A systematic approach for yielding a potential pool of enzymes: practical case for chiral resolution of (R,S)-ketoprofen ethyl ester

A systematic approach for the selection of potential biocatalysts from a natural source was developed and then a practical application was addressed. The approach that involves systematically combined conventional screening methods and current tools comprises the following consecutive steps: strain enrichment for activity screening, identification of positive strains, choosing whole genome-sequenced strains as candidates, gathering information about responsible enzymes, bioinformatic analyses and gene mining, probing genetic molecules and then functional expression. The target compound (R,S)-ketoprofen ethyl ester was to be resolved into an enantiomer, and a potential esterase from Pseudomonas fluorescens KCTC 1767 was prepared by the proposed procedure. The enzyme had a high activity and also strict selectivity for the enantiomer (S)-ketoprofen and was suitable therefore as a biocatalyst for practical use. The result achieved by the combined approach could not easily be obtained using other approaches with typical procedures. Hence the approach proposed here should be of considerable use for the screening of potential enzymes, particularly for enzymes with desired activity to unnatural substrates, from conditionally expressed and/or repressed proteins that are distributed widely in natural pools under normal conditions.     

Screening and characterization of a novel esterase from a metagenomic library

Metagenomes from various environmental soils were screened using alpha-naphthyl acetate and Fast Blue RR for a novel ester-hydrolyzing enzyme on Escherichia coli. Stepwise fragmentations and subcloning of the initial insert DNA (30-40 kb) using restriction enzymes selected to exclude already known esterases with subsequent screenings resulted in a positive clone with a 2.5-kb DNA fragment. The cloned sequence included an open reading frame consisting of 1089 bp, designated as est25, encoding a protein of 363 amino acids with a molecular mass of about 38.3 kDa. Amino acid sequence analysis revealed only moderate identity (< or = 48%) to the known esterases/lipases in the databases containing the conserved sequence motifs of esterases/lipases, such as HGGG (residues 124-127), GxSxG (residues 199-203), and the putative catalytic triad composed of Ser201, Asp303, and His333. Est25 was functionally overexpressed in a soluble form in E. coli with optimal activity at pH 7.0 and 25 degrees C. The purified Est25 exhibited hydrolyzing activity toward p-nitrophenyl (NP)-fatty acyl esters with short-length acyl chains (< or = C6) with the highest activity toward p-NP-acetate (Km=1.0 mM and Vmax = 63.7 U/mg), but not with chain lengths > or = C8, demonstrating that Est25 is an esterase originated most likely from a mesophilic microorganism in soils. Est25 efficiently hydrolyzed (R,S)-ketoprofen ethyl ester with Km of 16.4 mM and Vmax of 59.1 U/mg with slight enantioselectivity toward (R)-ketoprofen ethyl ester. This study demonstrates that functional screening combined with the sequential uses of restriction enzymes to exclude already known enzymes is a useful approach for isolating novel enzymes from a metagenome.     

拆分获得(R)-酮基布洛芬酯酶基因的筛选、克隆与表达

以自筛选出的具有一定不对称拆分外消旋酮基布洛芬氯乙酯能力的野生菌Bacillus megaterium NK13为材料,通过构建其基因文库,筛选得到一个阳性克隆重组子pUC18-NK-HYD3。分析测序结果发现外源片段中包含一段完整的741 bp的开放阅读框,其编码的蛋白中含有酯酶的GXSXG保守序列。经在NCBI的BLAST系统中比对,证明该酯酶基因属于首次发现(GenBank Accession Number: GU143552)。将酯酶基因克隆到载体pET21b(+)中,转化E. coli BL21(DE3),经IPTG诱导后在宿主菌得到表达。SDS-PAGE电泳检测证明该酯酶蛋白分子量约为28 kDa。TLC和HPLC检测结果显示,该酯酶优先水解(R)-型底物,在重组菌菌液体系,转化率为15%时,酯酶拆分获得(R)-酮基布洛芬的过量值(e.e.%)最高,达62.74％;改用重组菌湿菌体的PBS体系后,在转化率为10%~50%时,酯酶拆分获得(R)-酮基布洛芬的过量值(e.e.%)一直保持在73%~76%之间。     

Isolation of an esterase-producing Trichosporon brassicae and its catalytic performance in kinetic resolution of ketoprofen

A yeast strain CGMCC 0574, identified as Trichosporon brassicae, was selected from 92 strains for its high (S) selectivity in the hydrolysis of ketoprofen ethyl ester. The effective strains of the microorganisms were isolated from soil samples with the ester as the sole carbon source. The ethyl ester proved to be the best substrate for resolution of ketoprofen among several ketoprofen esters examined. The resting cells of CGMCC 0574 could catalyze the hydrolysis of ketoprofen ethyl ester with an enantiomeric ratio of 44.9, giving (S)-ketoprofen an enantiomeric excess of 91.5% at 42% conversion.     

