Cloning,sequence analysis,and expression of a gene encoding <Emphasis Type="Italic">Chromobacterium</Emphasis> sp. DS-1 cholesterol oxidase

Chromobacterium sp. strain DS-1 produces an extracellular cholesterol oxidase that is very stable at high temperatures and in the presence of organic solvents and detergents. In this study, we cloned and sequenced the structural gene encoding the cholesterol oxidase. The primary translation product was predicted to be 584 amino acid residues. The mature product is composed of 540 amino acid residues. The amino acid sequence of the product showed significant similarity (53–62%) to the cholesterol oxidases from Burkholderia spp. and Pseudomonas aeruginosa. The DNA fragment corresponding to the mature enzyme was subcloned in the pET-21d(+) expression vector and expressed as an active product in Escherichia coli. The cholesterol oxidase produced from the recombinant E. coli was purified to homogeneity. The physicochemical properties were similar to those of native enzyme purified from strain DS-1. K _m and V _max values of the cholesterol oxidase were estimated from Lineweaver–Burk plots. The V _max/K _m ratio of the enzyme was higher than those of commercially available cholesterol oxidases. The circular dichroism spectral analysis of the recombinant DS-1 enzyme and Burkholderia cepacia ST-200 cholesterol oxidase showed that the conformational stability of the DS-1 enzyme was higher than that of B. cepacia ST-200 enzyme at higher temperatures.     

Purification and characterization of a maltooligosaccharide-forming amylase that improves product selectivity in water-miscible organic solvents, from dimethylsulfoxide-tolerant Brachybacterium sp. strain LB25

A bacterium that secretes maltooligosaccharide-forming amylase in a medium containing 12.5% (vol/vol) dimethylsulfoxide (DMSO) was isolated and identified as Brachybacterium sp. strain LB25. The amylase of the strain was purified from the culture supernatant, and its molecular mass was 60 kDa. The enzyme was stable at pH 7.0–8.5 and active at pH 6.0–7.5. The optimum temperature at pH 7.0 was 35°C in the presence of 5 mM CaCl₂. The enzyme hydrolyzed starch to produce maltotriose primarily. The enzyme was active in the presence of various organic solvents. Its yield and product selectivity of maltooligosaccharides in the presence of DMSO or ethanol were compared with those of the industrial maltotriose-forming amylase from Microbacterium imperiale. Both enzymes improved the production selectivity of maltotriose by the addition of DMSO or ethanol. However, the total maltooligosaccharide yield in the presence of the solvents was higher for LB25 amylase than for M. imperiale amylase.     

A novel, extremely alkaliphilic and cold-active esterase from Antarctic desert soil

A novel, cold-active and highly alkaliphilic esterase was isolated from an Antarctic desert soil metagenomic library by functional screening. The 1,044 bp gene sequence contained several conserved regions common to lipases/esterases, but lacked clear classification based on sequence analysis alone. Moderate (<40%) amino acid sequence similarity to known esterases was apparent (the closest neighbour being a hypothetical protein from Chitinophaga pinensis), despite phylogenetic distance to many of the lipolytic “families”. The enzyme functionally demonstrated activity towards shorter chain p-nitrophenyl esters with the optimal activity recorded towards p-nitrophenyl propionate (C3). The enzyme possessed an apparent T_opt at 20°C and a pH optimum at pH 11. Esterases possessing such extreme alkaliphily are rare and so this enzyme represents an intriguing novel locus in protein sequence space. A metagenomic approach has been shown, in this case, to yield an enzyme with quite different sequential/structural properties to known lipases. It serves as an excellent candidate for analysis of the molecular mechanisms responsible for both cold and alkaline activity and novel structure–function relationships of esterase activity.     

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.     

A new esterase showing similarity to putative dienelactone hydrolase from a strict marine bacterium, <Emphasis Type="Italic">Vibrio</Emphasis> sp. GMD509

Vibrio sp. GMD509, a marine bacterium isolated from eggs of the sea hare, exhibited lipolytic activity on tributyrin (TBN) plate, and the gene representing lipolytic activity was cloned. As a result, an open reading frame (ORF) consisting of 1,017 bp (338 aa) was found, and the deduced amino acid sequence of the ORF showed low similarity (<20%) to α/β hydrolases such as dienelactone hydrolases and esterase/lipase with G–X₁–S–X₂–G sequence conserved. Phylogenetic analysis suggested that the protein belonged to a new family of esterase/lipase together with various hypothetical proteins. The enzyme was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme (Vlip509) showed the best hydrolyzing activity toward p-nitrophenyl butyrate (C₄) among various p-nitrophenyl esters (C₂ to C₁₈), and optimal activity of Vlip509 occurred at 30°C and pH 8.5, respectively. Kinetic parameters toward p-nitrophenyl butyrate were determined as K _m (307 μM), k _cat (5.72 s⁻¹), and k _cat/K _m (18.61 s⁻¹ mM⁻¹). Furthermore, Vlip509 preferentially hydrolyzed the S-enantiomer of racemic ofloxacin ester. Despite its sequence homology to dienelactone hydrolase, Vlip509 showed no dienelactone hydrolase activity. This study represents the identification of a novel lipolytic enzyme from marine environment.     

