Extremely Stable and Versatile Carboxylesterase from a Hyperthermophilic Archaeon

We have found that the hyperthermophilic archaeon Pyrobaculum calidifontis VA1 produced a thermostable esterase. We isolated and sequenced the esterase gene (est_Pc) from strain VA1. est_Pc consisted of 939 bp, corresponding to 313 amino acid residues with a molecular mass of 34,354 Da. As est_Pc showed significant identity (30%) to mammalian hormone-sensitive lipases (HSLs), esterase of P. calidifontis (Est) could be regarded as a new member of the HSL family. Activity levels of the enzyme were comparable or higher than those of previously reported enzymes not only at high temperature (6,410 U/mg at 90°C), but also at ambient temperature (1,050 U/mg at 30°C). The enzyme displayed extremely high thermostability and was also stable after incubation with various water-miscible organic solvents at a concentration of 80%. The enzyme also exhibited activity in the presence of organic solvents. Est of P. calidifontis showed higher hydrolytic activity towards esters with short to medium chains, with p-nitrophenyl caproate (C₆) the best substrate among the p-nitrophenyl esters examined. As for the alcoholic moiety, the enzyme displayed esterase activity towards esters with both straight- and branched-chain alcohols. Most surprisingly, we found that this Est enzyme hydrolyzed the tertiary alcohol ester tert-butyl acetate, a feature very rare among previously reported lipolytic enzymes. The extreme stability against heat and organic solvents, along with its activity towards a tertiary alcohol ester, indicates a high potential for the Est of P. calidifontis in future applications.     

Thermostable esterase from a thermoacidophilic archaeon: purification and characterization for enzymatic resolution of a chiral compound

Homolog to lipolytic enzymes having the consensus sequence Gly-X-Ser-X-Gly, from the Sulfolobus solfataricus P2 genome, were identified by multiple sequence alignments. Among three potential candidate sequences, one (Est3), which displayed higher activity than the other enzymes on the indicate plates, was characterized. The gene (est 3) was expressed in Escherichia coli, and the recombinant protein (Est3) was purified by chromatographic separation. The enzyme is a trimeric protein and has a molecular weight of 32 kDa in monomer form in its native structure. The optimal pH and temperature of the esterase were 7.4 and 80 degrees C respectively. The enzyme showed broad substrate specificities toward various p-nitrophenyl esters ranging from C2 to C16. The catalytic activity of the Est3 esterase was strongly inhibited by phenylmethylsulfonyl fluoride (PMSF) and diethyl p-nitrophenyl phosphate. Based on substrate specificity and the action of inhibitors, the Est3 enzyme was estimated to be a carboxylesterase (EC 3.1.1.1). The enzyme with methyl (+/-)-2-(3-benzoylphenyl)propionate-hydrolyzing activity to (-)-2-(3-benzoylphenyl)propionic acid displayed a moderate degree of enantioselectivity. The product, (-)-2-(3-benzoylphenyl)propionic acid, rather than its methyl ester, was obtained in 80% enantiomeric excess (e.e.(p)) at 20% conversion at 60 degrees C after a 32-h reaction. This result indicates that S. solfataricus esterase can be used for application in the synthesis of chiral compounds.     

Identification and characterization of a novel salt‐tolerant esterase from a Tibetan glacier metagenomic library

A salt‐tolerant esterase, designated H9Est, was identified from a metagenomic library of the Karuola glacier. H9Est gene comprised 1071 bp and encoded a polypeptide of 357 amino acids with a molecular mass of 40 kDa. Sequence analysis revealed that H9Est belonged to the family IV of bacterial lypolitic enzyme. H9Est was overexpressed in Escherichia coli and the purified enzyme showed hydrolytic activity towards p‐nitrophenyl esters with carbon chain from 2 to 8. The optimal esterase activity was at 40°C and pH 8.0 and the enzyme retained its activity towards some miscible organic solvents such as polyethylene glycol. A three‐dimensional model of H9Est revealed that S200, D294, and H324 formed the H9Est catalytic triad. Circular Dichroism spectra and molecular dynamic simulation indicated that the esterase had a wide denaturation temperature range and flexible loops that would be beneficial for H9Est performance at low temperatures while retaining heat‐resistant features. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:890–899, 2015     

