Classifications

• • 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 enzymauc apphcauon in commercial pharmaceuucal and chemical synthesis, DNA vec t ors for the producuon of recombinant ester hydrolyzing proteins, host cells transformed by such vectors, and recomDinant ester hydrolyzing proteins produced oy such vectors and transformed cells.
• Esterases and Lipases catalyze the hydiolysis of ester bonds to produce alcohols and carboxylic acids as shown below
• Es t erases and lipases can be characte ⁇ zed bv different substrate specificiues.
• R gioup or chain length preference, and unique inhibitors 1. 2
• the many esterases and hoases range from hydrolases such as the broad carboxyl esterases which preterenually hydrolvze esters with long carbon chain R groups, to choline esterases, and to acetyl esterases wh i ch ac t on very specific substrates. In many cases, these hydrolases are also known to show stereo- and regio-selective preferences resulting from the chiral nature inherent in pro t e i n active si t es.
• the products can then be separated by chromatograph y to prov i de pure R l .
• the availability of a large pool oi esterases and lipases with varying specificities would be useful for screening the enzymes tor specific reactions, and developing opumal protocols for specific chemical synthesis The expedience of this process would facilitate t he producuon 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 cany out reactions on a wide vanety of substrates, including esters containing cyclic and acyclic alcohols, mono- and di-esters, and lactams (3).
• esterases and lipases By carrying out the reactions in organic solvents (4, 5) where water is excluded, the reactions of esterases and lipases can be reversed. These enzymes can catalyze este ⁇ fication or acylation reacuons to form ester bonds (3, 6, 7). This process can also be used in die transeste ⁇ fication of esters and in ⁇ ng closure or opening reactions.
• Racemic drugs often contain one isomer which is therapeutically active and the other enantiomer which is at best inacuve and at worst a major cause of potenually harmful side effects.
• the non-useful isomer in a racemic drug is increasingly being viewed as a contaminant.
• Enzymatic synthesis of optically pure pharmaceuticals and intermediates Since it is often very difficult to generate optically memee 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 syndiesis procedures with more environmentally-f ⁇ endly 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).
• hydrolases All six major classes of enzymes (oxidoreductases, transferases, hydrolases, lyases, isomerases, and hgases) have been useful in the synthesis of optically pure compounds as described in several detailed reviews (3, 7).
• the 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.
• mesophihc hydrolases particularly esterases and lipases used in chemical synthesis or chiral resolution
• lipases have been used in the synthesis of propranolol (7), a beta-adrenergic blocking agent used in the treatment ol angina and hypertension.
• Ibuprofen a nonstearoidal antnnflammatory agent has been synthesized via stereo selective hydrolysis of its methyl ester using carboxyesteiase (7) While these enzymes have begun to demonstrate the utility of biocatalysts in chemical synthesis, there is still a profound need for a wider varie t y 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 industnal setung, there is a need for them to have increased stability, higher thermotoleiance and a longer "shelf life"
• Thermostable enzymes Thermophihc organisms have already provided a rich souice of useful proteins that catalyze reacuons at higher temperatures and are stable for much longei penods of time (21, 22).
• One example is die DNA Polymerase I from Thermus aquanciis and its use in polymerase chain reaction (PCR) (23, 24)
• PCR polymerase chain reaction
• Thermophihc enzymes have become the most commercially successful enzymes in industry because ot 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 vanous strains of Bacillus, has been widely used in laundry detergents and other cleaning soluuons.
• thermostable enzymes generally posses an increased shelf life which markedly improves handling conditions, especially by those not u-ained in biochemistry to work with the specific range of conditions used for mesophihc 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 muluple applications
• thermostable enzymes While most enzymes lose a significant portion of their activity in organic solvents, thermostable enzymes may prove more tolerant to the denatu ⁇ ng condiuons of many organic solvents. Highly thermostable esterases and lipases are necessary to expand the application of these biocatalysts in large scale industnal reactions.
• thermoalcalophi c lipase (35) was identified from a Bacillus species MC7 isolated by continuous culture and had a half-life of 3 hours at 70 ° C. Finally, NASAgisladottir et al. (6) have reported the isolauon 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 invenuon provides for the isolauon and characte ⁇ zation of commercial grade enzyme preparauons charactenzed by esterase activity, and corresponding to the data as disclosed in Table 1.
• die instant invention provides tor the isolation, and charactenzation of specifically punfied esterase which is characterized by esterase actiyity, and corresponding to the data as disclosed in Table 1.
• the instant invenuon provides for proteins generated by recombinant DNA technology which have esterase activity.
