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)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
  • C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
  • C12P41/005—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of carboxylic acid groups in the enantiomers or the inverse reaction
• • F—MECHANICAL ENGINEERING; LIGHTING; HEATING; WEAPONS; BLASTING
  • F02—COMBUSTION ENGINES; HOT-GAS OR COMBUSTION-PRODUCT ENGINE PLANTS
  • F02B—INTERNAL-COMBUSTION PISTON ENGINES; COMBUSTION ENGINES IN GENERAL
  • F02B75/00—Other engines
  • F02B75/02—Engines characterised by their cycles, e.g. six-stroke
  • F02B2075/022—Engines characterised by their cycles, e.g. six-stroke having less than six strokes per cycle
  • F02B2075/027—Engines characterised by their cycles, e.g. six-stroke having less than six strokes per cycle four

Definitions

• This invention relates to the preparation of (S)-6-methoxy- ⁇ -methyl-2-naphthaleneacetic acid by the enantioselective hydrolysis of racemic esters using microorganisms and enzymes derived therefrom.
• 6-Methoxy- ⁇ -methyl-2-napthaleneacetic acid which has the following structural formula:
• Naproxen is the USAN and INN nonproprietary name for (S)-6-methoxy- ⁇ -methyl-2-napththaleneacetic acid.
• naproxen and R,S-naproxen mean a mixture of the R- and S-enantiomers of 6-methoxy- ⁇ -methyl-2-napththaleneacetic acid, especially a racemic mixture; and "R-naproxen” and “S-naproxen” mean the two enantiomers individually.
• S-naproxen used in this application corresponds to the USAN/INN name "naproxen”.
• naproxen is an acid
• R,S-naproxen include not only the acid form of the compound, but also the anion form and pharmaceutically acceptable salts of the acid form, unless the context requires otherwise.
• intracellular esterase isolated from Pseudomonas fluorescens, which catalyzes the hydrolysis of methyl esters of short chain length.
• the intracellular esterase differs from known extracellular lipases in its sensitivity to inhibitors, molecular weight and substrate specificity.
• EP 0 153 474 describes the process of preparing S-naproxen from R,S-naproxen ester using microbial enzymes, but requires a two step hydrolysis process.
• the R,S-naproxen ester is first enantioselectively hydrolyzed to S-naproxen ester and R-naproxen with a microbial esterase, preferably from Aspergillus, and the R-naproxen separated.
• the S-naproxen ester is then nonselectively hydrolyzed by esterase from hog liver or Pleurotus ostreatus to form the desired S-naproxen.
• U.S. Patent No. 4,762,793 describes an enzymatic process in which enantioselective hydrolysis of R,S- ⁇ -arylalkanoic esters is carried out using a lipase enzyme isolated from Candida cylindracea. When used in the production of S-naproxen, this process took over two days at 32°C to convert 40% of R,S-naproxen ester to S-naproxen. Moreover, the enzyme loses about 80% of its activity over a 96 hour reaction period. (See also, EP 0 195 717).
• EP 0205 215 describes the process of preparing
• S- ⁇ -methylareneacetic acids from R,S-naproxen esters using extracellular lipases of microbial origin preferably Candida cylindracea.
• Candida cylindracea lipase required several days to convert 41% of methyl R,S-naproxen ester into S-naproxen. This rate of conversion is too slow to be suitable for a high yield, low cost industrial process.
• EP 0 328 125 describes a process for the enzymatically catalyzed enantioselective transesterification of racemic alcohols, such as (R,S)-6-methoxy- ⁇ -methyl-2-naphthaleneethanol, with an ester such as ethyl acetate, methyl acetate or methyl propionate, to afford the ester of the S-alcohol.
• racemic alcohols such as (R,S)-6-methoxy- ⁇ -methyl-2-naphthaleneethanol
• an ester such as ethyl acetate, methyl acetate or methyl propionate
• the resulting esters are said to be useful in the preparation of anti-inflammatory agents such as S-naproxen.
• Preferred enzymes are steapsin and the lipase from Pseudomonas fluorescens.
• EP 0 330 217 describes a continuous enzymatic process for the preparation of S-naproxen from an alkoxyethyl R,S-naproxen ester using a lipase isolated from Candida cylindracea.
• the enzymatic reaction gave a 37% conversion of R,S-naproxen ester at 35°C after 500 hours. This rate of conversion is too low for a high yield, low cost process.
• U.S. Patents Nos. 4,886,750 and 5,037,751 describe a process using microorganisms having the esterase ability for enantioselective hydrolysis of R,S-naproxen esters into S-naproxen having at least 60% ee.
• the patents describe an esterase that has the ability to enantioselectively hydrolyze R,S-naproxen ester into S-naproxen having at least 98.8% ee.
• the conversion of R,S-naprcxen ester to S-naproxen is limited to low substrate concentrations.
• esterases do not act in a biphasic aqueous/organic system or on insoluble R,S-naproxen ester. Moreover, the disclosed esterases require a surfactant, such as Tween ® , to be active; thereby restricting their use to a process requiring additional equipment and time to remove the surfactant.
• a surfactant such as Tween ®
• PCT/NL90/00058 describes the stabilization of esterases used to enantioselectively hydrolyze R,S-naproxen ester to S-naproxen.
• the enzymes being stabilized are disclosed in U.S. Patents Nos. 4,886,750 and 5,037,751.
• the described esterase is almost completely inactivated by the S-naproxen formed by the hydrolysis.
• these stabilizing agents include the preferred agent, formaldehyde
• these stabilizing agents are known carcinogens that must be removed by extensive processing for the product to be used in humans.
• the ability to run such a hydrolysis reaction without the need for carcinogenic stabilizing agents is a highly desirable characteristic.
• the rate of an enzymatic reaction depends on the reaction temperature.
• An enzyme exhibiting thermal stability permits running the reaction at a higher temperature which accelerates the rate, which in turn increases the production throughput.
• high temperature also drives the solid ester substrate towards its molten form, rendering the control of solid particle size less critical. It is, therefore, desirable to conduct the reaction at the highest temperature that can be tolerated by the enzyme. To this end, it is desirable to develop an enzyme that exhibits high thermal stability.
• surfactants in a manufacturing process can be quite costly and their removal requires additional process technology, equipment and labor. Also, many surfactants can be hydrolyzed by such enzymes; for example, soybean oil or Tween ® 80 are hydrolyzed by some esterases.
• hydrolysis without the need for surfactants is a highly desirable characteristic.
• a process for the production of S-naproxen comprising the enantioselective hydrolysis of A,S-naproxen ester by an ester hydrolase selected from ester hydrolases produced by a microorganism of the group Absidia griseola, Aspergillus sydowii,
• Heterocephalum aurantiacum, Pencillium roguefortii and Zopfiella latipes is described.
• a coding region of a gene encoding for an ester hydrolase capable of enantioselective hydrolysis of an A,S-naproxen ester which region comprise the nucleotide sequence as set forth in Sequence I.D. No. 2, Sequence I.D. No. 5, Sequence I.D.
• an ester hydrolase capable of the enantioselective hydrolysis of an A,S-naproxen ester to S-naproxen wherein said ester hydrolase hydrolyzes the reaction of R,S-naproxen ester at a temperature range from about 35°C to about 65°C is described.
• ester hydrolase capable of the enantioselective hydrolysis of ethyl R,S-naproxen ester to S-naproxen, which ester hydrolase comprises an amino acid sequence as set forth in Sequence I.D. No. 3, Sequence I.D. No. 6,
• an ester hydrolase capable of the enantioselective hydrolysis of n-propyl R,S-naproxen ester to S-naproxen, which ester hydrolase comprises an amino acid sequence as set forth in Sequence I.D. No. 3, Sequence I.D. No. 6, Sequence I.D. No. 9, Sequence I.D. No. 12 or Sequence I.D. No. 15.
• Figure 1(a) is a diagram illustrating cDNA synthesis for the ester hydrolase gene in E. coli.
• Figure 1(b) shows the construction of the yeast expression plasmid for the ester hydrolase gene.
• Figure 2 shows the degenerate oligonucleotide primers based on the partial amino acid sequences determined for the first 20 amino acids at the N-terminus as well as the four internal cyanogen bromide cleaved fragments of the Zopfiella ester hydrolase.
• Figure 3 shows the nucleotide junction sequences and the inferred amino acid sequences between the Zopfiella cDNA and the plasmid vector.
• Figure 4 shows the enhanced thermal tolerance of rec 780-m10 over rec 780.
• Figure 5 is a schematic flowsheet for an immobilized Zopfiella bioreactor system.
• Seq. I.D. No. 1 fusion protein sequence of the gene for ester hydrolase from Zopfiella rec 511 gene.