A novel thermostable esterase from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7

We have characterized an esterase expressed from the putative esterase gene (ST0071) selected from the total genome analysis from the thermoacidophilic archaeon Sulfolobus tokodaii strain 7. The ORF was cloned and expressed as a fusion protein in Escherichia coli. The protein was purified with heat treatment, affinity column chromatography, and size exclusion filtration. The optimum activity for ester cleavage against p-nitrophenyl esters was observed at around 70 degrees C and pH 7.5-8.0. The enzyme exhibited high thermostability and also showed activity in a mixture of a buffer and water-miscible organic solvents, such as acetonitrile and dimethyl sulfoxide. From the kinetic analysis, p-nitrophenyl butyrate was found to be a better substrate than caproate and caprylate.     

Cloning and identification of a new group esterase (Est5S) from noncultured rumen bacterium

The gene encoding an esterase enzyme was cloned from a metagenomic library of cow rumen bacteria. The esterase gene (est5S) was 1,026 bp in length, encoding a protein of 366 amino acid residues with a calculated molecular mass of 40,168 Da. The molecular mass of the enzyme was estimated to be 40,000 Da. The Est5S protein contains the Gly-X-Ser-X-Gly motif found in most bacterial and eukaryotic serine hydrolases. However, the Asp or Glu necessary for the catalytic triad [Ser-Asp-(Glu)-His] was not present, indicating Est5S represents a novel member of the GHSQG family of esterolytic enzymes. BlastP in the NCBI database analysis of Est5S revealed homology to hypothetical proteins and it had no homology to previous known lipases and esterases. Est5S was optimally active at pH 7.0 and 40 degrees C. Among the p-nitrophenyl acylesters tested, high enzymatic activities were observed on the short-chain p-nitrophenyl acylesters, such as p-nitrophenyl acetate, etc. The conserved serine residue (Ser190) was shown to be important for Est5S activity. The primers that amplified the est5S gene did not show any relative band with 49 species of culturable rumen bacteria. This implies that a new group esterase gene, est5S, may have come from a noncultured cow rumen bacterium.     

Special Rhodococcus sp. CR-53 esterase Est4 contains a GGG(A)X-oxyanion hole conferring activity for the kinetic resolution of tertiary alcohols

Rhodococci are highly adaptable bacteria, capable to degrade or transform a large number of organic compounds, including recalcitrant or toxic products. However, little information is available on the lipases of the genus Rhodococcus, except for LipR, the first lipase isolated and described from strain Rhodococcus CR-53. Taking into consideration the interest raised by the enzymes produced by actinomycetes, a search for new putative lipases was performed in strain Rhodococcus CR-53. We describe here the isolation, cloning, and characterization of intracellular esterase Est4, a mesophilic enzyme showing preference for short-chain-length acyl groups, without interfacial activation. Est4 displays moderate thermal and pH stability and low tolerance to most tested ions, being inhibited by detergents like sodium dodecyl sulfate and Triton X-100®. Nevertheless, the enzyme shows good long-term stability when stored at 4–20 °C and neutral pH. Amino acid sequence analysis of Est4 revealed a protein of 313 amino acids without a signal peptide, bearing most of the conserved blocks that define bacterial lipase family IV, thus being assigned to this family. Detection of a GGG(A)X oxyanion hole in the enzyme motivated the evaluation of Est4 ability to convert tertiary alcohol esters. The newly discovered esterase Est4 from Rhodococcus CR-53 successfully hydrolyzed the tertiary alcohol esters linalyl acetate, terpinyl acetate, and 1,1,1-trifluoro-2-phenylbut-3-yn-2-yl acetate.     