Isolation and characterization of Acinetobacter sp. ES-1 excreting a lipase with high enantioselectivity for (S)-ketoprofen ethyl ester

A new Acinetobacter sp. ES-1, grown on triolein, tryptone and Triton X-100, excreted a lipase that hydrolyzed 10 mM (R,S)-ketoprofen ethyl ester into (S)-ketoprofen. The crude lipase had an activity of 10 U ml(-1) and, at 30 degrees C and pH 7 over 48 h, gave a conversion yield of 35% with an enantiomeric excess for the product 96%.     

Significantly improved esterase activity of Trichosporon brassicae cells for ketoprofen resolution by 2-propanol treatment

The kinetic resolution of racemic ketoprofen was carried out by enantioselective hydrolysis of ketoprofen ethyl ester using intact cells of Trichosporon brassicae CGMCC0574 as a biocatalyst. After the yeast cells were pretreated by 2 vol.% of 2-propanol for 10 h, the esterase activity on the (S)-ketoprofen ester increased dramatically, by a factor of ca. 310% without reducing the enantioselectivity of enzymatic resolution.     

Crystal structure and characterization of esterase Est25 mutants reveal improved enantioselectivity toward (S)-ketoprofen ethyl ester

Esterases comprise a group of enzymes that catalyze the cleavage and synthesis of ester bonds. They are important in biotechnological applications owing to their enantioselectivity, regioselectivity, broad substrate specificity, and the fact that they do not require cofactors. In a previous study, we isolated the esterase Est25 from a metagenomic library. Est25 showed catalytic activity toward the (R,S)-ketoprofen ethyl ester but had low enantioselectivity toward the (S)-ketoprofen ethyl ester. Because (S)-ketoprofen has stronger anti-inflammatory effects and fewer side effects than (R)-ketoprofen, enantioselectivity of this esterase is important. In this study, we generated Est25 mutants with improved enantioselectivity toward the (S)-ketoprofen ethyl ester; improved enantioselectivity of mutants was established by analysis of their crystal structures. The enantioselectivity of mutants was influenced by substitution of Phe72 and Leu255. Substituting these residues changed the size of the binding pocket and the entrance hole that leads to the active site. The enantioselectivity of Est25 (E = 1.1 ± 0.0) was improved in the mutants F72G (E = 1.9 ± 0.2), L255W (E = 16.1 ± 1.1), and F72G/L255W (E = 60.1 ± 0.5). Finally, characterization of Est25 mutants was performed by determining the optimum reaction conditions, thermostability, effect of additives, and substrate specificity after substituting Phe72 and Leu255.

     

Generation of a broad esterolytic subtilisin using combined molecular evolution and periplasmic expression.

Concomitant activity improvement of an evolved enzyme toward two very different ester substrates was achieved when a unique combination of functional periplasmic enzyme expression in Escherichia coli, random mutagenesis, DNA shuffling and cell-based kinetic screenings was applied. Specifically, we focused on the conversion of subtilisin E into an enzyme with broader esterase activity as opposed to its native amidase activity. Cell-based microtiter assays were performed on N-acetyl-D,L-phenylalanine p-nitrophenyl ester (Phe-NPE) and sucrose 1'-adipate (S1'A), as well as on the tetrapeptide amide substrate N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide. After a single modified cycle of directed molecular evolution, we isolated a number of clones exhibiting increased activity toward Phe-NPE. In the following rounds of screenings, mutants with improved activity on Phe-NPE were also tested on S1'A. Three mutants were identified with increased esterolytic activity on Phe-NPE and S1'A, while having similar amidase activity to that of the parental enzymes. Because the two ester substrates are structurally distinct, we have evolved a more general esterolytic subtilisin and this may have important applications in synthesis.     

