposure to various temperatures.
  • the enzyme stock solution is diluted into 0.1 M sodium phosphate buffer pH 7.0 and placed in a temperature bath equilibrated to 25°C, 40°C or 60°C respectively under sealed conditions to avoid concentration effects due to evaporation. Residual activity is then determined by removing aliquots at regular intervals and measuring the rate of hydrolysis of p-nitrophenyl-proprionate as described above. Results are plotted as relative activity vs. time. The results (see Figure 4) indicate that all enzymes retain most of the initial activity for at least 48 hours when exposed to temperatures up to and including 40°C. Activity does decrease at 60°C particularly for enzymes isolated from organisms with optimal growth temperatures near 55°C.
  • Example 7 Method for determination of pH profile.
  • the pH profile of an esterase is determined as follows.
  • the rate of p-nitrophenylproprionate hydrolysis is determined under reaction conditions similar to those described for determination of specific activity in Example 2 with buffers of wide useful pH windows that overlap with at least one data point.
  • two buffers were selected that met the above criteria, Mes (useful range of 6-6.5) and Bis-tris propane (useful buffer range 6.5-9). All pH tests were corrected for spontaneous autohydrolysis by subtraction of experimental runs from controls substituting bovine serum albumen for esterase. This control data treatment becomes especially important for pH's greater than 7.5.
  • the number of esterase enzymes in each semi-pure sample is determined from native gel PAGE using 4-15% acrylamide gradient (precast gels purchased from Bio-Rad laboratories) separating proteins based on their charge to size ratio.
  • the gel shows trace contamination with other enzymes capable of indoylacetate hydrolysis that could not be detected easily with the HPLC because of column dilution effects. What is clear from the gel experiments is that most of the samples have a single major activity that have similar migration characteristics as shown in Figure 2.
  • the estimated native molecular weights for the protein of interest is determined by separation on a Pharmacia Superdex S200 FPLC column fitted to a Hitachi HPLC 6200 system. Proteins were separated by isocratic elution in 0.05 M sodium phosphate buffer at pH 7.0 containing 0.1 M NaCl. The solvent flow rate was maintained at 0.5 ml/min and protein was detected by UV at 280 nm. Esterase active fractions were detected initially by 5- bromo-3-chloro-3-indolyl-acetate plate assay with follow-up assay of most active fractions by p-nitrophenyl-proprionate hydrolysis (both methods are described in Example 2).
  • Molecular weights are estimated by comparison to standard elution profiles (plotted as the log of molecular weight vs. time in minutes) generated by use of the following proteins: ⁇ - amylase 200 Kd, alcohol dehydrogenase 150 Kd, bovine serum albumin 66 Kd, carbonic anhydrase 29 Kd, cytochrome c 12.3 Kd.
  • Example 11 Characterization of substrate specificities. Substrate preference of esterases for hydrolytic activity on various esters can be determined as follows. A grid of molecules is prepared on microtiter plates by dissolving each substrate (0.1 mM final concentration) in CH3CN and mixing with 0.1M phosphate buffer pH 7.5. Partially purified enzymes is then added to the wells and the reaction mixture is incubated for 30 minutes. Crude lysates can also be tested this way. Plates are checked after 10, 20 and 30 minutes to determine relative activities. For experiments with noncolored substrates, reactions are run in test tubes under the same conditions as described for the colored substrates except that the reactions are extracted three times with dichloromethane.
  • a new method was developed to rapidly screen for esterase activity based on the mechanism of the enzyme catalyzed hydrolysis reaction wherein the pH of the system is reduced by the release of protons upon ester hydrolysis.
  • the proton flux in the reaction can be monitored by use of indicator dyes that have pH-dependent color transitions in the desired pH range of enzyme activity.
  • the best indicators tested are phenol red for enzymes that function optimally at slightly elevated pHs (starting point pH 8.5) or bromothymol blue (starting point pH 7.2) for enzymes that operate well at more neutral conditions.
  • the indicator reactions are monitored by one of two methods. Spectroscopic studies are performed by measuring the UV/Vis maxima of a 0.001% solution of either phenol red or bromothymol blue dissolved in different pH buffers at 5 mM concentration. Hydrolytic reactions are then performed by adding the substrate (0.1 mM final concentration) to a 5 mM buffer solution (sodium phosphate pH 7.2 for bromothymol blue indicator and sodium borate pH 8.5 for phenol red indicator) and equilibrating the temperature at 25°C for five minutes followed by initiation of the reaction by addition of 0.1 U target enzyme.
  • Rapid assay of a variety of hydrolytic activities, in this cases esterases, is determined in a microtiter plate experiment using several different enzymes and substrates. Accurate comparison of commercially available enzymes can be insured by using the same specific activity for each enzyme determined from the total protein and the initial rate of hydrolysis of the common substrate p-nitrophenylproprionate. The data are recorded as the time required to visualize a pH dependent color change for the given indicator dye. Control experiments using BSA as the protein source cause no change in indicator color and establish that pH changes in solution are the result of an enzyme catalyzed hydrolysis. Control tests of reaction solutions containing enzymes and indicators without substrates established that color changes in the solutions are not the result of buffer salts or the enzymes alone.