Properties of free and immobilised lipase from Burkholderia cepacia in organic media

The purified lipase from Burkholderia cepacia was immobilised on a porous polypropylene support and its biocatalytic properties were compared with those of the free enzyme in organic media. For both lipase preparations, the rate of p-nitrophenyl ester hydrolysis in n-heptane was not restricted by mass transfer limitations. The immobilisation changed neither the temperature at which the reaction rate was maximal, nor the activation energy of the reaction. The enzyme stability was slightly decreased (1.3-fold) upon immobilisation. Moreover, the immobilised enzyme displayed fewer variations of activity with fatty acid chain length. Interestingly, for all the different p-nitrophenyl esters used, the immobilised enzyme was more active (from 5.8- to 18.9-fold) than the free enzyme. Therefore, it would be very useful to use B. cepacia lipase immobilised onto porous polypropylene for applications in organic media, as it displayed high activities on a larger range of substrates. Received: 8 February 1999 / Received revision: 19 March 1999 / Accepted: 20 March 1999     

Purification and properties of a highly enantioselective l-menthyl acetate hydrolase from Burkholderia cepacia

A highly enantioselective l-menthyl acetate esterase was purified to homogeneity from Burkholderia cepacia ATCC 25416, with a recovery of 4.8% and a fold purification of 22.7. The molecular weight of the esterase was found to be 37 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The N-terminal amino acid sequence was “MGARTDA”, and there was no homology in contrast to other Burkholderia sp. esterases. This enzyme preferentially hydrolyzed short-chain fatty acid esters of menthol with high stereospecificity and high hydrolytic activity, while long-chain l-menthyl esters were poor substrates. Considered its substrate specificity and N-terminal sequence, this esterase was concluded as a new enzyme belonging to the carboxylesterase group (EC 3.1.1.1) of esterase family. The optimum temperature and pH for enzyme activity using racemic menthyl acetate as substrate were 30 °C and 7.0, respectively. The esterase was more stable in the pH range of 7.0–9.0 and temperature range of 30–40 °C. Hydrolytic activity was enhanced by Ca²⁺, K⁺ and Mg²⁺, but completely inhibited by Hg²⁺, Cu²⁺, ionic detergents and phenylmethylsulfonyl fluoride (PMSF) at 0.01 M concentration.     

Cloning,expression and characterization of the esterase estUT1 from Ureibacillus thermosphaericus which belongs to a new lipase family XVIII

A new esterase gene from thermophilic bacteria Ureibacillus thermosphaericus was cloned into the pET32b vector and expressed in Escherichia coli BL21(DE3). Alignment of the estUT1 amino acid sequence revealed the presence of a novel canonical pentapeptide (GVSLG) and 41–47% identity to the closest family of the bacterial lipases XIII. Thus the esterase estUT1 from U. thermosphaericus was assigned as a member of the novel family XVIII. It also showed a strong activity toward short-chain esters (C2–C8), with the highest activity for C2. When p-nitrophenyl butyrate is used as a substrate, the temperature and pH optimum of the enzyme were 70–80 °C and 8.0, respectively. EstUT1 showed high thermostability and 68.9 ± 2.5% residual activity after incubation at 70 °C for 6 h. Homology modeling of the enzyme structure showed the presence of a putative catalytic triad Ser93, Asp192, and His222. The activity of estUT1 was inhibited by PMSF, suggesting that the serine residue is involved in the catalytic activity of the enzyme. The purified enzyme exhibited high stability in organic solvents. EstUT1 retained 85.8 ± 2.4% residual activity in 30% methanol at 50 °C for 6 h. Stability at high temperature and tolerance to organic solvents make estUT1 a promising enzyme for biotechnology application.