• the instant invention encompasses lambda phage expression vectors which contain an insert that can be used for the production of recombinant ester hydrolyzing proteins of the instant invenuon, from a transformed cell host.
• the insert contained on die lambda phage expression vector may be used in, for example, a phage-plasmid hybnd expression vector or other suitable expression vector such as, but not limited to, plasmids, YACs, cos ids, 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 protems, the transformed host cells, and the recombinant ester hydrolyzing protein.
• Approp ⁇ ate 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 charactenzed 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 conesponding protein from the onginal 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 conesponding punfied protein from the isolates
• the vector is a vector named in Table 7 or 8, or one which contains an insert which encodes tor a substantially similar recombinant protein
• a vector which encodes specific recombinant ester hydrolyzing protein is one of the vectois named and listed in Table 8.
• the instant invention is directed to the novel nucleic acids, and the proteins encoded for dierein, isolated from the expression vectors of the present invention.
• the present invenuon is directed towards die nucleic acid sequence for DNA insert of said vectors, and the die protein ammo acid sequence(s) expressible therefrom.
• Figure 1 Enzyme Characteristics.
• Figure 4 depicts a sample activity profile which characte ⁇ zes and enzyme of the instant disclosure
• Graph 1 depicts the Temperature Profile ot the enzyme plotting relative esterase activity versus temperature
• Graph 2 depicts the Residual Esterase Activity of the listed enzyme plotting relative remaining activity versus ume in hours, at 25°C, 40°C, and 65°C
• Graph 3 depicts the pH profile for die listed enzyme plotung Relauve Esterase Ac vity versus pH. Data for enzymes are summa ⁇ zed in Tables 1, 2 and 10.
• Residual acuvity 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.
• Type III and Type IV substrates can be considered subsets of Types I and II, but their unique properties dictate diat they be classified separately.
• Type III molecules require that the enzyme differentiates a prochiral substrate while Type IV compounds are meso structures.
• Figure 6 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 translated protein amino acid sequence. Alternative start codons are recognized in the an, however the encoded protein will comprise at minimum a core protein ORF.
• Figure 6A is an isolated nucleic acid sequence, and translated amino acid sequence which co ⁇ espond to E001 enzyme ORF, alternative start codons are underlined.
• Figure 6B is an isolated nucleic acid sequence, and translated amino acid sequence which correspond to E009 enzyme ORF, alternative start codons are underlined.
• Figure 6C is an isolated nucleic acid sequence, and translated amino acid sequence which correspond to E01 1 enzyme ORF, alternative start codons are underlined.
• Figure 6D is an isolated nucleic acid sequence, and translated amino acid sequence which correspond to E101 enzyme ORF, alternative start codons are underlined.
• Figure 6E is an isolated nucleic acid sequence, and translated amino acid sequence which corresponds to E019 enzyme ORF, alternative start codons are underlined.
• Figure 6F is an isolated nucleic acid sequence, and translated amino acid sequence which corresponds to E005 enzyme ORF, alternative start codons are underlined.
• Figure 6G is the cloned isolated nucleic acid sequence which contams the E004 ORF, alternative start codons are underlined.
• Figure 6H is the cloned isolated nucleic acid sequence which contams the E006 ORF, alternative start codons are underlined
• Figure 61 is the cloned isolated nucleic acid sequence which contains the E008 ORF, alternative start codons are underlined.
• Figure 6J is the cloned isolated nucleic acid sequence which contams the E010 ORF, alternative start codons are underlined
• Figure 6K is the cloned isolated nucleic acid sequence which contams the E013 ORF, alternative start codons are underlined.
• Figure 6L is the cloned isolated nucleic acid sequence which contains the E015 ORF
• alternative start codons are underlined
• Figure 6M is the cloned isolated nucleic acid sequence which contams the E016 ORF
• alternative start codons are underlined
• Figure 6N is the cloned isolated nucleic acid sequence which contams the E017 ORF
• alternative start codons are underlined
• Figure 60 is the cloned isolated nucleic acid sequence which contams the E020 ORF
• alternative start codons are underlined
• Figure 6P is the cloned isolated nucleic acid sequence which contams the E027 ORF
• alternative start codons are underlined
• Figure 6Q, 6R, 6S, 6T and 6U are partial sequences
• Figure 7A is a graph of data from a colorometnc esterase assay performed on the substrate bis-p-mtrophenyl-Carbonate
• Figure 7B is data from a colorometnc esterase assay performed on the substrate: p- nitrophenyl-Acetate
• Figure 7C the substrate bis-p-mtrophenyl-Propionate
• Figure 7D the substrate: bis-p-mtrophenyl-Butyrate
• Figure 7E the substrate: bis-p-mtrophenyl- Caproate.