• Seq. I.D. No. 2 nucleotide sequence of the coding region of the gene for ester hydrolase from Zopfiella rec 511 gene
• Seq. I.D. No. 3 inferred amino acid sequence of the coding region of the gene for ester hydrolase from Zopfiella rec 511 gene. Seq. I.D. No. 4 fusion protein sequence of the gene for ester hydrolase from Zopfiella rec 780 gene.
• Seq. I.D. No. 5 nucleotide sequence of the coding region of the gene for ester hydrolase from Zopfiella rec 780 gene
• Seq. I.D. No. 7 fusion protein sequence of the gene for ester hydrolase from Zopfiella rec 780-m10 gene.
• Seq. I.D. No. 9 inferred amino acid sequence of the coding region of the gene for ester hydrolase from Zopfiella rec 780-m10 gene.
• Seq. I.D. No. 10 fusion protein sequence of the gene for ester hydrolase from Zopfiella rec 780-165 gene.
• Seq. I.D. No. 11 nucleotide sequence of the coding region of the gene for ester hydrolase from Zopfiella rec 780-m165 gene.
• Seq. I.D. No. 12 inferred amino acid sequence of the coding region of the gene for ester hydrolase from Zopfiella rec 780-m165 gene.
• Seq. I.D. No. 13 fusion protein sequence of the gene for ester hydrolase from Zopfiella rec 780-m210 gene.
• Seq. I.D. No. 14 nucleotide sequence of the coding region of the gene for ester hydrolase from Zopfiella rec 780-m165r210 gene.
• Seq. I.D. No. 15 inferred amino acid sequence of the coding region of the gene for ester hydrolase from Zopfiella rec 780-m165r210 gene.
• Figure 4
• the present invention relates to a process for producing S-naproxen by presenting R,S-naproxen ester to the action of an ester hydrolase isolated from a microorganism selected from the group Absidia griseola, Aspergillus sydowii, Doratomyces stemonitis, Eupenicillium baarnenses, Graphium sp. , Heterocephalum aurantiacum, Pencillium roguefortii and Zopfiella latipes to enantioselectively catalyze the hydrolysis of
• this invention relates to the screening of a panel of microorganisms in order to identify a microorganism that produces an ester hydrolase of use in the high yield, low cost production of S-naproxen.
• the gene for the native enzyme is cloned and expressed in a suitable host.
• the recombinant enzyme is then used in the high yield, low cost production of S-naproxen.
• the Zopfiella latipes hereinafter
• this invention relates to a high yield, low cost process for the production of S-naproxen.
• R,S-naproxen ester or “racemic naproxen ester” mean a mixture of the R- and S-enantiomers of varying or equal ratios of an ester of 6-methoxy- ⁇ -methyl-2-naphthaleneacetic acid.
• R,S-naproxen ester is defined by the following formula: where R is alkyl, cycloalkyl, aralkyl or aryl. Preferably, R is lower alkyl, and more preferably R is ethyl or n-propyl.
• alkyl refers to both straight and branched chain alkyl groups having total of 1 to 12 carbon atoms, thus including primary, secondary and tertiary alkyl groups.
• Typical alkyls include, for example, methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, n-amyl, n-hexyl and the like.
• “Lower alkyl” refers to alkyl groups having 1 to 4 carbon atoms.
• Typical lower alkyls include, for example, methyl, ethyl, n-propyl and the like.
• Cycloalkyl refers to cyclic hydrocarbon groups having from 3 to 12 carbon atoms such as, for example, cyclopropyl, cyclopentyl, cyclohexyl, and the like.
• Lower cycloalkyl refers to cycloalkyl groups having 3 to 6 carbon atoms.
• Aryl refers to a monovalent unsaturated aromatic carbocyclic radical having a single ring (e.g., phenyl) or two condensed rings (e.g., naphthyl).
• Alkyl refers to an aryl substituted alkyl group, such as, for example, benzyl or phenethyl.
• An alkyl, cycloalkyl, aryl or aralkyl group can be optionally substituted with one or more non-interfering electron-withdrawing substituents, for example, halo, nitro, cyano, phenyl, hydroxy, alkoxy, alkylthio, or -C(O)R 1 wherein R 1 is lower alkyl, lower cycloalkyl, hydroxy, alkoxy, cycloalkoxy, phenoxy, benzyloxy, NR 2 R 3 (in which R 2 and R 3 are independently H, lower alkyl, lower cycloalkyl, or jointly form a 5- or 6-membered ring together with the nitrogen, the ring optionally including a hetero group selected from O, NH, or N-(lower alkyl)), or -OM wherein M is an alkali metal.
• non-interfering characterizes the substituents as not adversely affecting any reactions to be performed in accordance with the process of this
• Halo refers to iodo, bromo, chloro and fluoro.
• Alkoxy refers to the group having the formula -OR 4 , wherein R 4 is lower alkyl, as defined above. Typical alkoxy groups include, for example, methoxy, ethoxy, t-butoxy and the like.
• Alkylthio refers to the group having the formula -SR 5 , wherein R 5 is lower alkyl, as defined above. Typical alkylthio groups include, for example, thiomethyl, thioethyl and the like.
• Cycloalkoxy refers to the group having the formula -OR 6 , wherein R 6 is lower cycloalkyl, as defined above. Typical cycloalkoxy groups include, for example, cyclopropyloxy, cyclopentyloxy, cyclohexyloxy, and the like.
• Alkali metal refers to sodium, potassium, lithium and cesium.
• the electron-withdrawing substituents are preferably at the ⁇ - or ⁇ - position of the R group, to the extent consistent with the stability of the group.
• activated esters electron-withdrawing substituents are referred to as activated esters, since they generally hydrolyze more rapidly than those where the R group is not so substituted.
• alkyl groups are methyl, ethyl, n-propyl, t-butyl, n-hexyl, i-octyl, n-dodecyl, benzyl, 2-chloroethyl,
• Organic solvents includes solvents such as methanol, ethanol, acetic acid, methylene chloride, chloroform, tetrahydrofuran,
• Base refers to bases such as alkali metal hydroxides, alkali metal alkoxides, alkali metal hydrides, alkali metal di(lower alkyl)amines, alkali metal acetates, alkali metal bicarbonates, alkali metal, tri(lower alkyl)amines, and the like, for example, potassium hydroxide, sodium hydroxide, potassium methoxide, sodium carbonate, sodium salt of diethyl amine, sodium acetate, potassium bicarbonate, and the like.
• a “resolving agent” is an optical isomer of a chiral amine base such as ⁇ -methylbenzylamine, cinchonidine, cinchonine, quinine, quinidine, strychnine, brucine, morphine, ⁇ -phenylethylamine, arginine,
• dehydroabietylamine 2-amino-1-propanol
• amphetamine glucosamine
• conessine anabasine
• ephedrine ephedrine
• MeNPR Metal naproxen ester
• EtNPR methyl naproxen ester
• n-Propyl naproxen ester or “n-PrNPR” refers to the compound of Formula I when R is n-propyl.
• S-p-nitrophenyl ester or "S-PEN” refers to the compound of Formula I when R is p-nitrophenyl.
• Isolation and purification of the compounds and intermediates described herein can be effected, if desired, by any suitable separation or purification procedure such as, for example, filtration, extraction, crystallization, column chromatography, thin-layer chromatography, thick-layer (preparative) chromatography, distillation, or a combination of these procedures.
• suitable separation and purification procedures can be had by references to the examples herein.
• Recombinant enzyme refers to an ester hydrolase obtained by the cloning of a Zopfiella ester hydrolase gene into a suitable expression system. Whether used in the singular or plural form, “recombinant enzyme” refers to these particularly defined ester hydrolase enzymes, either as a group or individually.
• the identifier "rec” will be used with the name of the clone, for example, rec 511 refers to the recombinant enzyme from
• Fusion protein means a fusion protein of the recombinant ester hydrolase having a sequence expressed as a fusion between all or a portion of the sequence for the Zopfiella ester hydrolase and a heterologous protein.
• Regulatory region means the expression control sequence, for example, a promoter and ribosome binding site, necessary for transcription and translation.
• Stability means the retention of enzymatic activity under defined reaction conditions.
• High Thermal Stability refers to a temperature of about 10oC above T1 ⁇ 2, where T1 ⁇ 2 is defined as the temperature at which one-half of the enzymatic activity of a reference ester hydrolase is lost within one hour.
• Enantiomeric excess or "ee” means the excess of one enantiomer over the other in a mixture of two enantiomers, such as in the product of an enantioselective reaction; and is typically expressed as a percentage.
• the %ee of the S-naproxen reaction product refers to the percentage of S-naproxen present minus the percentage of R-naproxen present.
• Conversion in the enantioselective hydrolysis of R,S-naproxen ester to S-naproxen, means the ratio of S-naproxen produced to the initial R,S-naproxen ester present in a reaction mixture in a given time, and is usually expressed as a percentage.