Biochemical and genetic analysis of a cutinase-type polyesterase from a thermophilic Thermobifida alba AHK119

Recombinant polyesterase (Est119) from Thermobifida alba AHK119 was purified by two chromatography steps. The final protein was observed as a single band in SDS-PAGE, and the specific activity of Est119 for p-nitrophenyl butyrate was 2.30?u/mg. Purified Est119 was active with aliphatic and aliphatic-co-aromatic polyesters. Kinetic data indicated that p-nitrophenyl butyrate (pNPB) or hexanoate was the best substrate for Est119 among p-nitrophenyl acyl esters. Calcium was required for full activity and thermostability of Est119, which was stable at 50?°C for 16?h. Three-dimensional modeling and biochemical characterization showed that Est119 is a typical cutinase-type enzyme that has the compact ternary structure of an α/β-hydrolase. Random and site-directed mutagenesis of wild-type Est119 resulted in improved activity with increased hydrophobic interaction between the antiparallel first and second β-sheets (A68V had the greatest effect). Introduction of a proline residue (S219P) in a predicted substrate-docking loop increased the thermostability. The specific activity of the A68V/S219P mutant on pNPB was increased by more than 50-fold over the wild type. The mutant was further activated by 2.6-fold (299?u/mg) with 300?mM Ca(2+) and was stable up to 60?°C with 150?mM Ca(2+). Another identical gene was located in tandem in the upstream of est119.     

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.     

New thermophilic and thermostable esterase with sequence similarity to the hormone-sensitive lipase family, cloned from a metagenomic library

A gene coding for a thermostable esterase was isolated by functional screening of Escherichia coli cells that had been transformed with fosmid environmental DNA libraries constructed with metagenomes from thermal environmental samples. The gene conferring esterase activity on E. coli grown on tributyrin agar was composed of 936 bp, corresponding to 311 amino acid residues with a molecular mass of 34 kDa. The enzyme showed significant amino acid similarity (64%) to the enzyme from a hyperthermophilic archaeon, Pyrobaculum calidifontis. An amino acid sequence comparison with other esterases and lipases revealed that the enzyme should be classified as a new member of the hormone-sensitive lipase family. The recombinant esterase that was overexpressed and purified from E. coli was active above 30 degrees C up to 95 degrees C and had a high thermal stability. It displayed a high degree of activity in a pH range of 5.5 to 7.5, with an optimal pH of approximately 6.0. The best substrate for the enzyme among the p-nitrophenyl esters (C(4) to C(16)) examined was p-nitrophenyl caproate (C(6)), and no lipolytic activity was observed with esters containing an acyl chain length of longer than 10 carbon atoms, indicating that the enzyme is an esterase and not a lipase.     

Characterization of a thermostable esterase activity from the moderate thermophile Bacillus licheniformis

A new esterase activity from Bacillus licheniformis was characterized from an Escherichia coli recombinant strain. The protein was a single polypeptide chain with a molecular mass of 81 kDa. The optimum pH for esterase activity was 8-8.5 and it was stable in the range 7-8.5. The optimum temperature for activity was 45 degrees C and the half-life was 1 h at 64 degrees C. Maximum activity was observed on p-nitrophenyl caproate with little activity toward long-chain fatty acid esters. The enzyme had a KM of 0.52 mM for p-nitrophenyl caproate hydrolysis at pH 8 and 37 degrees C. The enzyme activity was not affected by either metal ions or sulfydryl reagents. Surprisingly, the enzyme was only slightly inhibited by PMSF. These characteristics classified the new enzyme as a thermostable esterase that shared similarities with lipases. The esterase might be useful for biotechnological applications such as ester synthesis.     

超嗜热羧酸酯酶的性质和应用研究

摘要 来源于超嗜热菌的超嗜热羧酸酯酶,结构上与激素敏感性脂肪酶(HSL)家族近似,属于α/β-水解酶,但是其空间结构比HSL更加紧密而有韧性,具有很强的热稳定性。性质研究表明,温度和有机溶剂对超嗜热羧酸酯酶的活性和对映选择性影响显著;其最适底物一般是对硝基苯酯,部分酯酶的基因含有GGGX基序,能够水解叔醇酯结构。由于超嗜热羧酸酯酶的独特结构和性质,其应用潜力巨大,尤其在拆分手性的外消旋酯方面独具优势。     