• Figure the substrate bis-p-nitrophenyl-Caprylate.
• Figure 7G the substrate bis-p-mtrophenyl-Laurate Note that E009 is an 80x dilution compared to the other enzymes in b, c, d, and f
• Figure 8A Entanriomer Substrate Specificity Figure 8A summanzes the results of colorometnc esterase acUvity assays for entantiomer specificity Figure 8B-D reports quantitative colorometnc assay data m terms of minutes required for detectable color change.
• the instant invention provides for isolated commercially useful protein preparations from themostable bactena which are selected for enzymatic activity, and charactenzed 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 charactenzation assays demonstrate that the esterase enzymes descnbed have a range of optimal parameters.
• ElOO and E101 have optimal operating temperatures above 70°C as would be consistent with enzymes isolated from an extreme thermophile
• E001 -E021 have optimal commercial temperatures in the range of 40-50°C as would be consistent with enzymes isolated from the more moderate thermophihc organisms. Both groups, however, provide added stability and functionality as compared to other known esterases from thermophihc bactena.
• 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 descnbed posses a vanety of pH optima including some with no apparent preference under the conditions of the expenment, however the trend for most of the proteins is to have pH optima near or slightly below neutral.
• strains - Thermus sp. T351 (ATCC 31674) is available from the Amencan Type Culture Collection (ATCC). All isolated strains and cultures are grown on TT medium 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 punfication are grown in 17 liter fermentors (LH Fermentation. Model 2000 senes 1 ).
• the fermentors have a working volume of 15 liters and cultures were grown m 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 with the stirred jars. E. coli strains are grown as descnbed in (37).
• Plates are incubated at 55°C or 65°C for one to two days and isolates then purified by numerous restreaks onto fresh plates for single colony isolation.
• the initial basis for differentiation is color, colony morphology, microscopic examination, temperature of growth, and lipase and esterase activities.
• Several hundred strains were initially isolated. 65 different microorganisms were chosen for further study.
• Esterase Plate assay - Organisms are grown in liquid cultures on TT media at either 55°C or 65°C. Cells are pelleted by centrifugation (3,000 RPM for 20 minutes) and die supernatants saved to be tested. Pellets are washed with 2 volumes of 10 mM Tris HCl pH 8.0 three times after which the cell pellets are resuspended in fresh Tris buffer and disrupted by sonication. Cell debris is removed by centrifugation and the crude extracts were tested for esterase acuvity on an esterase screening plate.
• 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 ac ⁇ vi ⁇ es) suspended in 0.7% agarose and 0. IM 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 posidve activity. Both cell extracts and culture supernatants are tested for esterase acuvity by this method.
• Isolates GP1, 27,28,29,30,31.32,34,62 appear to be thermophihc Actinomyces.
• E101 ⁇ Specific activity is the amount of p-nitrophenol produced in micromoles per minute per milligram of total protein at 40°C after purificauon to homogeneity (tor ElOO and E101) or se i-punficauon (for E001 -
• E021) as described 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 M phosphate buffer at pH 7.5 containing 200 mM KC1 and 0.1 M 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.
• DEAE Purification The protein solution is dialyzed against the resuspension buffer 3 umes 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 equilibrauon buffer and men eluted with a linear gradient from 0 to 0.5 M NaCl.
• Q Resin purification - Active tractions isolated from DEAE punficauon are pooled and dialyzed against three changes of equilibration buffer and dialysate was applied to a 50 ml bed volume of sepharose Q resm equilibrated widi the buffer above.
• the column is washed with 100 ml of 50 mM phosphate pH 6.5 containing 0.1M KC1 and 1 mM BME and then eluted with 150 ml of a KC1 gradient from 0.1 M to 0.6M added to the above buffer.
• Ultrafiltration Concentration - Active fractions are pooled and concentrated using an Amicon Ultrafiltration system fitted with a 30 Kd cut off membrane.
• Preparative SDS PAGE - Concentrated protein soluuons are loaded to a preparative 10% SDS-PAGE gel using the standard SDS loading buffer without boiling the sample. After development, die gel is treated with 0.7% agarose containing 0.1M phosphate pH 7.5 and 0.1 mg/ml 5-bromo-4-chloro- ⁇ ndoylacetate. The resulung blue band was excised from the gel, placed in dialysis tubing and die protein is recovered by electroeluuuon in 0.05M T ⁇ s buffer pH 8.5 for 1 hour. At this stage the protein is purified to homogeneity as observed by botii native- and SDS-PAGE stained with either coomassie or silver stain. Protein can be stored at 4°C for future use. Gel filtration - A gel filtrauon column can also be used as a further or substituted purification step.