  • Results are reported as the amount of time required to change indicator color. The data is indicative of variable substrate specificity between different environmental isolates. Of particular note is the suggestion of stereoselectivity as determined from the relative rates of hydrolysis for substrate enantiomers. Control reactions are similar to those described above in the substrate specificity studies with commercially available enzymes.
  • Example 13 Further characterization of substrate specificities.
  • FIG 10 Depicted in Figure 10 are examples of the substrates that can be tested with each enzyme activity. These molecules have been chosen specifically because of their importance as intermediates in the synthetic literature with the potential for industrial application. Experiments can be performed with crude lysates or proteins isolated from media broth in cases where the activities are known to rapidly assess the likely reaction chemistry including substrate preference and stereochemistry. All structure activity tests are compared to standard mesophile biocatalysts such as pig liver esterase. The reactions are monitored by TLC analysis to compare the products to standards purchased from commercial sources or prepared by chemical means (for example, base-catalyzed hydrolysis of esters).
  • the products of the reaction are isolated and analyzed for their enantiomeric excess (ee) by chiral phase HPLC (Diacel Chiralcel OD or OB) or ⁇ NMR of the corresponding diasteriomers prepared by derivatizing products to Mosher derivatives (alcohol products) or menthyl derivatives (carboxylate products).
  • Diastereomeric ratios determined from the NMR spectra are based on corresponding peak integrations and compared to either literature values or standards obtained from commercial sources using of chiral shift reagents when necessary.
  • Optical rotations and absolute configurations of the products are then determined by polarimetric analysis and compared to values found in the literature or determined from standards obtained from commercial suppliers.
  • Example 1 Strains from the identified sources as listed in Table 1 were isolated by growth in TT media at 65°C as described in Example 1 (ie. S 1 from soil, etc.). Specific esterase hydrolytic activity was identified by the methods described in Example 2 and the isolated esterase protein assigned the identifier as listed in Table 1 (ie. E001 etc.) To prepare enzyme, a 15 liter culture of isolate is grown and the cells are spun down and collected as described in Example 1. The cells are lysed and a isolated preparation of was purified according to the procedures outlined in Example 4. The protein was characterized using the methods described in Example 5 to determine the temperature profile, Example 6 to determine protein stability, and Example 7 to determine the pH profile, and the results are shown in Figure 4.
  • the protein was characterized by migration on Native gradient PAGE as described in Example 9 and the data is shown in Figure 2.
  • the specific activity was determined as described in Example 2 and the molecular weight was determined by chromatography as described in Example 10 and are presented in Table 1.
  • Substrate specificity for several proteins has been demonstrated and are shown in Table 2.
  • esterases have been demonstrated to be useful, and to posesses unique activity at commercially useful purity. Certain results are summarized in Table 10.
  • Example 15 Characterization of ElOO Purification of ElOO - ElOO is purified from Thermus sp. T351 over 300 fold by a series of four steps described in Example 3: DEAE purification, Q Resin purification, Ultrafiltration concentration, and preparative SDS PAGE.
  • the specific activity could not be measured in the crude lysate since there was a secondary esterase activity present (ElOl).
  • the secondary activity could be completely removed from the target esterase during the first chromatographic step in which the secondary esterase passed through the DEAE column unbound.
  • DEAE purification alone is sufficient to yield ElOO enzyme substantially purified away from any other contaminating activity.
  • Q Resin purification and ultrafiltration allow for higher purity product to be produced as required by specific applications.
  • a final SDS PAGE purification step allows the protein to be purified to homogeneity for determination of molecular characteristics.
  • Protein Characterization The active band is collected by electroelution on a preparative SDS-PAGE gel and rerun on 10% SDS-PAGE under denaturing conditions. This shows a single band with a relative molecular mass of about -45 Kd ( Figure 5). Unboiled samples run on the same SDS-PAGE gels show multiple bands in approximate increments of the proposed monomeric molecular mass. Additionally, the nonboiled sample can be stained for activity, however only bands corresponding to multimeric forms of the enzyme are found to retain activity beginning with dimeric species.
  • the specific activity of the purified protein is approximately 3.2xlO- 6 Mmin 1 mg 1 using 4-methyl-umbelliferyl-butyrate (MUB) as the substrate.
  • MUB 4-methyl-umbelliferyl-butyrate
  • the rates of enzyme catalyzed hydrolysis are corrected for the spontaneous hydrolysis of the substrate.
  • Protein concentrations are determined by either the absorbance at 280 nm or by Lowery assay.