     

Diversity of polyester-degrading bacteria in compost and molecular analysis of a thermoactive esterase from Thermobifida alba AHK119

More than 100 bacterial strains were isolated from composted polyester films and categorized into two groups, Actinomycetes (four genera) and Bacillus (three genera). Of these isolates, Thermobifida alba strain AHK119 (AB298783) was shown to possess the ability to significantly degrade aliphatic-aromatic copolyester film as well as decreasing the polymer particle sizes when grown at 50°C on LB medium supplemented with polymer particles, yielding terephthalic acid. The esterase gene (est119, 903 bp, encoding a signal peptide and a mature protein of 34 and 266 amino acids, respectively) was cloned from AHK119. The Est119 sequence contains a conserved lipase box (–G-X-S-X-G-) and a catalytic triad (Ser129, His207, and Asp175). Furthermore, Tyr59 and Met130 likely form an oxyanion hole. The recombinant enzyme was purified from cell-free extracts of Escherichia coli Rosetta-gami B (DE3) harboring pQE80L-est119. The enzyme is a monomeric protein of ca. 30 kDa, which is active from 20°C to 75°C (with an optimal range of 45 to 55°C) and in a pH range of 5.5 to 7.0 (with an optimal pH of 6.0). Its preferred substrate among the p-nitrophenyl acyl esters (C₂ to C₈) is p-nitrophenyl hexanoate (C₆), indicating that the enzyme is an esterase rather than a lipase.     

Identification and characterization of a novel cold-adapted esterase from a metagenomic library of mountain soil

A novel lipolytic enzyme was isolated from a metagenomic library after demonstration of lipolytic activity on an LB agar plate containing 1% (w/v) tributyrin. A novel esterase gene (estIM1), encoding a lipolytic enzyme (EstIM1), was cloned using a shotgun method from a pFosEstIM1 clone of the metagenomic library, and the enzyme was characterized. The estIM1 gene had an open reading frame (ORF) of 936 base pairs and encoded a protein of 311 amino acids with a molecular mass 34 kDa and a pI value of 4.32. The deduced amino acid sequence was 62% identical to that of an esterase from an uncultured bacterium (ABQ11271). The amino acid sequence indicated that EstIM1 was a member of the family IV of lipolytic enzymes, all of which contain a GDSAG motif shared with similar enzymes of lactic acid microorganisms. EstIM1 was active over a temperature range of 1–50°C, at alkaline pH. The activation energy for hydrolysis of p-nitrophenyl propionate was 1.04 kcal/mol, within a temperature range of 1–40°C. The activity of EstIM1 was about 60% of maximal even at 1°C, suggesting that EstIM1 is efficiently cold-adapted. Further characterization of this cold-adapted enzyme indicated that the esterase may be very valuable in industrial applications.     

Production, purification, and properties of a lipase from a bacterium (Pseudomonas aeruginosa YS-7) capable of growing in water-restricted environments.

An extracellular lipase from the low-water-tolerant bacterium P. aeruginosa YS-7 was produced, purified, and characterized with respect to its functional properties in aqueous solutions and organic solvents. The enzyme was partially released from the cells during fermentation in defined medium with 5% (wt/vol) soybean oil. Approximately one-half of the total culture activity remained in solution after removal of cells. More than 95% of the activity was found in culture supernatant after mild detergent treatment (10 mM sodium deoxycholate) or after shifting the carbon source during the fermentation from triglyceride to a free fatty acid. The enzyme was recovered from an acetone precipitate of the whole culture and purified by hydrophobic interaction chromatography, yielding a preparation having a specific activity of about 1,300 mumol of fatty acid mg-1 h-1. The lipase (molecular size, approximately 40 kDa) hydrolyzes a variety of fatty acid esters and has an optimum pH of about 7. The enzyme retained its full activity at 20 to 55 degrees C, even after prolonged exposure (more than 30 days) to different concentrations of water-miscible organic solvents such as alcohols, glycols, pyridine, acetonitrile, dimethyl formamide, and dimethyl sulfoxide. The hydrolysis of 4-nitrophenyl laurate ester and of triglyceride emulsified in water was slightly accelerated with increasing concentrations of alcohols and glycols up to about 20% but was abolished with a further increase in alcohol concentration or in the presence of acetonitrile. In contrast, the rate of hydrolysis of these substrates in concentrated solutions of dimethyl formamide or dimethyl sulfoxide was markedly increased, by more than twofold and more than fivefold, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Production, purification, and properties of a lipase from a bacterium (Pseudomonas aeruginosa YS-7) capable of growing in water-restricted environments.