• the crude cell lysate is diluted by three fold with 50 mM Tris-HCl pH 7.5 and the mate ⁇ al 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.
• 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 industnal applications
• a further purification (such as an Ammonium sulfate salt precipitation, gel filtration, or other methods as descnbed in Example 3) can be applied it necessary The process can be scaled up or down as desired
• Example 5 Method for determination of temperature profile.
• Opumal temperature profiles for an esterase protein is performed by measunng the activity of the esterase diluted into 0. IM 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 measunng the rate of hydrolysis of p-mtrophenylpropnonate added to the equilibrated solution under reaction conditions described tor determination ot specific activity in Example 2 (modified by the vanous temperatures used in this experiment)
• Control reactions that substitute bovine serum albumin for esterase enzymes aie used to allow correction for temperature dependent autohydrolysis of the substrate The data is then plotted as relative acuvity versus the temperature of the reacuon
• the long term catalytic stability the esterase enzyme is evaluated by tesung the activity remaining after exposure to vanous 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 measunng the rate of hydrolysis of p-nitrophenyl-propnonate as described above Results are plotted as relauve acuvity vs. time. The results indicate that all enzymes tested retain most of the initial activity for at least 48 hours when exposed to temperatures up to and including 40°C.
• the pH profile of an esterase is determined as follows The rate of p-mtrophenylpiop ⁇ onate hydrolysis is determined under reaction conditions similar to those described for determinauon of specific acuvity in Example 2 with buffers of wide useful pH windows that overlap with at least one data point. For the purposes of these expe ⁇ ments two buffers were selected that met the above cnte ⁇ a, Mes (useful range of 6-6.5) and Bis-t ⁇ s 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
• Expenments are run in the presence of vanous organic solvents such as ethanol, acetonit ⁇ le, dimethylformamide, dioxane, toluene, hexane and detergents like SDS, tnton XlOO and Tween 20. Additional expe ⁇ ments are also performed to test the activity of isolated catalysts in nearly anhydrous solvent conditions in which the enzymes will be lyophihzed from buffers and pH's of optimal activity
• Example 9 Method for Protein Characterization by migration on Native PAGE
• 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 laborato ⁇ es) separaung proteins based on their charge to size rauo.
• the gel shows trace contamination with other enzymes capable of lndoylacetate hydrolysis that could not be detected easily with the HPLC because of column diluuon effects. What is clear from the gel expe ⁇ ments is that most of the samples have a single major acuvity band or zone mat have similar migration characte ⁇ stics .
• the estimated nauve molecular weights tor the protein of interest is determined by separauon 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.
• lime 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.
• Substrate preference of esterases for hydrolytic activity on vanous esters can be determined as follows A gnd of molecules is prepared on microtiter plates by dissolving each substrate (0.1 M final concentration) in CH3CN and mixing with 0.1M phosphate buffer pH 7.5. Partially punfied enzymes is dien 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 relauve acuviues.
• a new metiiod 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 tiiat have pH-dependent color transiuons in the desired pH range of enzyme acuvity.
• the best indicators tested are phenol red for enzymes that function optimally at slightly elevated pHs (starting point pH 8.5) or bromothymol blue (starung point pH 7.2) for enzymes that operate well at more neutral conditions
• the indicator reacuons are monitored by one of two methods. Spectroscopic studies are performed by measunng the UV Vis maxuna of a 0.001% soluuon of either phenol red or bromothymol blue dissolved in different pH buffers at 5 mM concentration Hydrolytic reacuons are then performed by adding the substrate (0 1 mM final concentration) to a 5 mM buffer soluuon (sodium phosphate pH 7.2 for bromotiiymol 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 ot 0 1U target enzyme
• 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 die suggestion of stereoselectivity as determined from the relative rates of hydrolysis for substrate enantiomers. Control reacuons are similar to those described above in die 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 die 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).. Investigations of stereochemical preference by each esterase can be evaluated by one of two methods.
• 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 die 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. SI 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.
• EI00 Enzyme Activity - Esterase activity is measured by monitoring the hydrolysis of p-nitrophenylproprionate (pNP), or in some cases MUB.
• pNP p-nitrophenylproprionate
• Each substrate is dissolved in acetonitrile and added to the reaction mixture (100 ⁇ M final concentration) which contain 50 mM Tris HCl pH 8.5 adjusted for temperature dependent pH variation.
• Reactions are thermally equilibrated at 37°C for 5 minutes prior to initiation of the reaction by addition of 10 ⁇ L of enzyme sample, while control reactions substituted equivalent amounts of BSA.