Overexpression of Serratia marcescens lipase in Escherichia coli for efficient bioresolution of racemic ketoprofen

Lipase from Serratia marcescens ECU1010 was cloned and overexpressed in E. coli. After optimization, the maximum lipase activities reached 5000–6000 U/l and this recombinant lipase could enantioselectively hydrolyze (S)-ketoprofen esters into (S)-ketoprofen. Among six alkyl esters of racemic ketoprofen investigated, this lipase showed the best enantioselectivity for the kinetic resolution of ketoprofen ethyl ester, with an ee_p (enantiomeric excess of product) of 91.6% and E-value of 63 obtained at 48.2% conversion. Twelve nonionic surfactants were tested for enhancing the enantioselectivity of this lipase in the bioresolution of ketoprofen ethyl ester. A very high E-value of 1084 was achieved, with an optical purity of >99% ee_p and a yield of 42.6% in the presence of 3% Brij 92V. Further studies showed that the selectivity of the lipase was improved with the increase of Brij 92V concentration. The substrate (ketoprofen ethyl ester) does not inhibit the lipase activity, while the product (S)-ketoprofen inhibits the lipase activity to some extent. These results indicate that the S. marcescens lipase is very useful for biocatalytic production of chiral profens such as (S)-ketoprofen.     

Enantioselective hydrolysis of insoluble (R,S)-ketoprofen ethyl ester in dispersed aqueous reaction system induced by chiral cyclodextrin

The enantioselective hydrolysis of insoluble (R,S)-ketoprofen ethyl ester to the optically active (S)-ketoprofen was carried out in a dispersed aqueous lipase reaction system induced by the inclusion of chiral cyclodextrins for complexation of the substrate. Hydroxypropyl-beta-cyclodextrin was the most effective chiral selector and disperser giving an enantiomeric excess and conversion yield of 0.99 and 0.49, respectively.     

A carboxylesterase from the hyperthermophilic archaeon Sulfolobus solfataricus: cloning of the gene, characterization of the protein

A genomic library of the hyperthermophilic archaeon Sulfolobus solfataricus strain MT4 was constructed in Escherichia coli using a cloning vector not designed for heterologous gene expression. One positive clone exhibiting acquired thermophilic acetylesterase activity was directly detected by an in situ plate assay using a colony staining procedure with the chromogenic substrate beta-naphthyl acetate. The plasmid isolated from the clone contained a 3.3 kb genomic fragment from S. solfataricus and a full-length esterase coding sequence could be identified. Expression of the active thermostable esterase in E. coli was independent of isopropyl-beta-D-thiogalactopyranoside and of the kind of vector, suggesting that the archaeal esterase gene was controlled by fortuitous bacterial-like sequences present in its own 5' flanking region, not by the bacterial lac promoter or other serendipitous vector-located sequences. The protein, partially purified by thermoprecipitation of the host proteins at high temperature and gel exclusion chromatography, showed a homo-tetrameric structure with a subunit of molecular mass of 32 kDa which was in perfect agreement with that deduced from the cloned gene. The same protein was revealed in S. solfataricus cell extracts, thus demonstrating its functional occurrence in vivo under the cell culture conditions tested. The recombinant enzyme exhibited high thermal activity and thermostability with optimal activity between pH 6.5 and 7.0. The hydrolysis of p-nitrophenyl esters of fatty acids (from C(2) to C(8)) allowed the enzyme to be classified as a short length acyl esterase.     

A novel recombinant ethyl ferulate esterase from Burkholderia multivorans

AIMS: Isolation and identification of bacterial isolates with specific ferulic acid (FA) esterase activity and cloning of a gene encoding activity. METHODS AND RESULTS: A micro-organism with ethyl ferulate hydrolysing (EFH) activity was isolated by culture enrichment techniques. Detailed molecular identification based on species-specific primers and two conserved genes (16S rRNA and recA) led to the identification of the isolate as Burkholderia multivorans UWC10. A gene (designated estEFH5) encoding an EFH enzyme was cloned and its nucleotide sequence determined. Translational analysis revealed that estEFH5 encoded a polypeptide of 326 amino acids with an estimated molecular weight of 34.83 kDa. The EstEFH5 primary structure showed a typical serine hydrolase motif (G-H-S-L-G). The estEFH5 gene was over-expressed in Escherichia coli in an insoluble form. Following urea denaturation and in vitro refolding, the enzyme was purified using one-step His Select Nickel chromatographic column. CONCLUSION: Purified EstEFH5 showed a preference for short-chain rho-nitrophenyl esters (C2 and C3) a typical feature for carboxylesterase. Furthermore, the recombinant enzyme also retained the activity against ethyl ferulate (EF). SIGNIFICANCE AND IMPACT OF THE STUDY: A biocatalytic process for the production of FA from EF as a model substrate was demonstrated. This is the first report that describes the cloning and expression of a gene encoding FA esterase activity from the genus Burkholderia.     