Gene cloning and characterization of a cold-adapted esterase from Acinetobacter venetianus V28

Acinetobacter venetians V28 was isolated from the intestine of righteye flounder, Poecilopsetta plinthus caught in Vietnam seawater, and the esterase gene was cloned using a shotgun method. The amino acid sequence deduced from the nucleotide sequence (1,017 bp) corresponded to a protein of 338 amino acid residues with a molecular weight of 37,186. The esterase had 87% and 72% identities with the lipases of A. junii SH205 and A. calcoaceticus RUH2202, respectively. The esterase contained a putative leader sequence, as well as the conserved catalytic triad (Ser, His, Asp), consensus pentapeptide GXSXG, and oxyanion hole sequence (HG). The protein from the strain V28 was produced in both a soluble and an insoluble form when the Escherichia coli cells harboring the gene were cultured at 18 degrees C. The maximal activity of the purified enzyme was observed at a temperature of 40 degrees C and pH 9.0 using p-NP-caprylate as substrate; however, relative activity still reached to 70% even at 5 degrees C with an activation energy of 3.36 kcal/mol, which indicated that it was a cold-adapted enzyme. The enzyme was a nonmetalloprotein and was active against p-nitrophenyl esters of C4, C8, and C14. Remarkably, this enzyme retained much of its activity in the presence of commercial detergents and organic solvents. This cold-adapted esterase will be applicable as catalysts for reaction in the presence of organic solvents and detergents.     

Novel thermophilic and thermostable lipolytic enzymes from a Thailand hot spring metagenomic library

Functional screening for lipolytic enzymes from a metagenomic library (origin: Jae Sawn hot spring, Thailand) resulted in isolation of a novel patatin-like phospholipase (PLP) and an esterase (Est1). PLP contained four conserved domains similar to other patatin-like proteins with lipid acyl hydrolase activity. Likewise, sequence alignment analysis revealed that Est1 can be classified as a family V bacterial lipolytic enzyme. Both PLP and Est1 were expressed heterologously as soluble proteins in E. coli and exhibited more than 50% of their maximal activities at alkaline pH, of 7-9 and 8-10, respectively. In addition, both enzymes retained more than 50% of maximal activity in the temperature range of 50-75 degrees C, with optimal activity at 70 degrees C and were stable at 70 degrees C for at least 120 min. Both PLP and Est1 exhibited high V(max) toward p-nitrophenyl butyrate. The enzymes had activity toward both short-chain (C(4) and C(5)) and long chain (C(14) and C(16)) fatty acid esters. The isolated enzymes, are therefore, different from other known patatin-like phospholipases and esterases, which usually show no activity for substrates longer than C(10). We suggest that PLP and EstA enzymes are novel and have a; b potential use in industrial applications.     

An esterase on the outer membrane of Pseudomonas aeruginosa for the hydrolysis of long chain acyl esters.

A new esterase activity which hydrolyzes palmitoyl-CoA was found in the membrane fraction of Pseudomonas aeruginosa. All the 11 strains of P. aeruginosa tested possessed this esterase activity. The esterase was constitutive and was fully active on the intact cell bodies toward substrates in the medium. It was located on the outer membrane of the cell envelope, and was not released into the culture medium. This activity was designated as OM (outer membrane) esterase. OM esterase was solubilized from the cell envelope with EDTA-Triton X-100 and purified 690-fold. It was a minor component of the outer membrane. Its molecular weight was approximately 55,000. The activity was rather stable to heat, a wide range of pH, and treatment with detergents and organic solvents. No cofactors were required. The pH optimum of the reaction was 8.5. Among various acyl-CoAs, only long chain (C12--C18) thioesters were hydrolyzed. OM esterase also hydrolyzed some kinds of oxy-esters such as p-nitrophenyl acyl esters, monoacyl esters of sucrose and Tween 80 (polyoxyethylene sorbitan monooleate). On the other hand, triglycerides, phospholipids, or hydrophobic monoesters were not hydrolyzed at all. Thus, this enzyme seems to have specificity for long chain acyl esters with hydrophilic groups, whether thio- or oxy-ester. Mutants deficient in this esterase activity were isolated. These mutants were unable to grow on Tween 80 as a sole carbon source. This suggests a possible role of OM esterase in the utilization of acyl esters as carbon sources.     