• 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 expe ⁇ mental above except for the addition of specified components. Relative rates are corrected for the spontaneous rate of hydrolysis of the uncatalyzed reaction.
• ElOO displays a broad substrate specificity catalyzing the hydrolysis of a number of nitrophenyl, coumaryl and alkyl esters.
• the enzyme displays hydrolytic activity towards both straight chain and aromatic moieties on the carboxylate side of substrates however, carboxylate R groups of long alkyl chains >C8 or tiiose containing naphthyl leaving groups are not substrates.
• the enzyme displays no significant activity towards either casein or milk as assayed by clearing zones on agar plates.
• Structure activity assay of partially purified esterase ElOO from Thermus species (++) highest activity as determined by (a) color formation in less then 10 min D ⁇ 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 measunng the concentration dependent initial rates of enzyme catalyzed hydrolysis ot nitrophenyl prop ⁇ onate. Reactions are run at pH 8.5 in 50 mM Tns-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 dunng die course of the reacuon. Concentration vs.
• N-Term ⁇ nal Sequencing of ElOO - Punfied proteins are run on 10% SDS-PAGE gels and then transferred to PVDF membranes by electroblotung Membranes are washed witii seveial changes of doubly disulled water to remove any remaining SDS or other contaminants and tiien stained with coomassie blue. Membranes were then destained with several changes of 50:40:10 MeOH:H 0:AcOH followed by one wash of 10% MeOH. Membranes are then air dried and then submitted for sequencing. The N-terminal sequence of ElOO was determined at the University of Illinois Urbana Champaign genetic engineenng facility.
• ElOO The N-terminus of ElOO was determined by automated sequencing of the polypeptide punfied 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 me "?” refers to an indeterminate amino acid. Thus, ElOO has been demonstrated to be a useful esterase with unique activity at commercially useful punty.
• E101 is one of two esterase activities that are isolated from Thermus sp T351. E101 can be punfied away from a second esterase, ElOO, in an early punfication step. Purification ofElOl - A Thermus sp. T351 supernatant prepared as descnbed in Examples 1 and 2 is fractionated with NH 4 SO 4 and the piecipnated proteins are collected between 20- 60% saturation. Pellets are redissolved in 30 ml of buffer (50 mM Tns-HCl pH 8.0. 1 mM BME) and dialyzed against the same buffer using 30 Kd cutoff dialysis tubing.
• buffer 50 mM Tns-HCl pH 8.0. 1 mM BME
• Dialysate is loaded to 100 ml bed volume of DEAE res equilibrated with the buffer above and the column was washed with 150 ml of the equilibration buffer. Active protein is observed in the load and wash fractions, pooled, and concentrated with the use of an Amicon concentratoi 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.
• E101 can be purified over 35 fold by these methods and possesses characteristics dramaucally different from ElOO, the other esterase which is isolated from this suain Attempts to use ion exchange chromatography result in subtractive punfication since in no instance was the protein retained Resins investigated include DEAE, Q sepharose, CM cellulose, SP sepharose and hydroxyappatite under conditions that va ⁇ ed from pH 6 0 to 9.0, and buffers from phosphate to borate including Tns and Hepes After two ion exchange steps the protein is punfied 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 denatunng SDS-PAGE respectively
• El 01 Characteristics The specific activity of the enzyme is ten fold greater than observed for ElOO with 4-methyl-umbelhferyl butyiate (MUB) as the substrate.
• E101 is inhibited by PMSF but is insensitive to metal ions or metal ion chelators.
• the specific activity ot the punfied protein was found to be 3.2x10 s 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 vanous substances on E101 activity TABLE 5.
• Reaction conditions are those descnbed in the general expenmental above except for the addition of specified components. Relative rates are conected for the spontaneous rate of hydrolysis of the uncatalyzed reaction.
• Substrate specificity of El 01 - The substrate specificity of E 101 was determined as descnbed in Example 11. The results from the structure activity expenments 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.
• St cture activity assay of partially punfied 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, methylunmbelliferyl, pN, p-nitrophenyl, oN, o-nitrophenyl, PA, phenylacetate.
• 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 me 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 11910), 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/chloroforrn/iso-amyl 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 witii 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.
• One ⁇ l of the ligauon 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 Petn plates containing LB media.
• Se ⁇ al dilutions of die packaging mixture are produced in order to determine the cloning efficiency which is generally about 1.0 x 10 7 hybrid phages/ ⁇ g of cloned DNA.