• KNPR means the potassium salt of S-naproxen.
• microorganisms that produce the enzymes of this invention were discovered after selecting over 600 Class 1, i.e. non-pathogenic, microorganisms for screening.
• the panel of microorganisms screened included 284 fungi, 180 true bacteria, 69 yeasts, 51 filamentous bacteria, 8 algae and 16 unclassified strains.
• the microorganisms were obtained from the American Type Culture Collection ( "ATCC" ) .
• ATCC American Type Culture Collection
• Of the 600 carefully selected microorganisms only eleven demonstrated the level of enzyme activity suitable for the enantioselective conversion of R,S-naproxen esters to S-naproxen with at least a 95% ee for use in the high yield, low cost production of S-naproxen.
• the eleven microorganisms identified appear in Table 1. TABLE 1
• the dehydrated microorganism is rehydrated and plated out to assess growth and purity of the transported culture. If the microorganism passes a visual inspection for purity, the microorganism is transferred onto slants of a culture medium for initial growth.
• the microorganisms can be kept on agar slants, in 50% glycerol at -20oC or lyophilized.
• the culture media used contain an assimilable carbon source, for example glucose, lactate, sucrose and the like; a nitrogen source, for example ammonium sulphate, ammonium nitrate, ammonium chloride and the like; with an agent for an organic nutrient source, for example yeast extract, malt extract, peptone, meat extract and the like; and an inorganic nutrient source, for example phosphate, magnesium, potassium, zinc, iron and other metals in trace amounts.
• an assimilable carbon source for example glucose, lactate, sucrose and the like
• a nitrogen source for example ammonium sulphate, ammonium nitrate, ammonium chloride and the like
• an agent for an organic nutrient source for example yeast extract, malt extract, peptone, meat extract and the like
• an inorganic nutrient source for example phosphate, magnesium, potassium, zinc, iron and other metals in trace amounts.
• the preferred medium for a particular microorganism is defined by the slant with the best growth and is used for the liquid culture and assay. Following identification of the preferred medium for liquid culture and assay, a 5% (v/v) inoculum was grown in the defined medium for 24-48 hours, depending on the growth rate of the organism.
• a preferred growth medium for Absidia griseola is 28
• Aspergillus sydowii is 325
• Doratomyces stemonitis is 323
• Eupenicillium baarnenses is 28,
• Graphium sp. is 323, Heterocephalum aurantiacum is 28, Penicillium roguefortii is 325, Zopfiella latipes(ATCC #22015 and #44575) is 200 and Zopfiella latipes(ATCC #26183) is 325.
• the descriptions of these media can be found in R. Cote, ATCC Media Handbook, First Edition, 1984, which is incorporated by reference.
• a temperature between about 10°C and about 40°C and a pH between 4 and 10 is maintained during the growth of the microorganism.
• the microorganisms are grown at a temperature between about 23°C and about 36°C and at a pH between 5 and 9.
• microorganisms can be provided according to any of the well-established procedures, provided that the supply of oxygen is sufficient to meet the metabolic requirement of the microorganisms. This is most conveniently achieved by exposing the slant to air.
• the microorganisms might be in a growing stage using a culture medium, as described above, or might be preserved in any system (buffer or medium) preventing degradation of enzymes.
• an ordinary culture medium as described above, can be used.
• the preferred medium for the enantioselective hydrolysis of R,S-naproxen ester with a particular microorganism is the preferred medium used for growth of that
• microorganisms can be kept in the non-growing stage, for example, by exclusion of the assimilable carbon source or by exclusion of the nitrogen source.
• a preferred storage medium for Absidia griseola is 336, for Aspergillus sydowii(ATCC #1017) is 312, for Aspergillus
• Heterocephalum aurantiacum is 325, for Penicillium roguefortii is 336, for Zopfiella latipes(ATCC #22015 and #44575) is 200 and for Zopfiella latipes(ATCC #26183) is 340.
• the descriptions of these media can be found in ATCC Media Handbook, supra. A temperature between about 10°C and about 40°C and a pH between about 4 and 9 is maintained during the storage.
• a temperature between about 10°C and about 40°C and a pH between about 4 and 10 is maintained during the assay of the enantioselective hydrolysis of A,S-naproxen ester.
• the microorganisms are kept at a temperature between about 23°C and about 36°C and at a pH between 5 and 9.
• the aerobic conditions required during the assay can be provided according to any of the well-established procedures, provided that the supply of oxygen is sufficient to meet the metabolic requirement of the microorganisms. This is most conveniently achieved by supplying oxygen, suitably in the form of air by agitating the reaction liquid.
• Racemic naproxen ester preferably lower alkyl naproxen ester
• a sterile organic solvent preferably sterile soybean oil
• aqueous culture medium containing the microorganism is added the R,S-naproxen ester solution to obtain a concentration of 0.20-0.30 mg/ml, most preferably 0.25 mg/ml.
• Aliquots are removed from the mixture at defined intervals from duplicate cultures.
• the processing is preferably done by robotic sample preparation. Processing of the samples includes extraction into an organic solvent, preferably ethyl acetate, centrifugation, sampling of the organic layer and evaporation. The sample is then derivatized with a resolving agent, preferably (S)- ⁇ -methylbenzylamine, to form
• the amides are then dissolved in the desired solvent, preferably a mixture of acetonitrile/water, for HPLC analysis to assess S-naproxen concentration and enantioselectivity in the hydrolysis of racemic naproxen esters.
• desired solvent preferably a mixture of acetonitrile/water
• Isolated standards are run of R,S-naproxen, S-naproxen and the media in which the assay is run. Each sample can be run in duplicate. Standard organisms can also be run to check on the reproducibility of the analysis.
• a recombinant enzyme is obtained by isolating the ester hydrolase enzyme from a suitable microorganism, e.g., Absidia griseola, Aspergillus sydowii, Doratomyces stemonitis,
• a suitable microorganism e.g., Absidia griseola, Aspergillus sydowii, Doratomyces stemonitis,
• Pencillium roguefortii or Zopfiella latipes preferably from a Zopfiella strain, more preferably Zopfiella Strain 780: ATCC #44575, determining the amino acid sequence of the isolated enzyme and thereafter cloning and expressing the recombinant enzyme using an E. coli, yeast, such as
• Saccharomyces cerevisiae or other expression system.
• Other expression systems that can be used include Bacillus subtilis, Aspergillus niger, and Pichia pastoris.
• the E. coli bacterium is used for the production of the recombinant enzyme. Cloning and expression can be obtained rapidly in E. coli and high levels of gene expression are common. In addition, production in E. coli results in a system easily scaled up for large scale fermentation and protein purification.
• the ester hydrolase from the microorganism can be isolated and purified by standard techniques.
• the enzyme is purified by a straight forward series of purification steps, i.e. cell disruption, ammonium sulfate precipitation, gel filtration, anion exchange
• the purified enzyme is then used for determination of the internal amino acid sequence.
• the internal amino acid sequence of the enzyme is determined by CNBr cleavage and the isolation of the resulting polypeptide fragments, preferably by reverse phase-HPLC.
• the results provide a partial amino acid sequence (first 10 amino acids at the N-terminal), which aids in the cloning of the ester hydrolase gene.
• E. coli promoters in addition to the lac promoter which may be used in the practice of the invention include, for example, trp, tac, lambda P L , lambda P R and T7 phage promoter.
• microorganism can be carried out using standard molecular cloning techniques as described in Maniatis, et.al. Molecular Cloning: A
• Double stranded cDNA may be prepared from mRNAs isolated from the microorganism in the manner more completely described in Examples 4 and 6.
• the cDNA is ligated with ⁇ gt-11 phage DNA suitable for use as a cloning plasmid. After in vitro packaging, the recombinant phage DNA is used to infect an E. coli strain devoid of detectable basal enzyme activity.
• Plasmid DNA is prepared as more fully described in Examples 4 and 6.
• a gene encoding the recombinant enzyme is identified from a cDNA library prepared from microorganism by DNA hybridization with polymerase chain reaction (PCR) generated probes using oligonucleotide primers that were based on the partial amino acid sequence previously determined for that microorganism's native enzyme.
• PCR polymerase chain reaction
• a subset of the clones, which hybridized positively with the PCR generated probes, should also test positive in the agar-overlay esterase/lipase activity assay as described in Higerd and Spizizen, J.Bacteriol.. 114:1184(1973), which is
• pGEM-13Zf(+) Promega Corp., Madison, WI.
• the resulting fusion protein (Seq. I.D. No. 1 and 4) is expressed in high levels in an overnight fermentation of E. coli.
• the correct identity of a recombinant ester hydrolase gene can be confirmed by determining the DNA sequence of the insert and comparing its inferred amino acid sequence with that previously partially determined for the purified enzyme.