Cloning and characterization of thermostable esterase from <Emphasis Type="Italic">Archaeoglobus fulgidus</Emphasis>

Thermostable esterase gene was cloned (Est-AF) from extremophilic microorganisms, Archaeoglobus fulgidus DSM 4304. The protein analysis result showed that Est-AF is monomer with total 247 amino acids and molecular weight of estimated 27.5 kDa. It also showed repeating units G-X-S-X-G (GHSLG) (residues 86 approximately 90) which is reported as active site of known esterases, and the putative catalytic triad composed of Ser88, Asp198 and His226. The esterase activity test with various acyl chain length of rho-nitrophenol resulted that Est-AF showed highest specific activity with rho-nitrophenylbutyrate (pNPC4) and rapidly decrease with rho-nitrophenyl ester contain more than 8 carbon chain. These results represent that cloned enzyme is verified as a carboxylesterase but not a lipase because esterase activity is decreased with rho-nitrophenyl ester contains more than 8 carbon chains but lipase activity does not affected with carbon chain length. Optimum temperature of esterase reaction with rho-nitrophenylbutyrate (pNPC4) was 80 degrees C. When ketoprofen ethyl ester was used as a substrate, activity of Est-AF showed the highest value at 70 degrees C, and 10% of activity still remains after 3 h of incubation at 90 degrees C. This result represents Est-AF has high thermostability with comparison of other esterases that have been reported. However, Est-AF showed low enantioselectivity with ketoprofen ethyl ester. Optimum pH of Est-AF is between pH 7.0 and pH 8.0. Km value of ketoprofen ethyl ester is 1.6 mM and, Vmax is 1.7 micromole/mg protein/min. Est-AF showed similar substrate affinity but slower reaction with ketoprofen ethyl ester compare with esterase from mesophilic strain P. fluorescens.     

一种具有立体选择性新酯酶的菌种筛选和基因克隆

从土样分离到一株产生具有立体选择性酯水解酶的恶臭假单孢菌(Pseudomonas putida NH33)。构建P.putida NH33的基因组文库,并在E.coli中进行酯酶活性筛选,得到一个含有4.7kb插入片段的阳性克隆。对这个克隆的DNA片段进行序列分析,表明它含有一个1142个碱基的开放阅读框,为编码381个氨基酸的酯酶。推测的酯酶氨基酸序列与其它丝氨酸酯水解酶具有共同的保守基序GXSXG。把该蛋白在E.coli BL21(DE3)中进行表达,并用金属亲和层析纯化至单一条带。利用纯化的酶水解2-芳基丙酸乙酯制备2-芳基丙酸的S型异构体,产物的光学纯度ee_p>99%,说明此酶可8用于手性药物的合成。该酯酶是一个新酶,其基因序列已递交GenBank,登记号为AY896293。     

Biocatalytic preparation of enantiopure ( R)-ketoprofen from its racemic ester by a new yeast isolate Citeromyces matriensis CGMCC 0573

The yeast strain CGMCC 0573 was identified as Citeromyces matriensis and shown to be capable of enantioselectively hydrolyzing ethyl ester of ( R)-Ketoprofen (2-(3-benzoylphenyl)propionic acid). The strain was isolated for the first time from soil samples through a new and efficient screening procedure in which the probability of obtaining active strains was greatly increased by using ethanol and Tween-80 alternatively as additives during the enrichment culture. Studies of the culture conditions and catalytic performance of Citeromyces matriensis CGMCC 0573 showed that the enzyme occurs constitutively in the cells and its production is enhanced by feeding with Tween-80 during the early period of cultivation. Yeast extract was found to be beneficial both for growth and for esterase production. The optimal temperature and pH for the bioconversion were 40 degrees C and pH 8.0, respectively. Biotransformation using resting cells cultured in a flask with baffles and magnetic stirring and in the presence of 50 mM substrate resulted in the production of ( R)-ketoprofen at 93% ee (enantiomeric excess) and at 42.6% conversion.     

Purification and properties of arginine esterases from Bitis arietans (puff adder) venom

An arginine esterase (FT1) was purified from B. arietans venom by gel-filtration and ion-exchange chromatography. The purified enzyme contains 21.6% of carbohydrate, 240 amino acids including 12 half-cystine residues and has a mol. wt of approximately 43,000. The purified enzyme has a high esterolytic activity towards N-alpha-benzoyl-L-arginine ethyl ester but shows no proteolytic activity against Azocoll and no clotting activity with fibrinogen. The N-terminal sequence of the arginine esterase from B. arietans venom shares a significant degree of sequence homology with the arginine esterase of B. nasicornis, the thrombin-like enzyme of C. adamanteus and the kallikrein-like enzymes of C. atrox venoms. It would appear that the arginine esterase from B. arietans venom exists in various multiple forms of the enzyme.     