• Hyb ⁇ d 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 p ⁇ mary plates are treated with indicator agar containing 5-bromo-4-chloro-3- indolyl-acetate (X- Acetate) as descnbed below, to find hybnd plaques carrying esteiase genes Screening of gene banks for esterase activity -
• the products of the above packaging reacuons are infected into E coli XL1 blue MRF' (Stratagene)
• Pnmary 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
• O t her 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
• MUP 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.
• the plates are preheated at 65 ° C for 20 minutes. Hybrid phages surviving mis 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.
• 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 E. coli esterase activities.
• Approximately two ml of 0.7% top agar (0.8% NaCl, 10 mM MgSO 4 ) 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- methyl-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 die 4-methylumbelliferyl substates).
• a typical result for this process can yield a ratio of 1: 3000 positive colonies to hybrid phages.
• a single plaque from each clone is resuspended in 20 ⁇ l of an overnight culture of E. coli XL1 Blue (grown in LB medium with the presence of 10 mM of MgSO 4 ), incubated for 20 min at 24°C in one well of a 96-well microtiter plate to allow adsorption, transferred into 15-ml test tube containing 2 ml of LB, and grown overnight at 37°C in a New Brunswick Environmental Shaking incubator set at approximately 300 rpm. Cell debris can be removed by centrifugation at 12,000 g for 10 min.
• Phage lysates from the clones are then subjected to 4- 15% gradient Native polyacrylamide gel electrophoresis (PAGE) for comparison to the native proteins purified from the original organisms.
• Precast gradient gels are purchased from BioRad Laboratories (catalog number 161-0902) and used according to the manufacturer's instructions for native gels .
• 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.
• 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 heiper phage results in excision of a multicopy 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 M13 helper phage (about 10 10 CFU) are used to infect 20 ⁇ l of an overnight culture of die E.
• coli XL1 Blue grown in LB. After 20 min at 24°C, the cell suspension is transfe ⁇ ed 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 M13 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. Preliminary experiments are performed by growing plates at 37°C to demonstrate that a significant phenotypic segregation occurs with the transductant E. coli colonies expressing cloned thermophihc esterases.
• thermophilic esterase activity is lethal or partially lethal to the host cell
• the growth temperature of the strain should be lowered to 30°C or even room temperature.
• the recombinant strains harboring plasmids with active esterase proteins often exhibited a phenotypic segregation of the esterase activity on X-acetate plates.
• coli cells are plated in a medium containing X-Acetate to detect expression of cloned esterase by the plasmid, and a degree of segregation in or between primary colonies.
• growth of the transformed cells at a temperature which reduces the activity of the cloned esterase is important to the effective isolation of productive plasmids.
• eight bacterial colonies derived from each of the phage clones are picked from the plates without X-Acetate, transfe ⁇ ed into 100 ml of LB supplemented with 40 mg/ml Km in a 96-well plate and grown overnight. 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 further 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 ot the somewhat lethal indole product ot the colonmet ⁇ c reaction.
• Colonies aie selected by their phenotype in general giving a modest growth rate and intensive blue color
• Cell pellets are resuspended in 500 ml of 0.1 M Phosphate buffer pH 7.0 and sonicated using a Somes & Mate ⁇ als Vibra Cell 375 Watt sonicator at 4°C. Somcation is performed using a microtip, 40% max capacity, 50% time pulse for 45 sec.
• Lysates are cent ⁇ fuged 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 ot plating followed by growth in liquid medium and acuvity assays are performed to verify the stability of the clones.
• Deviauons in specific esterase activity among vanants trom 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 ot stabilization This could mdicate that E. coli host mutations are being selected which allow higher tolerance of the cloned protein, rathei 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 se ⁇ es of restnction endonucleases such as ⁇ coRI. BamHI, Hindlll. PstI, ⁇ coRV, 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 pioduction clones weie determined The insert sizes tor each clone are calculated from this data and is summarized in Table 8.
• Insert sizes are esumated from the agarose gel
• the estimated insert size is based on a vector size of 4.5 kb and the accuracy which could be achieved anaiyzing each of the six digesuon patterns.
• the DNA sequences of die ends of the insert fragment carrying esterase genes can be determined by sequencing the ends of the inserts using standard T7 and S6 p ⁇ mers to produce unique tags of the cloned DNA. Sequence analysis can be earned out to design PCR p ⁇ mers 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 die enzyme of interest.
• LB 10 gm/1 tryptone. 5gm/l yeast extract and lOgm/l NaCl
• Ternfic Broth 12gm/l tryptone. 24gm/l yeast extract
• 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 mote expensive. For instance, in die 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.
• Fermentation at 30°C, 600 RPM, and 0.5 vvm air flow.