• the cloned enzyme upon comparison with the natural enzyme isolated from the microorganism, should show identical substrate preference for the S- versus the R-naproxen ester.
• the expression of the ester hydrolase gene in E. coli is driven by the lac P-O promoter of the lac operon.
• IPTG isopropylthio-galactoside
• Strain 780 enzyme (Seq. I.D. No. 5). Activity staining of a recombinant enzyme of this invention, preferably a Zopfiella recombinant enzyme, and other commercially available enzymes in a non-denaturing system verifies the enzyme activity of the recombinant enzyme while indicating that the enzymes of the invention are distinctly different from known commercially available enzymes.
• A,S-naproxen esters a subset of the positive recombinant clones from a microorganism selected from the group Absidia griseola, Aspergillus sydowii, Doratomyces stemonitis, Eupenicillium baarnenses, Graphium sp. , Heterocephalum aurantiacum, Pencillium roguefortii and Zopfiella latipes, preferably Zopfiella latipes, shows activity and evidences enantioselectivity.
• the enantioselectivity of a recombinant enzyme is confirmed by analyzing hydrolysis products of racemic mixtures of R,S-naproxen esters.
• the enantioselectivity of the recombinant enzyme from Zopfiella Strain 511 was confirmed by analyzing hydrolysis products of racemic mixtures of methyl, ethyl, and n-propyl naproxen esters.
• the rec 511 Zopfiella Strain 511
• enantioselectivity of the recombinant enzyme from Zopfiella Strain 780 was confirmed by analyzing hydrolysis products of racemic mixtures of methyl, ethyl, and n-propyl naproxen esters.
• Table 2 shows that the enantioselectivity of the ester hydrolase enzyme isolated from the Zopfiella Strain 780 (the "780 enzyme”), the ester hydrolase enzyme isolated from the Zopfiella Strain 511 (the “511 enzyme”), the rec 511 enzyme, rec 780 enzyme and rec 780-m165r210 all yield an average ee of greater than 99%.
• rec 511, rec 780 and rec 780-m165r210 enzymes compare favorably with the native enzymes on the basis of their ability to hydrolyze a broad range of naproxen esters with a high enantioselectivity.
• the Zopfiella enzymes both native and recombinant have been found to be less sensitive towards S-naproxen inactivation than other untreated commercial enzymes.
• Strain 780 enzyme was shown to be more stable than Strain 511 enzyme towards KNPR
• a non-ionic stabilizer preferably bovine serum albumin ("BSA") or polyethylene glycol (“PEG”), more preferably PEG 8000, optionally can be added to the reaction mixture containing Zopfiella enzymes (both native and recombinant).
• BSA bovine serum albumin
• PEG polyethylene glycol
• surfactants such as Tween or soybean oil, did not stabilize the Zopfiella enzymes from inactivation by naproxen and formaldehyde treatment did not stabilize the Zopfiella enzymes from inactivation by naproxen.
• a non-ionic stabilizer preferably PEG or BSA
• a concentration of about 0.05% to about 2.0%, preferably to a concentration of about 0.2% has a stabilizing effect, nearly doubling the half-life of enzymes denatured by S-naproxen.
• a non-ionic stabilizer preferably PEG or BSA
• Table 3 the addition of a non-ionic stabilizer is not required for the Zopfiella ester hydrolase to enantioselectively hydrolyze
• Mutagenesis experiments, as described herein, have been carried out with rec 780 enzyme in order to minimize the extent of inactivation by S-naproxen. Inactivation by S-naproxen is also a highly temperature-dependent process. Mutagenesis experiments, as described herein, have also been carried out for rec 780 enzyme in order to minimize the extent of thermal denaturation.
• Chemical mutagenesis for example, nitrous acid mutagenesis and hydroxylaraine mutagenesis, and other forms of mutagenesis can be employed, including site-directed mutagenesis (Smith, Ann. Rev. Genet, 19:423
• ester hydrolase recovered from a mutant with high thermal stability (“rec 780-m10”) was purified and sequenced to determine the genotype changes. At position 443 of rec 780 the threonine was changed to an isoleucine to give the rec 780-m10 enzyme (Seq. I.D. No. 8 and 9). This specific change gave high thermal tolerance of the rec 780-m10 enzyme as shown in Figure 4.
• ester hydrolase recovered from a mutant with improved stability to S-naproxen (“rec 780-m165”) was purified and sequenced to determine the genotype changes.
• the threonine was changed to alanine
• the alanine was changed to threonine
• at position 133 the lysine was changed to arginine
• at position 330 the valine was changed to leucine
• at position 400 the threonine was changed to isoleucine in the rec 780 enzyme (Seq. I.D. No. 11 and 12).
• these specific changes were those which gave the rec 780-m165 enzyme high tolerance to KNPR inactivation as shown in Table 4, Example 10.
• Ester hydrolase recovered from a mutant (identified as rec 780-m165r210) with improved stability to KNPR was purified and sequenced to determine the genotype.
• the threonine was changed to alanine
• the alanine was changed to threonine
• at position 133 the lysine was changed to arginine
• at position 210 the serine was changed to arginine
• at position 330 the valine was changed to leucine
• these specific changes were those which gave the rec 780-m165r210 enzyme high resistance to KNPR
• the recombinant enzymes of the invention are especially suitable for use in the high yield, low cost production of S-naproxen.
• the recombinant enzymes can be purified from an E. coli or a yeast culture using various standard protein purification techniques, for example, affinity, ion exchange, size exclusion or hydrophobic interaction chromatography.
• An exemplary preparation and purification scheme comprises 1) growing the transformed E. coli cells in LB broth and inducing with IPTG; 2) harvesting the culture by centrifugation; 3) resuspending the cell pellet in buffer followed by cell disruption; 4) centrifuging the cell lysate; and 5) purifying the soluble enzyme by passage of the cell lysate over a two-step chromatographic column.
• Immobilized Enzymes Introduction and Application in Biotechnology, John Wiley, Chichester, UK, (1980)).
• immobilize cells which contain the enzyme, thereby indirectly immobilizing the enzyme.
• Such techniques are well known in the art and are described, e.g. Wood, L.L. and Calton, G.J.,"A Novel Method of Immobilization and Its Use in Aspartic Acid Production,” Biotechnology, 12:1081 (1984).
• an ester hydrolase as purified enzyme from the microorganism or as a recombinant enzyme
• the Zopfiella enzyme in the production of S-naproxen can be carried out in many formats.
• the enzyme can be added into a continuous stirred tank reactor.
• it can be immobilized onto matrixes either as immobilized enzyme or as host cells containing the enzyme.
• the enzyme is immobilized on a solid support.
• the immobilization is carried out by glutaraldehyde binding of the recombinant enzyme to an inert substance, such as silica or the like.
• the inert substance is Manville Celite ® R-648, R-649 or R-685.
• Figure 5 shows a schematic of the immobilization process and a description of the immobilization procedure for the isolated enzyme is set forth in Examples 11 and 12. The items identified in Figure 5 are as follows:
• host cells such as E. coli, preferably E. coli Strain JM109 or Strain BL 21 DE 3 , that express the recombinant ester hydrolase gene are immobilized without isolating the enzyme.
• the use of whole cells is less expensive and time-consuming than the use of isolated enzyme.
• the rate of hydrolysis may be stimulated by a biphasic system having organic solvents at about 5% - about 40%(v/v), preferably about 20% to about 25% (v/v).
• hexane or toluene is used.
• DMSO may also be used in a monophase system.
• Example 13 sets forth a description of the intact cell immobilization procedure.
• Table 9 shows the activity of intact E. coli carrying rec 780-m165r210 immobilized with Polymer 1195 and Polyazetidine.
• a reactor configuration for using immobilized enzyme to hydrolyze R,S-naproxen esters to S-naproxen in an organic/aqueous solution is in theory, relatively easy to operate. In practice, however, the
• the R,S-naproxen ester concentration is approximately 100-500 g/1 in the organic phase and the relative amounts of organic and aqueous phase are approximately 3:1.
• A,S-naproxen ester preferably a lower alkyl ester, more preferably ethyl or n-propyl naproxen ester
• A,S-naproxen ester is introduced continuously into the reactor as a slurry, preferably 50-250 gm per liter.
• a non-ionic surfactant preferably PEG
• PEG poly(ethylene glycol)
• the actual residence time of the enzyme in the enzyme reactor will depend on the substrate infusion rate, the removal rate of the final product and the reaction volume. Preferably the residence time is 12-36 hours.
• the enzymatic hydrolysis can be conducted in a continuous or batch mode.
• the reaction is generally carried out at the temperature range between about 30°C and about 65°C, preferably between about 40°C and about 55°C.
• the incubation temperature should be between about 40°C and about 55°C.
• a feed reservoir contains water as the aqueous phase and R,S-naproxen ester dissolved in an organic solvent, preferably in an aliphatic solvent.