Molecular cloning,over expression and characterization of thermoalkalophilic esterases isolated from <Emphasis Type="Italic">Geobacillus</Emphasis> sp.

Due to potential use for variety of biotechnological applications, genes encoding thermoalkalophilic esterase from three different Geobacillus strains isolated from thermal environmental samples in Balçova (Agamemnon) geothermal site were cloned and respective proteins were expressed in Escherichia coli (E.coli) and characterized in detail. Three esterases (Est1, Est2, Est3) were cloned directly by PCR amplification using consensus degenerate primers from genomic DNA of the strains Est1, Est2 and Est3 which were from mud, reinjection water and uncontrolled thermal leak, respectively. The genes contained an open reading frame (ORF) consisting of 741 bp for Est1 and Est2, which encoded 246 amino acids and ORF of Est3 was 729 bp encoded 242 amino acids. The esterase genes were expressed in E. coli and purified using His-Select HF nickel affinity gel. The molecular mass of the recombinant enzyme for each esterase was approximately 27.5 kDa. The three esterases showed high specific activity toward short chain p-NP esters. Recombinant Est1, Est2, Est3 have exhibited similar activity and the highest esterase activity of 1,100 U/mg with p-nitrophenyl acetate (pNPC₂) as substrate was observed with Est1. All three esterase were most active around 65°C and pH 9.5–10.0. The effect of organic solvents, several metal ions, inhibitors and detergents on enzyme activity for purified Est1, Est2, Est3 were determined separately and compared.     

A thermostable esterase from Thermoanaerobacter tengcongensis opening up a new family of bacterial lipolytic enzymes

An unidentified α/β hydrolase gene lipA3 from thermostable eubacterium species Thermoanaerobacter tengcongensis MB4 was cloned and heterologously expressed by Escherichia coli BL21(DE3)pLysS. The purified recombinant enzyme EstA3 turned out to be a monomeric thermostable esterase with optimal activity at 70°C and pH 9.5. The enzyme showed lipolytic activity towards a wide range of ester substrates including p-nitrophenyl esters and triacylglycerides, with the highest activity being observed for p-nitrophenyl caproate at 150 U/mg and for Triacetin at 126U/mg, respectively. Phylogenetic analysis revealed that EstA3 did not show homology to any identified bacterial lipolytic hydrolases. Sequence alignment showed that there was a common pentapeptide CHSMG with a cysteine replacing the first glycine in most esterase and lipase conserved motif GXSXG. The catalytic triad of EstA3 is Ser92, Asp269 and His292, which was confirmed by site directed mutagenesis. Based on the enzymatic properties and sequence alignment we concluded that the esterase EstA3 represented a novel bacterial lipolytic enzyme group and in chronological order this group was assigned as Family XIV.     

Human liver aldehyde dehydrogenase. Esterase activity.

Human liver aldehyde dehydrogenase has been found to be capable of hydrolyzing p-nitrophenyl esters. Esterase and dehydrogenase activities exhibited identical ion exchange and affinity properties, indicating that the same protein catalyzes both reactions. Competitive inhibition of esterase activity by glyceraldehyde and chloral hydrate furnished evidence that p-nitrophenyl acetate was hydrolyzed at the aldehyde binding site for dehydrogenase activity. Pyridine nucleotides modified esterase activity; NAD+ accelerated the rate of p-nitrophenyl acetate hydrolysis more that 5-fold, whereas NADH increased activity by a factor of 2. Activation constants of 117 muM for NAD+ and 3.5 muM for NADH were obtained from double reciprocal plots of initial rates as a function of modifier concentration at pH 7. The kinetics of activation of ester hydrolysis were consistent with random addition of pyridine nucleotide modifier and ester substrate to this enzyme.     