• 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 Kirtoi Production of substantially purified preparations from a cell paste of strains producing the recombinant enzymes are earned out similar to the methods descnbed in Example 4 and the specific protocols descnbed in Examples 14-34 for the native proteins.
• Acuvity Cell mass Total Acuvity Cell mass Total Growth media
• 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) ot N-methyl-N-mtro-ZV-nitrosoguanidine (MNNG)
• EMS ethylmethane sulfanoate
• MNNG ot N-methyl-N-mtro-ZV-nitrosoguanidine
• MNNG ot N-methyl-N-mtro-ZV-nitrosoguanidine
• 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 tor 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 plate screen an agar overlay containing a colo ⁇ met ⁇ c or fluorogenic substrate such as 5-bromo-4-chloro-3- ⁇ ndolyl-acetate or 4-methyulumbell ⁇ feryl acetate will be applied
• Candidate mutants are then analyzed by native polyacrylamide gel electrophoresis and compared to the parental strain. Standard assay methods descnbed in Example 2 or the rapid esterase/lipase screen descnbed 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, ve ⁇ fying that the increase is in d e enzyme of interest It is then confirmed that the mutants have unalteied kinetic and substrate properties as the parental enzyme.
• mutauons identified by dns approach are expression mutauons which can be isolated in eidier a promotei region, repressor molecule, or other controlling element. Most mutations m the enzyme structural genes will likely inacuvate the enzyme, however, an enhanced activity may also be isolated If it is apparent that the mutauon increases the acuvity of the desired protein band but not the intensity of the band on a coomassie stained gel, the mutant is lecharacte ⁇ zed 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 lyophihzed form if possible near its optimal buffer and reaction conditions.
• kits for many different formats for the kit aie possible, from a senes 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 mictotiter plates are made ot polystyrene however, which will not stand up to most organic solvents. For expenments which utdize aqueous solvent, the polystyrene is not a problem
• 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 ot a stu bai, buffer (0 IM Na phosphate pH 7 0) and 1U of each enzyme to each well of a 24 well polypropylene tray (Tomtec). Enzymes aie ahquotted into each well or vial in set amounts so that it can be assured that an equal amoun t of activity is provided for companson
• the entire kit is then lyophihzed, sealed with heat seal foil (3M) and labeled. Separate expenments found that there was no significant loss in enzyme activity when proteins were lyophihzed in the kit trays as suggested by earlier experiments compa ⁇ ng glass to plastic.
• each kit contains toui control wells that are composed of buffers at pH's trom 6-9 since it was found that some ot 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 mediod descnbed in Example 12 can also be included in each well or vial. This makes a preliminary qualitative determination of enzyme effectiveness simple and fast.
• Figure 6B is an isolated nucleic acid sequence, and translated amino acid sequence which correspond to E009 enzyme ORF, alternative start codons are underlined.
• Figure 6C is the cloned isolated nucleic acid sequence which contams the E011 ORF, alternative start codons are underlined.
• Figure 6D is the cloned isolated nucleic acid sequence which contains the ElOl ORF, alternative start codons are underlined.
• Figure 6E is the cloned isolated nucleic acid sequence which contains the E019 ORF, alternative start codons are underlined.
• Figure 6F is the cloned isolated nucleic acid sequence which contams the E005 ORF, alternative start codons are underlined
• Figure 6G is the cloned isolated nucleic acid sequence which contains the E004 ORF, alternative start codons are underlined
• Figure 6H is the cloned isolated nucleic acid sequence which contams the E006 ORF, alternative start codons are underlined
• Figure 61 is the cloned isolated nucleic acid sequence which contains the E008 ORF
• alternative start codons are underlined
• Figure 6J is the cloned isolated nucleic acid sequence which contains the E010 ORF, alternative start codons are underlined.
• Figure 6K is the cloned isolated nucleic acid sequence which contains the E013 ORF, alternative start codons are underlined.
• Figure 6L is the cloned isolated nucleic acid sequence which contains the E015 ORF, alternative start codons are underlined.
• Figure 6M is the cloned isolated nucleic acid sequence which contains the E016 ORF, alternative start codons are underlined.
• Figure 6N is the cloned isolated nucleic acid sequence which contams the E017 ORF, alternative start codons are underlined
• Figure 60 is the cloned isolated nucleic acid sequence which contains die E020 ORF, alternative start codons are underlined.
• Figure 6P is the cloned isolated nucleic acid sequence which contains the E027 ORF, alternative start codons are underlined.
• Figure 6Q contams the nucleic acid sequence of the 5' end
• Figure 6R contains the 3' end of the insert which contams the E003
• Figure 6S contains the nucleic acid sequence of the 5' end
• Figure 6T contains me 3' end of the insert which contains the E004 ORF
• Figure 6U contams the nucleic acid sequence of the 3' end of the insert which contains the E014 ORF.