• the solvent should have a normal boiling point equal to or greater than water, such as heptane, octane, decane, and dodecane.
• the preferred solvent is heptane. This biphasic mixture is agitated to keep the phases well mixed.
• the biphasic mixture is fed to the hydrolysis reactor where the S-naproxen ester in the organic phase is hydrolyzed to s-naproxen.
• the S-naproxen then transfers to aqueous phase and both phases return to the feed reservoir.
• a base preferably an alkali metal salt, more preferably potassium hydroxide, is added to the feed reservoir to maintain a constant pH of 6-10, preferably 8.0-9.5.
• R,S-naproxen ester, and organic solvent can be continuously added to the feed reservoir while a portion of the reactor effluent is withdrawn from the system and sent to product recovery, wherein a phase separation technique is used to separate the S- naproxen (KNPR) from any R-naproxen ester and unhydrolyzed S-naproxen ester.
• KNPR S- naproxen
• the use of a lower alkyl naproxen ester is preferable.
• ethyl or n-propyl naproxen ester is more preferable in the hydrolysis reaction of the invention.
• the use of the ethyl ester results in the highest enantioselectively as shown in Example 9, Table 2 and the n-propyl ester is an oil at low temperatures, allowing greater freedom in the design of a hydrolysis bioreactor.
• Ethylene glycol based esters such as the ethoxyethyl ester, can also be used, as can other esters previously described in the specification.
• S-naproxen the product of the ester hydrolysis, is preferably removed from the process stream by passing through a series of filtration membranes that have different and specific molecular weight cut-offs.
• the final product can then be further purified by crystallization.
• Potential impurities such as, naproxen esters, ester hydrolase, proteins, DNA associated with production of the ester hydrolase, etc.
• impurities such as, naproxen esters, ester hydrolase, proteins, DNA associated with production of the ester hydrolase, etc.
• the unreacted R-naproxen ester, as well as any residual S-naproxen ester, can be recycled through a separate reactor in which both are racemized chemically.
• the resultant 50-50 racemic mixture of naproxen ester, as well as fresh R,S-naproxen ester, can again be introduced into the bioreactors and the processing cycle repeated.
• the dehydrated Zopfiella(ATCC# 26183) microorganism purchased from ATCC, was rehydrated and plated out on medium 325 to assess growth and purity of the culture by visual inspection.
• the medium is described in R. Cote, ATCC Media Handbook, First Edition, 1984, which is incorporated by reference.
• Zopfiella was then transferred to slants of medium 340 for initial growth.
• Several media were also screened at this point in an attempt to define the optimum liquid medium in which to conduct the enzymatic assay. The preferred medium was determined by the slant with the beet growth and was used for the liquid culture and assay.
• a 5% (v/v) inoculum was then added to 25 ml of medium 200 for the assay.
• 25 ⁇ l of a suspension of 2.5g racemic naproxen ethyl ester in 10 ml sterile soybean oil was added to the medium to a final concentration of 0.25 mg/ml. This mixture was then agitated at 150 r.p.m. at approximately 25oC for 48 hours.
• Processing consisted of extraction into ethyl acetate, centrifugation, sampling of the ethyl acetate layer, evaporation, derivatization with (S)- ⁇ -methylbenzylamine to form the diastereomeric amides and dissolution in a mixture of 80% acetonitrile and 20% water for liquid chromatography.
• the sample containing 10 ⁇ g/ml was then assessed for KNPR concentration and enantioselectivity by HPLC analysis (Hypersil, 3 micron, 4.6 ⁇ 100 mm, C-18 or equivalent, UV at 235 nm, 0.2 alssd).
• the dehydrated Absidia griseola microorganism purchased from ATCC, was rehydrated and plated out on medium 325 to assess growth and purity of the transported culture by visual inspection and for determination of the optimum culture medium using the procedure essentially as described in Example 1(a).
• Absidia griseola the S-amide peak was 28.7 unite and the R-amide was 0.4 unite with an ee of 97%.
• Isolated standards were also run of R,S- naproxen, S-naproxen and medium 28.
• Enzyme from Zopfiella (ATCC #26183: Strain 511) was prepared from 3-day cultures by cell lysis in a bead beater, removal of cell wall debris by centrifugation, and concentration by ammonium sulfate precipitation (40-60% saturation). The pellet was redissolved in 10 ml of 20 mM
• Trie HCl/1 mM EDTA pH 8 buffer loaded on and eluted from a Sephacryl HR300 gel filtration column (Pharmacia, Piscataway, NJ) with 50 mM Tris HCl/1 mM EDTA pH 8.
• Enzyme was then adsorbed to a 2.5 ⁇ 20 cm column of anion exchange Q-Sepharose-Fast Flow (Pharmacia, Piscataway, NJ) equilibrated in 20 mM Trie HCl/1 ⁇ M EDTA pH 8, and was eluted in a 0.35 - 0.5 M NaCl gradient. Following elution, hydrophobic interaction chromatography was used as the next purification step.
• Enzyme was adsorbed to a 1.6 ⁇ 20 cm column of Phenyl Sepharose (Pharmacia, Piscataway, NJ) equilibrated in 0.2 M NaCl/20 mM Tris HCl/1 mM EDTA pH 8, and was eluted with a 0-50% ethylene glycol gradient in 10 mM Tris HCl/1 mM EDTA pH 7.5 buffer. The activity peak was pooled and was concentrated 40-fold in a Centriprep 30 (Amicon ultrafiltration devices, 30 kD cut off).
• Activity gels showed a single, active protein band that corresponded to the protein on the SDS-PAGE gels at a molecular weight of approximately
• Identification of the N-terminal sequence and the internal sequences from four peptide fragments provided sufficient information for the design of oligonucleotide probes for screening a cDNA library prepared from the Strain 511 gene. Construction of the cDNA library was as described in Example 4. The internal amino acid sequence of the Strain 511 enzyme was determined by CNBr cleavage and the isolation of the nine resulting polypeptide fragments by reverse phase-HPLC. In particular, a purified preparation of the enzyme was electrophoresed on a SDS-polyacrylamide gel.
• the resolved protein band(s) were then electro-blotted onto an Immobulon filter (Millipore Corporation, Medford, MA) and the protein band of interest was cut out and subjected to the standard micro-sequencing technique as described in Matsudira, J. Biol . Chem. 262 :10035 (1987), which is incorporated by reference.
• the products were then analyzed on an automated gas-phase microsequentor (Applied Biosystem Inc., Foster City, CA) using the methods as described by Hunkapellier et al. , Meth. Enz. ,
• Zopfiella (ATCC #26183: Strain 511) was propagated in YM broth using sheared glass broken mycelia as seed to provide uniform growth. The mycelia were harvested by filtering through sterile gauze after 2-3 days of growth and prior to asci formation according to the methods of Davis and DeSerres, Methods of Enzymology, Vol.17a (1970) and Weigel et al . , J. of Bacteriol . , 170 (9 ):3187 (1988), which are incorporated by reference. 2. mRNA preparation
• mRNA was prepared according to Chirgwin, Biochem. , 18: 5294
• Frozen Zopfiella cells were resuspended in buffer containing 4 M guanidinium thiocyanate, 0.5% sodium N-laurylsarcosine, 25 mM sodium citrate and 0.1 mM ⁇ -mercaptoethanol.
• the suspension was Polytron (Brinkmann Instruments Inc., Westbury, N.Y.) treated twice at 30 seconds each.
• the lysate was repeatedly drawn into a hypodermic syringe fitted with a 18 gauge needle and then expelled into polypropylene tubes. This was repeated 10 times to shear the cellular DNA.
• RNA pellet was resuspended in 10 mM Trie HCl/0.1 mM EDTA pH 7.4 buffer containing 0.1% SDS and
• RNA recovered from the aqueous phase was extracted with phenol and chloroform. The RNA was then precipitated with ethanol.
• PolyA RNA was selected from the bulk of cellular RNA by affinity chromatography on oligo(dT)-cellulose as described in Edmonds, Proc. Natl . Acad. Sci . USA, 68: 1336 (1971) and Aviv, Proc. Natl . Acad. Sci. USA, 69:1408 (1972), which are incorporated by reference.
• a cDNA library was constructed using a Promega Riboclone Kit
• Ethanol precipitated first strand synthetic product was resuspended in buffer containing 20 mM Tris HCl, pH 7.5, 5 mM MgCl 2 , 10 mM NH 4 SO 4 , 100 mM KCl, 0.5 mM BSA, 0.2 mM NAD, 0.2 mM dNTP's.
• 18 Unite of RNaseH/JE. coli ligase (1:1) and 30 Units of E. coli DNA polymerase were added to the reaction and the mixture was first incubated at 15°C for 90 min, and subsequently at room temperature for an additional 30 min. The reaction was then heat denatured at 70°C for 10 min and set at room temperature for 2 min. After the addition of T 4 DNA polymerase (27)
• the reaction was further incubated for 10 min at 37°C.