  • Crude activity is determined by a colorimetric assay based on the hydrolysis of 5-bromo-4-chloro-3-indoyl esters suspended in a 0.7% agar matrix on microtiter plates.
  • a 0.1 mg/ml solution of the indolyl derivative is dissolved in a minimal volume of acetonitrile and added to a warm solution of 0.7% agar containing 0.1M phosphate buffer pH 7.5. 10 ⁇ L of this solution is distributed to microtiter plates which, when cooled, could be used with as much as 100 ⁇ L of enzyme sample and incubated at temperatures from ambient to >65°C.
  • Reaction conditions are those described in the general experimental above except for the addition of specified components. Relative rates are corrected for the spontaneous rate of hydrolysis of the uncatalyzed reaction.
  • Structure activity assay of partially purified esterase ElOO from Thermus species (++) highest activity as determined by (a) color formation in less then 10 min or significant product formation on (b)TLC. The remaining activity measurements follow the order: + > +/- > - > - -.
  • Structure abbreviations are as follows: I, chloro-bromo-indoyl, N, a-napthyl, U, methylumbelliferyl, pN, p-nitrophenyl, oN, o-nitrophenyl, PA, phenylacetate.
  • Kinetic characteristics are determined by measuring the concentration dependent initial rates of enzyme catalyzed hydrolysis of nitrophenyl proprionate. Reactions are run at pH 8.5 in 50 mM Tris-HCl buffer equilibrated to 37°C and initiated by addition of enzyme. Rates are determined from the absorbance changes due to formation of product nitrophenol at 405 nm. Rates are corrected for the spontaneous hydrolysis of substrate during the course of the reaction. Concentration vs. rate data are analyzed by both double reciprocal plots and by Hanes Wolff plots to determine Km, Vmax and Vmax/Km. The kinetic characteristics of ElOO determined from plots of the initial rates of hydrolytic reactions are shown in Figure 6.
  • ElOO The N-terminus of ElOO was determined by automated sequencing of the polypeptide purified by 10% SDS-PAGE and transferred to a PVDF support. The sequence obtained was: MKLLEWLK7EV, where the letters refer to the standard amino acid single letter code and the "?” refers to an indeterminate amino acid. Thus, ElOO has been demonstrated to be a useful esterase with unique activity at commercially useful purity.
  • ElOl is one of two esterase activities that are isolated from Thermus sp T351. ElOl can be purified away from a second esterase, ElOO, in an early purification step.
  • Active protein is observed in the load and wash fractions, pooled, and concentrated with the use of an Amicon concentrator fitted with a YM30 membrane. Concentrated proteins are then loaded directly to a 25 ml bed volume of sepharose SP resin equilibrated with the above buffer. Active fractions appear in the load and wash fractions which are pooled and concentrated as above. Concentrate is then loaded to a Sephracryl HR200 gel filtration column (1x40 cm) and 0.5 ml fractions are collected at a flow rate of 2 ml/hr. Active fractions are collected and analyzed by SDS- PAGE. In order to perform N-terminal sequencing, fractions considered to be homogeneous are concentrated and submitted to a protein sequencing service center. The enzyme is stored at 4°C for future use.
  • E101' can be purified over 35 fold by these methods and possesses characteristics dramatically different from ElOO, the other esterase which is isolated from this strain. Attempts to use ion exchange chromatography result in subtractive purification since in no instance was the protein retained. Resins investigated include DEAE, Q sepharose, CM cellulose, SP sepharose and hydroxyappatite under conditions that varied from pH 6.0 to 9.0, and buffers from phosphate to borate including Tris and Hepes. After two ion exchange steps the protein is purified to homogeneity by gel filtration chromatography however, the protein appears to have an interaction with the column as retention is considerably longer than the molecular weight would suggest. The molecular weight of the protein appears to be approximately 135 Kd with a monomer mass of -35 Kd as determined from native and denaturing SDS-PAGE respectively ( Figure 9).
  • ElOl Characteristics The specific activity of the enzyme is ten fold greater than observed for ElOO with 4-methyl-umbelliferyl butyrate (MUB) as the substrate. ElOl is inhibited by PMSF but is insensitive to metal ions or metal ion chelators. The specific activity of the purified protein was found to be 3.2xl0' 5 mol min ⁇ mg' 1 and was determined from initial rates of hydrolysis using methyl umbelliferyl butyrate as a substrate. Table 5 outlines the inhibitory effect of various substances on ElOl activity.
  • MUB 4-methyl-umbelliferyl butyrate
  • Reaction conditions are those described in the general experimental above except for the addition of specified components. Relative rates are corrected for the spontaneous rate of hydrolysis of the uncatalyzed reaction.
  • Substrate specificity of ElOl was determined as described in Example 1 1. The results from the structure activity experiments for ElOl are shown in Table 6. The hydrolytic activity of the enzyme is similar to that observed for ElOO and has no observable protease activity toward milk or casein.