• nucleic acid sequences allow one of ordinary skill in the art, practicing routine methods to complete charactenzation of the full length nucleic acid sequence of the insert, the detection of clones via hybndization, and the creation of amplification pnmers for detecting, amplifying and generating full length homologous genes Table 10.
• 'broad pH range refers to > 50% activity through all pH tested (6.0-8.5)
• the enzymes ot the instant invention can be further charactenzed by testing tot enzymatic specificty for substrate esters of different chain length. Such assays can be conducted using die methods descnbed above, selecung die appropnate substrates.
• Figure 7 depicts die result of colormetric esterase acuvity assays of the vanous enzymes.
• the graphed data was obtained where the reacuon condiuons were estimated to be approximately 0.1 U/l ml reacuon, wim 500 ug/ml substrate, where 1 Unit (U) is calculated for each enzyme stock prepara ⁇ on in relauon to esterase activity where 1 Unit is the amount of enzyme needed to hydrohze approximately 1 umol of p-nitrophenyl prop ⁇ onate per minute.
• the data is reported as approximate maximum OD4i() n m dunng incubation.
• Figure 7A graphs data using the substrate bis-p-nitrophenyl-carbonate. The highest activity was found with enzyme E019, which showed an OD4 i ⁇ nm of 0.30 atter 4 houts 5 incubauon.
• Figure 7B graphs data using the substrate p-nitrophenyl-acetate. The highest activity was found with enzyme E020, which showed an OD4i ⁇ n of 3 571 atter 400 seconds incubation.
• Figure 7C graphs data using the substrate bis-p-nitrophenyl-propionate The highest activity was found with enzyme E003, which showed an OD4i ⁇ nm of 1 4 Jt lt; ⁇ 600 seconds incubauon.
• Figure 7D graphs data using the substrate bis-p-nitrophenvl butyrate. The highest activity was found with enzyme E020, which showed an OD4iQ nm of
• Figure 7E graphs data using the substrate bis-p- mtrophenyl-caproate. The highest acuvity was found with enzyme E009, which showed an OD4i ⁇ nm of 0-37 after 560 seconds incubation.
• Figure 7F graphs data using the substrate bis-p-nitrophenyl-caprylate. The highest activity was found with enzyme E003, which showed an OD4i ⁇ nm of 0.07 after 360 seconds incubation.
• Figure 7G graphs data using the substrate bis-p-nitrophenyl-laurate. The highest activity was found with enzyme E016, which showed an OD4i ⁇ nm of 0.1 1 after 480 seconds incubauon.
• the enzymes of the invention can be further characterized by testing for enzymatic specificity for specific entanuomer substrate esters of different chiral structure. Such assays can be performed using the methods descnbed above, selecting the appropnate substrate. The results of screening are depicted in Figure 8.
• Figure 8A summanzes the results of colorometnc esterase activity assays for entamomer specificity.
• Figure 8B depicts quantita ve colorometnc assay data results in terms of minutes required for detectable color change, indicating pH change. The numbers report time in minutes following addition of enzyme. NH indicates no hydrolysis was detected after 3 days, and o/n indicates no hydrolysis after overnight incubauon (approximately 6-15 hours).
• the esterases tested were added in the amount of 1 U per well, as determined by hydrolysis of PNP-propionate.
• the control reaction was the substrate solution, widi no added enzyme.
• the enzymes of the invention can be further characterized by testing for enzymatic specificity for alternative substrates which are similar to esters. Such assays can be performed using the methods described above, selecting the approp ⁇ ate substrates.
• the enzymes of the invention were charactenzed against the ani des and esters listed below and the results depicted in Figure 9. The assays were performed according to the general formula:
• Test reactions were run in microtiter plates with each reaction in a total volume o ⁇ about 100 ul. Each reaction consisted of about 75 ul of pH7.0 phosphate buffer, 5 ul of 5mM substrate, and 20 ul of enzyme adjusted to 50 U/ml (where 1 U is approximatly the amount needed to hydrolize 1 uM of p-nitrophenyl-propionate in 1 minute). The final reaction mixture contained about 1U enzyme and 0.25 mM substrate in each well. The reactions were incubated for about 2.5 hours at 37C. Control reactions, lacking enzyme, were run in adjacent wells. A control containing no substrate was also run on each plate. Following incubation, the plates were read at 405 nm in a BIORAD Model 3550 microplate reader. Values of the controls were subtracted from the experimental well values to determine net acuvity.

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