• the reaction was then extracted with phenol/CHCl 3 .
• cDNA products were size-selected on agarose gels. DNA molecules greater than 0.8 Kb were electro-eluted and concentrated by ethanol precipitation. The cDNA molecules (2.5 ⁇ g) were resuspended in buffer containing 30 mM Tris HCl, pH 7.8, 10 mM MgCl 2 , 10 mM DTT, 0.5 mM ATP and 100 ⁇ g/ml BSA. 10 pmoles of synthetic Eco RI adaptors and 7.5 Weiss unite of T 4 DNA ligase were added to the mixture and the reaction was incubated at 15°C overnight.
• reaction was adjusted to IX Not I buffer (NEB, Beverly, MA), 100 ⁇ g/ml BSA, and 5 ⁇ M ATP.
• T 4 polynucleotide kinase 10 Unite
• Not I 30 Unite
• DNA samples following phenol/CHCl 3 extractions were purified using Promega CE802 Spin columns (Madison, WI) and ethanol-precipitated.
• In vitro phage packaging was carried out using a "Gigapack Gold Extract" according to the vendor's protocol (Stratagene, La Jolla, CA). E. coli LE392 infected with the in vitro packaged phages were plated onto NZY plates in NZY soft agar. Alternatively, E. coli Y1090 cells were used and plated onto LB plates in LB soft agar containing 1.2 mM IPTG and 0.07% X-gal. The packaging efficiency of recombinant phages was about 1-2 ⁇ 10 6 pfu per ⁇ g of lambda arms. 7. Screening of recombinant library
• Partial amino acid sequences were previously determined for the first 20 amino acids at the N-terminus and four internal cyanogen bromide cleaved fragments (order unknown) of the Zopfiella ester hydrolase (Example 3). Based on these sequences, degenerate oligonucleotide primers were synthesized.
• Oligonucleotides 1 to 3 correspond to the sense strand of the mRNA that encodes the amino-terminal sequence.
• Oligonucleotides 4 to 7 correspond to the antisense strand of the mRNA that encode the internal tryptic fragments.
• Primers 1, 2 and 3 were independently paired with 4, 5, 6 and 7 anti-sense primers to generate polymerase chain reaction (PCR) products using the Zopfiella 511 genomic DNA as template.
• PCR products were generated using an USB GeneAmp Kit (Perkin Elmer Cetus, through United States Biochemical, Cleveland, OH).
• Oligonucleotide primers (10 pmoles each) were mixed with 250 ng of genomic DNA in buffer containing 10 mM Trie HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl 2 , 0.01% gelatin and 250 ⁇ M dNTPs. 1.25 units of AmpliTaq DNA polymerase was added to the reaction. Temperatures for annealing were increased step-wise: 4 cycles at 37°C, 3 cycles at 43°C and 26 cycles at 50°C. All extensions were performed at 72°C for 3 min except for the last 50°C annealing cycle which was for 10 min. Between cycles, reactions were denatured at 94°C for 2.5 min in the very first cycle and 1 min in all subsequent cycles. The products were analyzed on 1.8% agarose gels.
• PCR products generated by one set of primers were probed with radioactively labelled PCR products generated by another set of primers, certain common fragments showed positive hybridization. Based on the intensity and simplicity of hybridization pattern, PCR products generated by primers 1 + 6 and 2 + 7 were regarded as most likely to be specific for the Zopfiella ester hydrolase gene. Four phage plaque lifts were made per plate using
• Hybond-N membranes (Amersham Corp., Arlington Hts., Il.) Filters in duplicates were hybridized to the primer 1 + 6 and 2 + 7 generated probes.
• the generation of radioactively labelled PCR probes, conditions for hybridization (at 42oC, in 50% formamide, 750 mM NaCl, 250 mM Tris HCl, pH 8, 5 mM EDTA and 0.01% NaPi, and 100 ⁇ g salmon sperm DNA), and washing (at 60°C in 0.5 X SSC, 0.1% SDS) were according to Strub and Walter, Proc. Natl. Acad. Sci. USA, 86:9747-9751 (1989), which is incorporated by reference. 179 plaques from a pool of 1.2 million showed positive hybridization to the two PCR generated probes.
• the phage plaques were induced with IPTG and assayed in situ for ester hydrolase activity. If the open reading frame of the ester hydrolase gene was in the same translational reading frame as the lac gene and without interruption by stop codons within the 5' untranslated region, a functional ester hydrolase would be produced, as evidenced by the development of purple color when the phage plaques were overlaid with soft agar containing ⁇ -naphthyl-acetate and fast blue BB salt (Sigma, St. Louis, MO) (Higerd and Spizizen, supra, (1973)).
• Three ester hydrolase positive phages were identified among 5,000 plaques screened. It was subsequently shown that these three phages also hybridized positively to the radioactive PCR generated probes (1 + 6 and 2 + 7). The three phages, designated Zl-2, Z2-5 and Z3-2, were extensively purified and phage DNAs prepared.
• DNA inserts from the three ester hydrolase positive phages were excised using restriction enzyme Sfi I and Not I.
• the DNA inserts which were about 1.55 kb in length, were transferred onto pGEM-13Zf(+) plasmid DNA
• FIG. 3 shows the junction sequences between the Zopfiella cDNA and the plasmid vector.
• the cDNA inserts are in the same translational reading frame as the lac sequence.
• the 5' portion of the cDNA molecule encodes amino acids which correspond to those previously determined for the N terminus of the purified Zopfiella ester hydrolase.
• the complete DNA sequence for the Zopfiella ester hydrolase gene (clone 1-2) was subsequently determined using an Applied Biosystem DNA sequenator (Applied Biosystems, Inc., Foster City, CA). The complete DNA sequence as identified is set forth as Seq. I.D. No. 12.
• E. coli cells harboring the pGEM-13Zf(+) /enzyme plasmids were propagated overnight in LB broth in the presence of 1 mM IPTG. The cells were harvested by centrifugation and disrupted by sonication. After centrifugation at 10,000 rpm for 30 min (JA20 rotor), the supernatants were assayed for enzyme activity. Enzyme activity was measured using the S-enantiomer of p-nitrophenyl naproxen ester (5-PEN).
• clone (1-2) had the highest enzyme activity.
• clone (1-2) also showed a prominent protein band at about 46 kD.
• the rec 511 enzyme has a major protein band of approximately 46.5 kD which is slightly larger than the naturally occurring Strain 511 enzyme. Also, there is a major protein band in the native (non-reduced) gels of rec 511 enzyme that corresponds to the migration pattern of the activity stain.
• Example 9 Upon further purification as described in Example 2(a), the 46.5 kD protein was subsequently shown to have good enzyme activity. Moreover, it preferentially hydrolyzed S-naproxen esters as described in Example 9.
• the cDNA library was screened using a radioactively labelled rec 511 DNA as hybridization probe and approximately 30 (out of half a million plaques screened) positive plaques were identified. 10% of these plaques were also positive for ester hydrolase activity as evidenced by their
• Enzyme activity was assayed using methodologies identical to that described in Example 5.
• the purified Strain 780 enzyme like the Strain 511 enzyme had a major protein band that migrated at a rate consistent with a 46.5 kD size protein.
• the two activity bands associated with the purest preparation migrated with an R f number of 0.67 and 0.56.
• Yeast shuttle plasmid pSRF137 was constructed to allow galactoseinducible expression of the Zopfiella 511 enzyme in Saccharomyces cerevisiae.
• Figure 1(b) sets forth a diagram of the expression plasmid construction.
• cDNA from clone 1-2 ( Figure 3) was first subcloned into the Sma I site of pUC18, creating pSRF115, by digesting with Eco RI and Not I, and treating with the Klenow fragment of DNA polymerase.
• the cDNA was excised from pSRF114 as a Bam HI-Asp718I fragment and inserted between the BAM HI and Asp718I sites of pSEY303 to create pSRF16, as described by Emr, Douglas, J. Cell Biol. , 102:523 (1986), which in incorporated by
• pYRF102 is a 2 ⁇ -based shuttle plasmid that contains LEU2 and URA3 selectable markers, the GAL4 gene, and the GAL1 regulatory region promoter with a unique Bam HI site about 65bp distal to the transcription initiation site as described in U.S. Patent No. 4,661,454, which is incorporated by reference.
• the Bam HI-SnaB I CDNA-SUC2 fragment from pSRF16 was inserted at the Bam HI site of pYRF102, to create pSRF137.