An extracellular lipase from the low-water-tolerant bacterium P. aeruginosa YS-7 was produced, purified, and characterized with respect to its functional properties in aqueous solutions and organic solvents. The enzyme was partially released from the cells during fermentation in defined medium with 5% (wt/vol) soybean oil. Approximately one-half of the total culture activity remained in solution after removal of cells. More than 95% of the activity was found in culture supernatant after mild detergent treatment (10 mM sodium deoxycholate) or after shifting the carbon source during the fermentation from triglyceride to a free fatty acid. The enzyme was recovered from an acetone precipitate of the whole culture and purified by hydrophobic interaction chromatography, yielding a preparation having a specific activity of about 1,300 mumol of fatty acid mg-1 h-1. The lipase (molecular size, approximately 40 kDa) hydrolyzes a variety of fatty acid esters and has an optimum pH of about 7. The enzyme retained its full activity at 20 to 55 degrees C, even after prolonged exposure (more than 30 days) to different concentrations of water-miscible organic solvents such as alcohols, glycols, pyridine, acetonitrile, dimethyl formamide, and dimethyl sulfoxide. The hydrolysis of 4-nitrophenyl laurate ester and of triglyceride emulsified in water was slightly accelerated with increasing concentrations of alcohols and glycols up to about 20% but was abolished with a further increase in alcohol concentration or in the presence of acetonitrile. In contrast, the rate of hydrolysis of these substrates in concentrated solutions of dimethyl formamide or dimethyl sulfoxide was markedly increased, by more than twofold and more than fivefold, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 degrees C), but also at ambient temperature (1,050 U/mg at 30 degrees 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 (C(6)) 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.     

Catalytic activity and stability of xanthine oxidase in aqueous-organic mixtures

In the present study, bovine milk xanthine oxidase activity in various aqueous-organic mixtures and the effects of pH, temperature, and lyophilization on the enzyme activity have been investigated. The enzyme was incubated with xan-thine as the substrate in Sorenson’s phosphate buffer (pH 7.0) containing 0.1 mM EDTA, and the activity was determined spectrophotometrically in the absence and presence of different fractions of nine water-miscible organic solvents at 27–50°C and at different pH values ranging from 6 to 9. The organic solvents reduced the enzyme activity to different extents. In spite of these inhibitory effects, the enzyme showed relatively good stability in the aqueous-organic mixtures compared with the aqueous medium. A significant increase in the activity of the lyophilized enzyme was observed in pure organic solvents. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 1, pp. 124–130.     

Isolation and characterization of a thermostable esterase from a metagenomic library

A novel esterase gene was isolated by functional screening of a metagenomic library prepared from an activated sludge sample. The gene (est-XG2) consists of 1,506 bp with GC content of 74.8 %, and encodes a protein of 501 amino acids with a molecular mass of 53 kDa. Sequence alignment revealed that Est-XG2 shows a maximum amino acid identity (47 %) with the carboxylesterase from Thermaerobacter marianensis DSM 12885 (YP_004101478). The catalytic triad of Est-XG2 was predicted to be Ser₁₉₂-Glu₃₁₃-His₄₁₂ with Ser₁₉₂ in a conserved pentapeptide (GXSXG), and further confirmed by site-directed mutagenesis. Phylogenetic analysis suggested Est-XG2 belongs to the bacterial lipase/esterase family VII. The recombinant Est-XG2, expressed and purified from Escherichia coli, preferred to hydrolyze short and medium length p-nitrophenyl esters with the best substrate being p-nitrophenyl acetate (K _m and k _cat of 0.33 mM and 36.21 s^?1, respectively). The purified enzyme also had the ability to cleave sterically hindered esters of tertiary alcohols. Biochemical characterization of Est-XG2 revealed that it is a thermophilic esterase that exhibits optimum activity at pH 8.5 and 70 °C. Est-XG2 had moderate tolerance to organic solvents and surfactants. The unique properties of Est-XG2, high thermostability and stability in the presence of organic solvents, may render it a potential candidate for industrial applications.     

Cloning,expression and characterization of a halotolerant esterase from a marine bacterium <Emphasis Type="Italic">Pelagibacterium halotolerans</Emphasis> B2<Superscript>T</Superscript>

An esterase PE10 (279 aa) from Pelagibacterium halotolerans B2^T was cloned and overexpressed in Escherichia coli Rosetta in a soluble form. The deduced protein was 29.91 kDa and the phylogenetic analysis of the deduced amino acids sequence showed it represented a new family of lipolytic enzymes. The recombinant protein was purified by Ni–NTA affinity chromatography column and the characterization showed its optimal temperature and pH were 45 °C and pH 7.5, respectively. Substrate specificity study showed PE10 preferred short chain p-nitrophenyl esters and exhibited maximum activity toward p-nitrophenyl acetate. In addition, PE10 was a halotolerant esterase as it was still active under 4 M NaCl. Three-dimensional modeling of PE10 suggested that the high negative electrostatic potential on the surface may relevant to its tolerance to high salt environment. With this halotolerance property, PE10 could be a candidate for industrial use.     