  • Structure activity assay of partially purified esterase ElOl from Thermus species (++) highest activity as determined by (a) color formation in less then 10 min or significant product formation on (b)TLC. The remaining activity measurements follow the order: + > +/- > - > - -.
  • Structure abbreviations are as follows: I, chloro-bromo-indoyl, N, a-napthyl, U, methylu ⁇ mbelliferyl, pN, p-nitrophenyl, oN, o-nitrophenyl, PA, phenylacetate.
  • ElOl has been demonstrated to be a useful esterase with unique activity at commercially useful purity.
  • Example 17 Cloning of Esterase.
  • General Cloning Strategy The ⁇ ZAP cloning system from StratageneTM can be used for the library constructions and detection of esterase activity. Other cloning systems can also be used to yield similar results.
  • the usual efficiency of cloning in ⁇ vectors vary from 10 5 to 10 7 hybrid clones per mg of cloned DNA and is sufficient to produce a representative gene library from a convenient amount of size-selected chromosomal DNA fragments.
  • detection of esterase activity in phage plaques, as opposed to bacterial colonies, is more efficient due to the easier access of substrate to the enzyme.
  • Phages are generally less sensitive to the toxic action of cloned proteins and are also able to survive at the temperatures up to 70°C.
  • the ability of the cloning system to tolerate elevated temperatures and potential toxicity of the cloned proteins is necessary for the detection of the activity of thermophilic proteins, such as the esterases described here.
  • Genomic DNA is prepared from a culture of the appropriate strain containing the esterase of interest as described in Example 1.
  • Cells of different strains are grown to late log phase in 100 ml TT broth (8 g Polypeptone (BBL 1 1910), 4 g yeast extract, 2 g NaCl, per liter) at 55°C or 65°C overnight shaking at 250 RPM.
  • Cells are recovered by centrifugation and the pellet is resuspended in 5 ml of lysis buffer (10 mM Tris-HCL, pH 7.0, 1 mM EDTA, and 10 mM NaCl). Lysozyme is added to a final concentration of 2 mg ml.
  • Cells are incubated at 37°C for 15 minutes followed by the addition of SDS to 1%.
  • the lysate is gently extracted three times with phenol/chloroform/iso-amyi alcohol (25/24/1) and the DNA spooled from a 95% ethanol overlay of the aqueous phase.
  • Lysozyme- generated spheroplasts are lysed by the addition of 1% SDS and partially deproteinased by addition of 100 ⁇ g/ml of proteinase K at 24°C for 10 min.
  • Chromosomal DNA is further purified by three phenol/chloroform extractions, precipitated with 2.5 volumes of ethanol and resuspended in 1 ml of TE (10 mM Tris pH 8.0; 1 mM EDTA), after washing in 20 ml of 75% ethanol.
  • the extracted fraction consists of DNA fragments larger than 50 kb, with a concentration of about 0.5 ng/ ⁇ l, as detected by gel electrophoresis using a 0.7% agarose gel run at 10 V/cm for 4 hours.
  • Genomic DNA is partially digested with the restriction enzyme Sau3A and then ligated to predigested Lambda ZAP Express (Stratagene Cloning Systems). Products of ligation reactions are packed in vitro using ⁇ packaging extracts which are purchased from Promega. This vector accommodates DNA up to 12 kb in length and allows identification of clones both by expression off the T3 and T7 promoters and by probe hybridization to plaques. The library is retained and screened for esterase activity. Other methods for generating genomic DNA libraries are also well known in the art.
  • Fractions with an average fragment size of 5 kb are chosen for cloning.
  • native strains containing E001, E002, E003, E006, E007, E008, E009, E010, E012, E016, E020 these are the second of the five samples of digested chromosomal DNA with the concentration of Sau3A of about 0.02 u/ ⁇ g of the DNA.
  • the proper degree of partial digestion is achieved in the first test tube with 0.1 u of Sau3A / ⁇ g of the DNA.
  • chromosomal DNA fragments Fifty ng of chromosomal DNA fragments are ligated with equimolar amounts of dephosphorilatyed BamHI-arms of the lambda ZAP phage vector (Stratagene) in 5 ⁇ l with 1 unit of ligase (New England Biolabs). Ligation reactions are performed at 18°C for 8 hours and stopped by heat inactivation at 70°C for 10 min. One ⁇ l of the ligation reaction, containing approximately 10 ng of DNA insert, is used for in vitro packaging with 10 ⁇ l of lambda proheads (produced by Promega Corp). The packaging reaction is performed at 28°C for 90 min, combined with 100 ⁇ l of an overnight culture of E.
  • coli XL1 Blue and plated using 2 ml of 0.7% top agar (0.8%NaCl, 10 mM MgSO4) per plate onto five 90-mm Petri plates containing LB media.
  • Serial dilutions of the packaging mixture are produced in order to determine the cloning efficiency which is generally about 1.0 x IO 7 hybrid phages ⁇ g of cloned DNA.