• Yeast cells (DA2102) Barnes, D.A. and J. Thorner, Mol. Cell. Biol . 6:2828 (1986) were grown and plasmid pSRF137 selection was maintained in media lacking uracil (0.67% Yeast Nitrogen Base without amino acids (Difco), 0.5% vitamin-assay Casamino acids (Difco), 50 ⁇ g/ml adenine sulfate, 40 ⁇ g/ml histidine hydrochloride, and 25 ⁇ g/ml tryptophan).
• Non-inducing media was supplemented with 2% glucose whereas inducing media was supplemented with 2% galactose plus 0.1% glucose.
• Extracts were prepared by disrupting cells with glass beads, as modified from a previously described procedure in Asubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, ed., Current Protocols in Molecular Biology. , New York: Greene publishing and Wiley-Interscience, 1991, which is incorporated by reference.
• Cell pellets were resuspended in ice cold MOPS buffer (1 ml for each 10,000-12,000 klett unite) and 0.25 to 0.6 ml aliquots were placed in 1.5 ml microfuge tubes and 1 ⁇ l antifoam A. (Sigma, St. Louis, MO) was added.
• the protein concentration of the extracts was determined either by a Bradford Bio-Rad Protein Assay (BioRad Laboratories, Richmond, CA) or Pierce BCA Protein Assay Kit (Pierce Chemical Co., Rockford, IL) assay using BSA as a standard. Enzyme activity was measured using either the S-enantiomer of p-nitrophenyl naproxen ester (S-PEN) or racemic naproxen ethyl ester.
• S-PEN S-enantiomer of p-nitrophenyl naproxen ester
• racemic naproxen ethyl ester racemic naproxen ethyl ester
• Non-denaturing gels contained 12.5% acrylamide (acrylamide:bis is 30:0.8) and 370 mM Trie HCl pH 8.8 in the running buffer and 4%
• Running buffere contained 37.7 mM Trie HCl, 40 mM glycine pH 8.9 in the top reservoir; and 62.5 mM Trie HCl pH 7.5 in the bottom reservoir.
• Gels were stained for lipase activity using a ⁇ -naphthyl acetatefast blue assay.
• the gels were incubated for 15 min at room temperature in 100 ml of NaPi pH 7.4, 5 ml isopropanol, 0.4 mg/ml fast blue (Sigma F-0500), 0.03% ⁇ -naphthyl acetate (Sigma N-6875, 1.5 ml of a prepared/2% solution in acetone). Gels were subsequently destained in 7% acetic acid.
• Proteine were eluted from non-denaturing gels using an in situ gel assay, "Elutrap” (Schleicher and Scheull, Woburn, MA, ) with 25 mM Tris HCl/192 mM glycine pH 8.3 (Jacobs and Clad, Anal. Biochem. 154:583
• Polyclonal antisera to the Zopfiella ester hydrolase were prepared by injecting rabbits with recombinant or native enzyme (initial injection, 0.1 mg subcutaneous in complete Freund's adjuvant; subsequent boosts, 0.5 mg IM in incomplete Freund's adjuvant).
• Enzyme expression was induced in strain DA2102 carrying the pSRF137 by growth in galactose for 26 hrs. Extracts were prepared from these cells as well as a control strain carrying pYRF102. These extracts were subjected to native gel electrophoresis and stained with an ester hydrolase specific stain. A single band was stained in the pSRF137 lane but not in the control lane. This band was eluted and subjected to amino-terminal sequencing. Sequence data indicated that this band contained the yeast-derived Zopfiella enzyme.
• the eluted band was subjected to SDS-polyacrylamide gel
• the eluted band contained a single 43 kD species, the molecular weight predicted from the DNA sequence of the Zopfiella enzyme. Together with the amino-terminal sequencing, these data suggest that the yeast-derived enzyme is unmodified and has the expected carboxy terminus. Following SDS gel electrophoresis these samples were immunoblotted with anti-ester hydrolase antibodies. A protein of approximately 43 KD, present in both the eluted band and extracts from the pSRFl37-containing strain, but absent in the control strain, reacted with the antibodies. This is a further confirmation that the eluted band contained the yeast-derived enzyme. Also, the yeast-derived enzyme was shown to be slightly smaller than the recombinant enzyme produced in E. coli which is expressed with a 3 kilodalton fusion partner, as expression in yeast results in a full length authentic enzyme. EXAMPLE 9
• the hydrolysate was diluted 1:3 in 50 mM KH 2 PO 4 and ultrafiltered through a 3000 MWCO membrane (Centricon microconcentrator, Amicon,
• Strain 511 enzyme was harvested from a 3-day culture and prepared by 30%-60% ammonium sulfate fractionation, followed by DEAE and hydrophobic interaction chromatography as described in Example 2(A).
• Strain 780 enzyme was harvested from a 2-day culture and prepared by 30%-60% ammonium sulfate fractionation, followed by DEAE and hydrophobic interaction chromatography as described in Example 2(B).
• Rec 511, rec 780 and rec 780-ml65r210 enzymes were obtained from an overnight E. coli fermentation in LB broth and were concentrated with a 30%-60% ammonium sulfate precipitation, followed by purification with DEAE and size exclusion HPLC.
• the ee values given in Table 2 are not corrected for background levels of racemic acid.
• the levels of background acid are methyl ester»n-propyl ester>ethyl ester and may well account for the differences in ee's between these naproxen alkyl esters.
• the Zopfiella enzymes studied showed an enantioselectivity of greater than 98%.
• a comparison of the ability of Zopfiella Strain 511 ester hydrolase to hydrolyze ethyl and n-propyl naproxen ester was carried out.
• a 2.5 ml solution of heptane containing 2% ethyl A,S-naproxen ester was mixed with 2.5 ml of 0.1 M Tris HCl, pH 8.0.
• the reaction was started by adding 200 ⁇ l of Zopfiella Strain 511 unpurified ester hydrolase solution (16 mg dry weight/ml) to the appropriate reaction mixture.
• the 780 ester hydrolase gene was cloned into the vector pSELECT-1 (Promega) .
• the pSELECT-1 vector containing the 780 gene will be
• pS780 pSELECT-1 DNA contains lac operon sequences and transformants show ⁇ -galactosidase activity.
• pS780 was transformed into E. coli JM109 cells. The cells were then infected with helper phage R408 (Promega) to generate single stranded DNA copies of pS780. The single stranded pS780 was packaged into phage, harvested, and isolated.
• the isolated DNA was treated with 0.2 M nitrous acid for 15 minutes at room temperature.
• the single stranded DNA was primer extended using a T7 primer and AMV reverse transcriptase in the presence of deoxynucleotides.
• the mutated 780 gene was excised by Hind III/Bam HI digestion and gel purified and then ligated into gel purified Hind III/Bam HI digested p-SELECT-1.
• the mutagenized single stranded DNA was then transformed into E. coli JM109.
• Transformants generated were replica plated into LB + tetracycline (15 ⁇ g/ml) + IPTG (1 mM) medium. The replica platea were allowed to grow overnight at 37oC.
• the pSELECT-1 plasmid offered an easy way to determine the
• Replica plates containing 200-700 colonies were heated at 55°C for several hours. The plates were then cooled to room temperature and overlaid with 0.5% agarose containing ⁇ -naphthyl acetate and the indicator fast blue. Ester hydrolase activity was indicated by the colonies turning a red-purple color. Colonies showing the most rapid color changes were restreaked onto the same medium. After outgrowth, the plates were replicated and the replates examined again for enzyme activity after heating at 55°C for up to 7 hours.
• Enzyme inactivation kinetics of cells transformed with mutant DNA were compared with those of non-mutant pS780 transformed cells.
• Cell extracts in 0.2% PEG 8000 and 0.1 M Trie HCl, pH 8.0
• samples were taken and assayed for activity using the S-PEN assay.
• the mutant enzyme retained approximately 45% of its original activity, while the non-mutant enzyme had lost almost all activity.
• the DNA from the mutant enzyme rec 780-m10 was then purified and sequenced to determine genotype changes. The thermal stability of rec 780-m10 at 54°C was assessed. Equal amounts of enzyme activity of rec 780 and rec 780-m10 were added to tubes containing 1 mg/ml of S-PEN solubilized in DMSO in 0.1 M MOPS buffer.
• thermostable mutants Resistance to KNPR inactivation of thermostable mutants was compared with that of non-mutants by incubating cell extracts at 45oC in the presence of KNPR at 20 g/l and 33 g/l. At various times, samples were taken and assayed for enzyme activity by the S-PEN method. Ester hydrolase activity was more stable with the mutant extracts than with the non-mutant extracts when incubated with 20 g/l KNPR. At 33 g/l KNPR, the mutant extract was rapidly inactivated.
• Thermostable mutants were subjected further to nitrous acid mutagenesis as described above.
• the mutated DNA was made double stranded, excised, and ligated into the appropriate vector.
• E. coli JM109 was transformed and about 200,000 transformants were obtained.