A novel esterase from Ralstonia sp. M1: gene cloning, sequencing, high-level expression and characterization

A newly isolated gene from Ralstonia sp. M1, encoding an esterase, was cloned in Escherichia coli and its nucleotide sequence determined. The 1.6kb insert revealed one complete open reading frame, predicted to encode an esterase (320 aa, 34.1kDa) with a pI of 9.86. EstR contained a putative oxyanion hole H36G37, a conserved pentapeptide G103HSLG107 and a conserved catalytic His265 and Asp237. The EstR sequence shared 64-70 and 44-48% identity with the hydrolases/acyltransferases from Burkholderia strains and from Ralstonia strains, respectively, 44 and 38% identity with the lactone-specific esterase from Pseudomonas fluorescens and Mesorhizobium loti, respectively. The esterase EstR was expressed with a high level of 41mg/g wet cells. The Ni-NTA-purified esterase EstR showed an optimal activity in the temperature range 60-65 degrees C and pH range 7.5-9.0 towards p-nitrophenyl caproate. The enzyme was found to be highly resistant to many organic solvents especially induced by ethanolamine. Metal ions showed slight effect on esterase activity. The inhibitor phenylmethanesulfonyl fluoride inhibited strongly the esterase. Triton X-45 induced the activation of EstR, but other detergents slightly to strongly decreased or completely inhibited. Among tested p-NP esters, caproate was the most preferential substrate of this esterase.     

Characterization of a cold-adapted and salt-tolerant esterase from a psychrotrophic bacterium Psychrobacter pacificensis

An esterase gene, est10, was identified from the genomic library of a deep-sea psychrotrophic bacterium Psychrobacter pacificensis. The esterase exhibited the optimal activity around 25 °C and pH 7.5, and maintained as high as 55.0 % of its maximum activity at 0 °C, indicating its cold adaptation. Est10 was fairly stable under room temperatures, retaining more than 80 % of its original activity after incubation at 40 °C for 2 h. The highest activity was observed against the short-chain substrate p-nitrophenyl butyrate (C4) among the tested p-nitrophenyl esters (C2–C16). It was slightly activated at a low concentration (1 mM) of Zn²⁺, Mg²⁺, Ba²⁺, Ca²⁺, Cu²⁺, Fe³⁺, urea and EDTA, but was inhibited by DTT and totally inactivated by PMSF. Interestingly, increased salinity considerably stimulated Est10 activity (up to 143.2 % of original activity at 2 M NaCl) and stability (up to 126.4 % after incubation with 5 M NaCl for 6.5 h), proving its salt tolerance. 0.05 and 0.1 % Tween 20, Tween 80, Triton X-100 and CHAPS increased the activity and stability of Est10 while SDS, CTAB had the opposite effect. Est10 was quite active after incubation with several 30 % organic solvents (methanol, DMSO, ethanediol) but exhibited little activity with 30 % isopropanol, ethanol, n-butanol and acetonitrile.     

Novel serine penicillocarboxypeptidase CPD-S3 from Penicillium janthinellum IBT 3991: Purification, characterization, and uses in peptide synthesis and modification

A novel carboxypeptidase (CPD-S3) from Penicillium janthinellum IBT 3991 has been isolated in a two-step purification procedure by cation exchange and affinity chromatography. The enzyme is a serine carboxypeptidase with a denatured molecular mass determined by SDS of 62 kDa of which 32% is carbohydrate. The isoelectric point is 5.1. CPD-S3 exhibits a high stability towards organic solvents and elevated temperatures. Besides the carboxypeptidase activity, CPD-S3 exhibits esterase, amidase, and carboxamidohydrolase activities. CPD-S3 favors substrates of -configuration with basic amino acid residues in either P₁ or P_1', and particularly dibasic substrates and medium-sized straight-chain alkyl esters for hydrolysis. In aminolysis of esters, amino acid amides and hydrazines coupled in good yield, but methyl esters poorly, and unlike other carboxypeptidases, free amino acids could not be coupled or transpeptidation effected to form amides. In ester semisynthesis, peptides with neutral, but not basic, residues in P₁ could be esterified. The scope of applicability for enzymatic peptide synthesis is limited.