  • Cloning efficiencies for each individual strain varied, the size of the library generated fell within a range of 0.5 to 2.5 x 10 ⁇ from which two to twelve positive clones were analyzed (data not shown).
  • Hybrid phages from one plate are harvested to collect the amplified library, which is stored in 3 ml of LB media with 25% glycerol.
  • the four other primary plates are treated with indicator agar containing 5-bromo-4-chloro-3- indolyl-acetate (X-Acetate) as described below, to find hybrid plaques carrying esterase genes. Screening of gene banks for esterase activity - The products of the above packaging reactions are infected into E. coli XL1 blue MRF (Stratagene). Primary plaques of an unamplified gene library are screened for enzyme activity by overlaying the plates with top agar containing X-Acetate for 30 minutes at 65°C.
  • the concentration of substrate in the indicator overlay is diluted from a 4% stock in ethanol or N,N-dimethyl formamide to a 95,963-D _ 28 concentration generally between 0.1 and 1 % (usually about 0.4% is used) in the final solution.
  • suitable substrates may be substituted in this procedure including, but not limited to, 5-bromo-4-chloro-3-indolyl-butyrate (X-butyrate), 5-bromo-4-chloro-3-indolyl- proprionate (X-proprionate), 5-bromo-4-chloro-3-indolyl-stearate (X-stearate), 4- methylumbelliferyl-acetate (MUA), 4-methylumbelliferyl-butyrate (MUB), 4- methylumbelliferyl-proprionate (MUP), or other 5-bromo-4-chloro-3-indolyl- or 4- methylumbelliferyl- esters which may be either synthesized or purchased from a commercial vendor such as Sigma Chemical.
  • X-butyrate 5-bromo-4-chloro-3-indolyl-butyrate
  • X-proprionate 5-bromo-4-chloro-3-indolyl-stearate
  • MUP 4- methylum
  • the plates are preheated at 65 * C for 20 minutes. Hybrid phages surviving this procedure are picked and re-screened three times. The extracts are then analyzed for the presence of a protein band with the same mobility as the native protein as described below.
  • the lambda ZAP cloning system permits an excision of smaller plasmid vector to simplify the insert characterization. While other methods may be employed for screening gene banks for esterase activity, i.e.
  • isolation, purification, and N-terminal sequencing of protein creation of degenerate nucleotide probes from N-terminal sequence; screening of gene bank with degenerate probes, the instant method is efficient and uniquely suited for the purpose of isolation of promising clones.
  • the four primary plates with phage colonies generated during the cloning described above are incubated at 65°C for 30 min. in order to inactivate some of the potential £. coli esterase activities.
  • Approximately two ml of 0.7% top agar (0.8% NaCl, 10 mM MgSO4) containing about 1 mg/ml of the colorimetric esterase substrate X-Acetate or other substrate (including but not limited to X-butyrate, X-proprionate, X-stearate, and 4- mcthyl-umbelliferyl based substrates) is overlaid onto each plate.
  • Expression of cloned esterases can be detected by blue halos around phage colonies (or fluorescent halos in the case of the 4-methylumbelliferyl substates).
  • the expression pattern observed for the gene library from strain isolate 28 (E009) is depicted in Figure 1 la.
  • a typical result for this process can yield a ratio of 1 : 3000 positive colonies to hybrid phages.
  • Precast gradient gels are purchased from BioRad Laboratories (catalog number 161 -0902) and used according to the manufacturer's instructions for native gels to generate the gels shown in Figure 12a-m .
  • An esterase preparation from the original strain, purified by HPLC to a single protein band is used as a control on the same gel.
  • a native protein preparation which has not been purified to homogeneity but is purified to a single esterase activity can be used as a control. Protein bands possessing an esterase activity can be detected by applying an X- Acetate overlay and incubating at room temperature for 5-20 min. The relative mobility of the clone candidates can be compared to the native esterase protein.
  • Figure 12a-z shows the results of the typical comparison of the esterase activities detected in lambda clones compared to the host strain.
  • the data generated for 107 hybrid phage clone candidates from 20 strains are summarized in Table 7.
  • Table 7 For each gene library screened, there is at least one clone candidate expressing an esterase protein with the mobility of the protein purified from the original strain.
  • Several of the ⁇ clone candidates express esterase activities which have mobilities that are different from the major component of the esterase specimens purified from the original strains. Similar sized bands possessing esterase activity are observed in the native organism as minor components (data not shown). These cloned ester hydrolyzing activities are given names depicted in Table 7.
  • the lambda ZAP vector allows the phage clone to be conveniently converted into a plasmid vector to allow better physical characterization of the DNA insert and regulated expression of cloned genes.
  • Induction of M13-specific replication by co-infection with the helper phage results in excision of a multi ⁇ copy plasmid carrying the cloned insert.