• Replicas were made onto LB plates supplemented with 15 ⁇ g/ml tetracycline and 0.5 mM IPTG. After grow out, the plates were incubated at 60°-65°C for various lengths of time and subsequently screened for enzyme activity using the ⁇ -naphthyl acetate overlay method. Mutants that exhibited strong
• thermostable mutants were prepared using the above procedure.
• Third-generation mutants exhibited enzyme stability in the presence of 40 g/l KNPR at 40°C.
• Fourth- generation mutants, similarly prepared, exhibited enzyme stability in the presence of 60 g/l KNPR at 40°C.
• E. coli JM109 containing rec 780, rec 780-ml65 and rec 780-ml65r210 were grown overnight in LB broth supplemented with IPTG (1 ⁇ m) and tetracycline (15 ⁇ g/ml).
• the cells were harvested, suspended in 1 ml of 0.1 M Trie HCl, pH 8.0, supplemented with 0.2% PEG 8000 and disrupted by vigorous agitation in the presence of glass beads. Cellular debris and glass beads were removed by centrifugation (10,000 ⁇ g for 10 min).
• Inoculum was started from frozen seed stocks of Zopfiella stored at -70°C in 20% glycerol.
• One vial was thawed and inoculated into the basal media containing 0.6% glucose (w/v), 5 g/l (NH 4 ) 2 PO 2 , 6 g/l Na 2 HPO 4 , 3 g/l KH 2 PO 4 , 1.1 g/l Na 2 SO 4 , 5 mg/1 thiamine, 500 mg/1 MgSO 4 7H 2 O, 100 mg/l ampicillin and 0.5 ml/l trace metal solution.
• the culture was incubated in a baffled flask on a rotary shaker at 37°C for 7-8 hours. The cells were then passaged into fresh medium containing 1% glucose and incubated 14-16 hours.
• the fermentor was inoculated with these cells at a
• concentration of 1 part to 20 parts of minimal medium Specifically, eight liters of basal medium are inoculated with 400 ml of the seed.
• Dissolved oxygen is maintained at 20-40% through control of agitation speed and addition of supplemental oxygen.
• the pH is regulated at 6.9-7.0 by addition of 5N NH 4 OH.
• Feed solution #1 of 400 g/l glucose, 10 g/1 MgSO 4 .7H 2 O and 100 mg/l thiamine is added at a rate to maintain the glucose concentration at 1-3 g/l.
• Feed solution #2 of 100 g/l (NH 4 ) 2 HPO 4 is added when dissolved oxygen starts to increase and feed rate is adjusted to maintain a steady growth rate based on dissolved oxygen status.
• coli culture was then induced (lac promoter of the plasmid is induced) with 1 mM IPTG when the cell density reached an absorbence of .20 at 550 nm.
• the feed streams were discontinued at this time and the culture was harvested five to six hours post induction.
• the cells were then concentrated by centrifugation.
• the cells can also be concentrated by cross filtration.
• the cell lysate including insoluble cellular debris, was extracted in 17% (w/v) PEG 1550, 8% (w/v) sodium phosphate and 20% (weight wet cells prior to disruption/v) biomass. After mixing for 20 minutes, the mixture was centrifuged at 2000 rpm. Eighty percent of the enzyme partitions to the upper PEG rich phase. The PEG was removed from the enzyme utilizing ultrafiltration (30,000 molecular weight cutoff, Amicon spiral cartridge).
• the enzyme can be added to wet support after washing with DI water. d. Attachment of enzyme to glutaraldehyde-grafted Celite ®
• the enzyme solution contained 1.0 mg/ml protein in 50 mM Bicine buffer at a pH of 8.5.
• the flask was evacuated several times to ensure that the pores were liquid filled. The mixture stood overnight for 12-15 hours at room temperature.
• the first immobilization technique used was glutaraldehyde linking of the Zopfiella enzyme to a silica support.
• the support used was
• Manville Celite ® R-648 comprised of spherical particles of -30+50 mesh with a surface area of 46 m 2 /g. The procedure for support preparation and enzyme attachment was as described above.
• Fraction II ammonium sulfate fraction
• the experiments were conducted by adding 8.0 g of the immobilized enzyme to 50 ml of 0.05 M KH 2 PO 4 containing 500 ppm of PEG 8000 (Sigma, St. Louis, MO). After the aqueous phase was heated to 40°C and the pH adjusted to 8.5, 10.0 ml of heptane containing 1.63 g PrNPR was added. The reaction was allowed to proceed for 24 hours. The aqueous phase was sampled for S-naproxen analysis, including concentration and ee. Table 6 summarizes the results of the hydrolysis experiments with the immobilized enzyme preparations.
• the reaction can be carried out using soluble enzyme.
• the optical purity of the KNPR was measured by chiral HPLC using a chiral AGP column(ChromTech, Sweden) .
• the unreacted ester was recovered from the hexane by evaporation and analyzed for optical purity using the same chiral HPLC column.
• the aqueous phase contained 13.2 g/l naproxen as the potassium salt with an enantiomeric excess of 99.0%. This represented an R,S-ester conversion of 35.0%.
• the unreacted n-propyl ester of naproxen contained 68.2% of the R-enantiomer and 31.8% of the S- enantiomer.
• E. coli cells carrying the rec 511 gene, and cells carrying the rec 780 gene were grown overnight in LB broth supplemented with 100 ⁇ g/ml ampicillin, harvested, and suspended in distilled water.
• the cells were permeabilized with 1% v/v toluene.
• the permeabilized cells were then mixed with an equal volume of polyazetidine.
• the pH was maintained around pH 8.0 by adding a email volume of 1.0 M NaOH.
• the mixture was then poured into a plastic container and a vacuum was pulled. After a short period of vigorous bubbling, the suspension solidified into a wafer.
• the wafer was ground into a powder using a coffee mill.
• the cells were assayed for n-propyl naproxen ester hydrolysis at 35°C in the presence of 25% v/v hexane. This was done by adding varying amounts of immobilized cells to flasks containing 15 ml of 0.1 M Tris HCl buffer, pH 8.0 containing 0.2% PEG 8000 and 25% v/v hexane containing 100 mg of PrNPR. Samples of the aqueous phase were taken at various times and analyzed for naproxen concentration by HPLC using a Hypersil C8 column. The results of these hydrolysis experiments are shown in Table 7. The immobilized cells hydrolyzed n-propyl naproxen ester at rates dependent upon catalyst concentration.
• Stability of the immobilized cells in KNPR was determined by adding 200 mg of immobilized cells to 13 ml of 1 mM Trie HCl buffer, pH 8.0 containing 0.2% PEG 8000 and 48.75 g/l KNPR, and 4.0 ml DMSO. After the enzyme was allowed to stand in this solution at room temperature for ten minutes, 2 ml of PrNPR was added to the flask and the pH monitored with time. The decrease in pH shown in Table 8 below indicates the formation of S-naproxen.
• E. coli JM109 carrying rec 780-ml65r210 was suspended to an optical density of 20 at 620 nm in 120 ml of 0.1 M Tris HCl containing 0.2% PEG 8000.
• 1.5 ml of polyazetidine (Hercules, Inc., Wilmington, DE) was added to the cell suspension while stirring.
• 1.5 ml of polymer 1195 (Betz, Trevose, PA) was added, while continuing to stir.
• the flocculated cells were then pelleted by low speed centrifugation and the resulting pellets were combined, pressed and dried overnight at 37°C. This material was then cut into thin strips, dried for an additional 24 hours at 37°C and cut into email pellets (approx. 1 mm).
• the reaction mixture contained 100 mM of 3 mm Trie HCl, 0.2% PEG 8000, 15 ml of PrNPR and 1 g of the pellets.
• the reaction was carried out at 37°C and a pH of 7.9 was maintained by adding 20% KOH to the reaction mixture.
• the results of this study are presented in Table 9.
• Example 11 the reaction can be carried out using soluble enzyme.
• a reaction flask containing 300 ml of 50 mM potassium phosphate buffer and 30.0 g of the ethyl ester of A,S-naproxen was heated to 50°C.
• To this flask was added 7,800 Unite of the recombinant enzyme, rec 780-ml65r210, in 22.4 ml of 30 mM Trie HCl buffer.
• the reaction mixture was maintained at 50°C and the pH was maintained at 8.5 by the addition of 1.0 M KOH.
• the reaction slurry was separated by filtration.
• the KNPR content in the aqueous phase was measured by HPLC using a Hypersil C8 column (Alltech, Deerfield, IL) and the optical purity of the KNPR was measured by chiral HPLC using a chiral AGP column
• CTCGCAACTG ACTCAACTTC CTCCTTCACT TTCTCCTCTG CCGTAGGCAC TCGCACTCTT 240

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• 1993
  • 1993-05-14 EP EP93911192A patent/EP0644940A1/en not_active Withdrawn
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