Enantioselective ester hydrolase from Sphingobacterium sp. 238C5 useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis

A novel enzyme, β-phenylalanine ester hydrolase, useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis was characterized. The enzyme purified from the cell free-extract of Sphingobacterium sp. 238C5 well hydrolyzed β-phenylalanine esters (S)-stereospecifically. Besides β-phenylalanine esters, the enzyme catalyzed the hydrolysis of several α-amino acid esters with l-stereospecificity, while the deduced 369 amino acid sequence of the enzyme exhibited homology to alkaline d-stereospecific peptide hydrolases from Bacillus strains. Escherichia coli transformant expressing the β-phenylalanine ester hydrolase gene exhibited an about 8-fold increase in specific (S)-β-phenylalanine ethyl ester hydrolysis as compared with that of Sphingobacterium sp. 238C5. The E. coli transformant showed (S)-enantiomer specific esterase activity in the reaction with a low concentration (30 mM) of β-phenylalanine ethyl ester, while it showed both esterase and transpeptidase activity in the reaction with a high concentration (170 mM) of β-phenylalanine ethyl ester and produced β-phenylalanyl-β-phenylalanine ethyl ester. This transpeptidase activity was useful for β-phenylalanine β-peptide synthesis.     

Genome-wide cloning and characterization of microbial esterases

We have isolated putative esterase genes from various bacterial chromosomes. Thirty open reading frames predicted to encode esterases were randomly selected from 13 sequenced bacterial chromosomes and were cloned into an expression vector. The esterase activity of the resulting clones was tested on a tributyrin plate at different pH values and temperatures. Nine out of thirty tested clones exhibited significant tributyrin hydrolyzing activity. The enzyme S5 from the gene b0494 of Escherichia coli, the enzyme S12 from the gene STM0506 of Salmonella typhimurium, and the enzyme S28 from the gene AF1716 of Archaeoglobus fulgidus exhibited high activity at an alkaline pH range. The esterase S11 encoded by the gene PA3859 of Pseudomonas aeruginosa PAO1 and the esterase S21 from the gene SMc01033 of Sinorhizobium meliloti 1021, both showed a sharp increase in enzyme activity above pH 8.0. Furthermore, the enzymes S5, S12, S21, and S28 retained the esterase activity when they were incubated at 50 degrees C, suggesting that these enzymes are thermostable. Subsequent pH vs. activity and temperature vs. activity experiments with selected enzymes in a solution assay system confirmed the validity of the above data. The genome-wide exploration strategy of proteins provided valuable information on the esterases by revealing subtle biochemical differences between the esterases of different sources.     

The nature of differences in amidase and esterase activities of some acyltrypsins]

Specific trypsin substrates (esters, anilides, amides, peptides) were shown to accelerate deacetylation of monoacetylated trypsin. The amidase activity of monoacetyl-, monopropyonyl-, and tetraformyl-trypsin was not manifested if the amidase activity of native enzyme was suppressed in these preparations by the ester substrates (benzoylarginine ethyl ester or p-nitrophenyl acetate). Therefore the differences in the residual amidase and esterase activities of these acylated trypsin preparations found earlier did not contradict the universality of the acylenzyme mechanism. These differences are due to the strong deacylating effect of specific substrate in its complex with the enzyme modified with nonspecific acyl residue. The latter fact is suggested to be an experimental confirmation of the "induced fit" hypothesis.     

Molecular cloning, expression and characterization of a novel feruloyl esterase enzyme from the symbionts of termite(Coptotermes formosanus) gut

Termites play an important role in the degradation of dead plant materials and have acquired endogenous and symbiotic cellulose digestion capabilities. The feruloyl esterase enzyme (FAE) gene amplified from the metagenomic DNA of Coptotermes formosanus gut was cloned in the TA cloning vector and subcloned into a pET32a expression vector. The Ft3-7 gene has 84% sequence identity with Clostridium saccharolyticum and shows amino acid sequence identity with predicted xylanase/chitin deacetylase and endo-1,4-beta-xylanase. The sequence analysis reveals that probably Ft3-7 could be a new gene and that its molecular mass was 18.5 kDa. The activity of the recombinant enzyme (Ft3-7) produced in Escherichia coli (E.coli) was 21.4 U with substrate ethyl ferulate and its specific activity was 24.6 U/mg protein. The optimum pH and temperature for enzyme activity were 7.0 and 37oC, respectively. The substrate utilization preferences and sequence similarity of the Ft3-7 place it in the type-D sub-class of FAE.