  • 10 ⁇ l phage stocks of the lambda hybrids (with about 10 7 Colony Forming Units (CFU)) and 1 ⁇ l of Exassist Ml 3 helper phage (about 10 10 CFU) are used to infect 20 ⁇ l of an overnight culture of the E.
  • coli XL1 Blue grown in LB. After 20 min at 24°C, the cell suspension is transferred from one of the wells of a 96-well microtiter plate into a 15-ml culture tube, diluted with 2 ml of LB, grown overnight at 37°C and 300 rpm, heated at 65°C for 10 min, and cleared by centrifugation at 3000 g for 20 min.
  • Excised plasmids packed in Ml 3 particles are transduced into a lambda resistant strain, XLOLR, that does not permit the development of the Ml 3 helper phage.
  • Ten ⁇ l of excised phage lysate are mixed with 30 ⁇ l of the overnight culture of the E.
  • coli XLOLR strain in one well of 96-well microtiter plate, incubated for 20 min at 37°C to allow adsorption, diluted with 100 ⁇ l of LB, and incubated at 37°C for 40 min to express the kanamycin (Km) resistance marker (neo) of the plasmid.
  • Km kanamycin
  • Cells are plated onto two LB plates supplemented with 40 mg/ml Km. One of the plates also contains 50 ⁇ l of a 4% X-Acetate stock solution.
  • bacterial colonies derived from each of the phage clones are picked from the plates without X-Acetate, transferred into 100 ml of LB supplemented with 40 mg/ml Km in a 96-well plate and grown ovemight. Progeny of these colonies are analyzed by a spot-test using X-Acetate containing agar.
  • Several plasmid clones derived from each phage are chosen for fu ⁇ her study by picking ones producing brightest blue halos and least amount of the esterase" segregants.
  • E. coli cells carrying excised plasmids are purified using LB plates supplemented with Km and a limited amount of X- Acetate to reduce any potential negative growth impacts from production of the somewhat lethal indole product of the colorimetric reaction. Colonies are selected by their phenotype (in general giving a modest growth rate and intensive blue color) and grown in 2 mi of LB with Km in 15 ml test tube for 48 hours to reach OD600 of about 1.0 and harvested by centrifugation at 12,000 g for 1 min.
  • Cell pellets are resuspended in 500 ml of 0.1 M Phosphate buffer pH 7.0 and sonicated using a Sonics & Materials Vibra Cell 375 Watt sonicator at 4°C. Sonication is performed using a microtip, 40% max capacity, 50% time pulse for 45 sec. Lysates are centrifuged at 12,000 g for 5 min and tested for its relative esterase activity. Variants with the highest activity are selected for the next round of growth and analysis. Three rounds of plating followed by growth in liquid medium and activity assays are performed to verify the stability of the clones.
  • Deviations in specific esterase activity among variants from the same plasmid lineage can be reduced to a factor of three from over a factor of 100 by this procedure. Stabilization of the activity generally occurs at the level corresponding to the highest activity values detected in the first round of stabilization. This could indicate that E. coli host mutations are being selected which allow higher tolerance of the cloned protein, rather than simply suppressed activity of cloned toxic gene.
  • Plasmid DNA is extracted from E. coli cells using a standard alkali lysis procedure, or other procedures known in the art (37).
  • the DNA is digested with a series of restriction endonucleases such as EcoRI, BamHI, Hindlll, Pstl, EcoRV, and Xbal to establish digestion pattern of the clone and to determine a size of the cloned DNA fragment.
  • restriction endonucleases such as EcoRI, BamHI, Hindlll, Pstl, EcoRV, and Xbal to establish digestion pattern of the clone and to determine a size of the cloned DNA fragment.
  • the physical map patterns for the 24 selected production clones are depicted in Figure 15.
  • the insert sizes for each clone are calculated from this data and is summarized in Table 8.
  • 'insert sizes are esumated from the agarose _::.
  • the esumaied insert size is based on a vector size of 4.5 kb and the accuracy which could be acne-, ed analyzing each of the six digesuon patterns.
  • ⁇ Specific activity is calculated as the amoun :r D-nitrophenol produced in micromoles per minute per milligram of total protein as described in L:-imple 2. The numbers reported here are from a typical production batch and may vary. Generation of the tag sequences for PCR identification of esterase containing inserts
  • the DNA sequences of the ends of the insert fragment carrying esterase genes can be determined by sequencing the ends of the inserts using standard T7 and S6 primers to produce unique tags of the cloned DNA. Sequence analysis can be carried out to design PCR primers which can uniquely amplify the DNA inserts from both the clones and the host organisms. These tags can be potentially used to generate this DNA fragment from the chromosome of the studied organisms using PCR technique.
  • a degenerative probe is prepared to the N-terminal sequence of the protein and hybridized to plaques from the recombinant phage bank.