A Cold-Adapted Lipase of an Alaskan Psychrotroph, Pseudomonas sp. Strain B11-1: Gene Cloning and Enzyme Purification and Characterization

A psychrotrophic bacterium producing a cold-adapted lipase upon growth at low temperatures was isolated from Alaskan soil and identified as a Pseudomonas strain. The lipase gene (lipP) was cloned from the strain and sequenced. The amino acid sequence deduced from the nucleotide sequence of the gene (924 bp) corresponded to a protein of 308 amino acid residues with a molecular weight of 33,714. LipP also has consensus motifs conserved in other cold-adapted lipases, i.e., Lipase 2 from Antarctic Moraxella TA144 (G. Feller, M. Thiry, J. L. Arpigny, and C. Gerday, DNA Cell Biol. 10:381–388, 1991) and the mammalian hormone-sensitive lipase (D. Langin, H. Laurell, L. S. Holst, P. Belfrage, and C. Holm, Proc. Natl. Acad. Sci. USA 90:4897–4901, 1993): a pentapeptide, GDSAG, containing the putative active-site serine and an HG dipeptide. LipP was purified from an extract of recombinant Escherichia coli C600 cells harboring a plasmid coding for the lipP gene. The enzyme showed a 1,3-positional specificity toward triolein. p-Nitrophenyl esters of fatty acids with short to medium chains (C₄ and C₆) served as good substrates. The enzyme was stable between pH 6 and 9, and the optimal pH for the enzymatic hydrolysis of tributyrin was around 8. The activation energies for the hydrolysis of p-nitrophenyl butyrate and p-nitrophenyl laurate were determined to be 11.2 and 7.7 kcal/mol, respectively, in the temperature range 5 to 35°C. The enzyme was unstable at temperatures higher than 45°C. The K_m of the enzyme for p-nitrophenyl butyrate increased with increases in the assay temperature. The enzyme was strongly inhibited by Zn²⁺, Cu²⁺, Fe³⁺, and Hg²⁺ but was not affected by phenylmethylsulfonyl fluoride and bis-nitrophenyl phosphate. Various water-miscible organic solvents, such as methanol and dimethyl sulfoxide, at concentrations of 0 to 30% (vol/vol) activated the enzyme.     

Isolation of a bacterium that degrades urethane compounds and characterization of its urethane hydrolase

A bacterium which degrades urethane compounds was isolated and identified as Rhodococcus equi strain TB-60. Strain TB-60 degraded toluene-2,4-dicarbamic acid dibutyl ester (TDCB) and accumulated toluene diamine as the degradation product. The enzyme which cleaves urethane bond in TDCB was strongly induced by acetanilide. The purified enzyme (urethane hydrolase) was found to be homogeneous on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The molecular weight was estimated to be 55 kDa. The optimal temperature and pH were 45°C and 5.5, respectively. The enzyme hydrolyzed aliphatic urethane compound as well as aromatic ones. The activity was inhibited by HgCl₂, p-chrolomercuribenzoic acid, and phenylmethylsulfonyl fluoride, suggesting that cysteine and/or serine residues play an important role in the activity. The enzyme catalyzed the hydrolysis of anilides, amides, and esters as well as TDCB. It was characterized as a novel amidase/esterase, differing in some properties from other known amidases/esterases.     

A Maltooligosaccharide-Forming Amylase Gene from Brachybacterium sp. Strain LB25: Cloning and Expression in Escherichia coli

Brachybacterium sp. strain LB25 produces a maltooligosaccharide-forming amylase that improves product selectivity in water-miscible organic solvents. The enzyme hydrolyzed starch to produce maltotriose primarily. The structural gene encoding the amylase from strain LB25 was cloned and sequenced. The amino acid sequence of the product showed significant similarity (45 to 49%) to amylases from the genus Streptomyces. The amylase gene was expressed in Escherichia coli, but the specific activity of the recombinant amylase was lower than that of the amylase purified from strain LB25.     

Isolation of the Volatile Components of Fresh Onion by Thermal Desorption Cold Trap Capillary Gas Chromatography

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°C and the half-life was 1 h at 64°C. Maximum activity was observed on p-nitrophenyl caproate with little activity toward long-chainfatty acid esters. The enzyme had a K_M of 0.52 mM for p-nitrophenyl caproate hydrolysis at pH 8 and 37°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.