  • degenerate PCR amplification probes can be made using the N-terminal sequence or sequences obtained from the n-termini of internal protein fragments which have been obtained after proteolytic digestion of the enzyme. Using these sequences, a probe can be made from an amplified region between the N-terminus and an internal fragment or between two internal fragment sequences to identify a clone carrying the DNA encoding for the enzyme of interest.
  • LB 10 gm/1 tryptone, 5gm/l yeast extract and lOgm/1 NaCl
  • Terrific Broth 12gm/l tryptone, 24gm/l yeast extract and 4ml/l glycerol supplemented with 100 ml of a sterile solution of 0.17 M KH2PO4, 0.72 M K2HPO4 after autoclaving
  • Each media is supplemented with 10-50 ⁇ g/ml kanamycin.
  • Optimal production media depends on a number of factors, including media cost and specific activity of the produced proteins.
  • TB media is a richer media and therefore more expensive. For instance, in the case of CE009, while more total units are produced in a single fermentation run, not enough is produced to justify the higher cost of the media.
  • specific activity is higher for the LB media preparation.
  • the seed train is established as follows. A loopful of a frozen production culture is used to inoculate 50 ml of production media in a 250 ml Erlenmeyer flask. The flask is incubated at 30°C for two days (250RPM) and then used to inoculate a 1 liter flask with 250 ml of production media. This flask is incubated 1 day at 30°C and 250 RPM. The 1 liter flask is used to inoculate the fermentor. Production of substantially purified preparations from a cell paste of strains producing the recombinant enzymes are carried out similar to the methods described in Example 4 and the specific protocols described in Examples 14-34 for the native proteins.
  • esterase production is performed by media studies in shake flasks followed by further optimization at the 1 liter to 20 liter scale.
  • final fermentation conditions can involve either a fed-batch or continuous fermentation process. Since the esterase activity being analyzed is intracellular, the use of a clear or defined media such as TT media is necessary.
  • Organisms of interest are grown and cell pellets are collected by centrifugation. Pellets are disrupted by sonication and enzymes can be purified using the standard techniques of ion exchange and gel permeation chromatography described in Examples 3 and 4. Growth conditions including media composition, pH, and temperature are optimized at the small scale (ie. shake flasks, and 1 liter fermentors) to give the highest cell density while retaining the highest amount of enzyme.
  • mutagenesis schemes are used to try and isolate high-producing mutants of the different activities of interest. These include mutagenesis with uv-light or chemical mutagens such as ethylmethane sulfanoate (EMS) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).
  • EMS ethylmethane sulfanoate
  • MNNG N-methyl-N'-nitro-N-nitrosoguanidine
  • the cells are treated with varying concentrations of the mutagen (or varying exposure times with uv light) according to methods described in Miller (38). Optimal concentrations of the different mutagens with different organisms vary. In general, killing concentrations allowing 80% survival for EMS, approximately 50% survival for MNNG, or 10-50% survival for uv light are desired. Mutagenized cultures are then grown up, allowing the mutagen to wash out and plated onto solid media.
  • Mutants are identified by applying an esterase plate screen to the cells.
  • an esterase screen an agar overlay containing a colorimetric or fluorogenic substrate such as 5-bromo-4-chloro-3-indolyl-acetate or 4-methyulumbelliferyl acetate will be applied.
  • Candidate mutants are then analyzed by native polyacrylamide gel electrophoresis and compared to the parental strain. Standard assay methods described in Example 2 or the rapid esterase/lipase screen described in Example 12 can then be applied to identify any differences in amounts of enzyme activity. If a production level increase is large an increased band on either a Native or SDS polyacrylamide gel after coomassie staining may be seen. Strains with multiple activities can also be differentiated in this way, verifying that the increase is in the enzyme of interest. It is then confirmed that the mutants have unaltered kinetic and substrate properties as the parental enzyme. The majority of mutations identified by this approach are expression mutations which can be isolated in either a promoter region, repressor molecule, or other controlling element.
  • the mutant is recharacterized to determine if it is a more efficient biocatalyst.
  • Example 19 Esterase Screening Kit A large subset of enzymes can be packaged into an easy to use screening kit to rapidly analyze a large number of enzymes at once.
  • the kits are formulated to eliminate as many potential errors as possible and each enzyme is provided in a lyophilized form if possible near its optimal buffer and reaction conditions.
  • kits Many different formats for the kit are possible, from a series of glass vials, to varying size microtiter plates constructed of different plastic materials.
  • the microtiter plate is favored because of its ease of handling and manipulating.
  • Most microtiter plates are made of polystyrene however, which will not stand up to most organic solvents. For experiments which utilize aqueous solvent, the polystyrene is not a problem.
  • Other more tolerant plastics such as polypropylene are available and are ideal for the kit.
  • Large size 24-well microtiter plates which allow 3 ml of sample to be assayed (allowing enough sample for multiple TLC or HPLC analysis) have been developed. Other formats may also be useful for different applications.
  • Each kit is prepared by addition of a stir bar, buffer (0.1M Na phosphate pH 7.0) and 1U of each enzyme to each well of a 24 well polypropylene tray (Tomtec). Enzymes are aliquotted into each well or vial in set amounts so that it can be assured that an equal amount of activity is provided for comparison. The entire kit is then lyophilized, sealed with heat seal foil (3M) and labeled.
  • each kit contains four control wells that are composed of buffers at pH's from 6-9 since it was found that some of the substrates tested tend to be unstable in buffered solutions which can confuse positive results with autohydrolysis.
  • the rest of the kit is composed of an instruction manual, a data sheet, a sample preparation vial a glass eye dropper and a plastic eye dropper.
  • the kit is formulated in such a way that only solvent and substrate need be added to each well.
  • the rapid-screen indicator dye method described in Example 12 can also be included in each well or vial. This makes a preliminary qualitative determination of enzyme effectiveness simple and fast.
  • Example 37 The cloning and characterization of recombinant proteins from strain isolates which produced the native isolated protein (as listed in Table 1) was carried out as described in Example 37. Lambda expression vectors were isolated as described above (specific named isolates are shown in Table 7). E. coli clones harboring the excised hybrid phage-plasmids were derived as summarized in Table 7, and were finally selected for esterase activity by subsequent screening, which after 3 rounds of stabilizing procedure was calculated to approximate units of activity per mg of total cell protein obtained. Esterase activity stain gel used to screen positive phage library candidates for the recombinant proteins are shown in Figure 12, which allowed the identification of alternative recombinant proteins as well. Production of the recombinant protein is carried out as described in Example 38, using TB for the media and purifying the enzyme as described for the native (nonrecombinant) protein in Example 4.
  • Example 21 Sequencing of Recombinant Proteins
  • ORF open reading frame
  • Figure 16A is an isolated nucleic- acid sequence, and translated amino acid sequence which correspond to EOOl enzyme ORF, alternative start codons are underlined.
  • Figure 16B is the cloned isolated nucleic acid sequence which contains the EOOl ORF.
  • Figure 16C is an isolated nucleic acid sequence, and translated amino acid sequence which correspond to E009 enzyme ORF, alternative start codons are underlined.
  • Figure 16D is the cloned isolated nucleic acid sequence which contains the E009 ORF.
  • Figure 16E is an isolated nucleic acid sequence, and translated amino acid sequence which correspond to EOl 1 enzyme ORF, altemative start codons are underlined.
  • Figure 16F is the cloned isolated nucleic acid sequence which contains the EOl 1 ORF.
  • Figure 16G is an isolated nucleic acid sequence, and translated amino acid sequence which correspond to ElOl enzyme ORF, alternative start codons are underlined.
  • Figure 16H is the cloned isolated nucleic acid sequence which contains the ElOl ORF.
  • 'broad pH range refers to > 50% activity through all pH tested (6.0-8.5)
  • thermophilic enzymes studies on 6-phosphogluconate dehydrogenasc from Bacillus stearothermophilus and yeast. J Appl Biochem. 6:39-47.

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Abstract

La présente invention concerne des enzymes du type estérase stables, isolées, caractérisées par leur aptitude à rester stables à certaines températures, leurs spécificités de substrat, et leur profil d'activité.
EP96929678A 1996-01-11 1996-08-02 Biocatalyseurs stables pour l'hydrolyse d'esters Withdrawn EP0952847A4 (fr)

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PCT/US1996/013065 WO1997025058A1 (fr) 1996-01-11 1996-08-02 Biocatalyseurs stables pour l'hydrolyse d'esters

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CA2286481A1 (fr) * 1997-04-11 1998-10-22 Thermogen, Inc. Catalyseurs biologiques stables pour hydrolyse d'ester
CA2332638A1 (fr) * 1998-07-07 2000-01-13 Thermogen, Inc. Visualisation de reactions catalysees par des enzymes utilisant des indicateurs de ph: criblage rapide de bibliotheques d'hydrolases pour detecter des enzymes enantioselectives
AU2001238407A1 (en) * 2000-02-16 2001-08-27 Thermogen, Inc. Esterase enzymes having selective activity
JP2009000002A (ja) * 2007-06-19 2009-01-08 Nissui Pharm Co Ltd 微生物検出用培地
ES2347398B1 (es) * 2008-06-11 2011-09-15 Universidade Da Coruña Esterasa termofila de thermus thermophilus.
CN103740668B (zh) * 2014-01-17 2016-02-10 江苏八巨药业有限公司 一种提高葡萄糖脱氢酶释放量的方法

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WO1996002653A1 (fr) * 1994-07-20 1996-02-01 Novo Nordisk Biotech, Inc. Systeme d'expression fongique thermophile

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CA2242864A1 (fr) 1997-07-17

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