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1.
A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His 6-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase
containing a proline–serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights
of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V
max and K
m of 3.63 ± 0.16 μmol min −1 mg −1 and 0.79 ± 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50–55°C and pH 7–8. Surface plasmon resonance (SPR) analysis demonstrated that
PhaZ-Th catalyzed the degradation of poly-[( R)-3-hydroxybutyrate] (PHB) films, which was accelerated in ( R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm −2 h −1 for poly[( R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films
was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique
will be of utility for further study of bioplastic degradation. 相似文献
2.
A novel esterase gene, estDL30, was isolated from an alluvial metagenomic library using function-driven screening. estDL30 consisted of 1,524 nucleotides and encoded a 507-amino acid protein. Sequence analysis revealed that EstDL30 is similar to
many type B carboxylesterases, containing a G-E-S-A-G pentapeptide with a catalytic Ser residue. Phylogenetic analysis suggested
that EstDL30 belongs to the family VII lipases, together with esterases from Bacillus subtilis (P37967), Streptomyces coelicolor A3(2) (CAA22794), and Arthrobacter oxydans (Q01470). Purified EstDL30 showed its highest catalytic efficiency toward p-nitrophenyl butyrate, with a k
cat of 2293 s −1 and k
cat/ K
m
of 176.4 s −1mM −1; however, little activity was detected when the acyl chain length exceeded C 8. Biochemical characterization of EstDL30 revealed that it is an alkaline esterase that possesses maximal activity at pH 8
and 40° C. The effects of denaturants and divalent cations were also investigated. EstDL30 tolerated well the presence of
methanol and Tween 20. Its activity was strongly inhibited by 1 mM Cu 2+ and Zn 2+, but stimulated by Fe 2+. The unique properties of EstDL30, its high activity under alkaline conditions and stability in the presence of organic solvents,
may render it applicable to organic synthesis. 相似文献
3.
A thermostable esterase from the hyperthemophilic archaeon Sulfolobus solfataricus was partially purified 590-fold with 16.2% recovery. The partially purified esterase had a specific activity of 29.5μmol
min −1 mg −1 when the enzyme activity was determined using p-nitrophenyl butyrate as a substrate. The apparent molecular weight was about 100 kDa, while the optimum temperature and pH
for esterase were 75°C and 8.0, respectively. The enzyme showed high thermal stability and solvent tolerance in comparison
to its mesophilic counterpart. The enzyme also showed chiral resolution activity for (S)-ibuprofen, indicating that S. solfataricus esterase can be used for the production of commercially important chiral drugs. 相似文献
4.
The present study was conducted to investigate the capability of Haloarcula marismortui to synthesize esterases and lipases, and the effect of physicochemical conditions on the growth and the production of esterases
and lipases. Finally, the effect of NaCl concentration and temperature on esterase and lipase activities was studied using
intracellular crude extracts. In order to confirm the genomic prediction about the esterase and lipase synthesis, H. marismortui was cultured on a rich medium and the crude extracts (intra- or extracellular) obtained were assayed for both activities
using p-nitrophenyl esters and triacylglycerides as substrates. Studies on the kinetics of growth and production of esterase and
lipase of H. marismortui were performed, reaching a maximum growth rate of 0.053 h −1 and maximal productions of intracellular esterase and lipase of 2.094 and 0.722 U l −1 using p-nitrophenyl valerate and p-nitrophenyl laurate, respectively. Both enzymes were produced as growth-associated metabolites. The effects of temperature,
pH, and NaCl concentration on the growth rate and production of enzymes were studied by using a Box–Behnken response surface
design. The three response variables were significantly influenced by the physicochemical factors and an interaction effect
between temperature and NaCl concentration was also evidenced. The surface response method estimated the following maximal
values for growth rate and productions of esterase and lipase: 0.086 h −1 (at 42.5°C, pH 7.4, and 3.6 mol l −1 NaCl), 2.3 U l −1 (at 50°C, pH 7.5, and 4.3 mol l −1 NaCl), and 0.58 U l −1 (at 50°C, pH 7.6, and 4.5 mol l −1 NaCl), respectively. Esterases were active at different salt concentrations, showing two optimal activities (at 0.5 and 5 mol l −1 NaCl), which suggested the presence of two different esterases. Interestingly, in the absence of salt, esterase retained
50% residual activity. Esterases and lipase activities were maximal at 45°C and inactive at 75°C. This study represents the
first report evidencing the synthesis of esterase and lipase by H. marismortui. 相似文献
5.
The gene encoding carboxylesterase from the hyperthermophilic bacterium Thermotoga maritima ( tm0053) was cloned. The recombinant protein (EST53) was overexpressed in Escherichia coli without its NH 2-terminal hydrophobic region, and with a C-terminal hexahistidine sequence. The enzyme was purified to homogeneity by heat
treatment, followed by Ni 2+ affinity chromatography, and then characterized. Among the p-nitrophenyl esters tested, the best substrate was p-nitrophenyl decanoate with K
m and k
cat values of 3.1 μM and 10.8 s −1, respectively, at 60°C and pH 7.5. The addition of O,O′-bis(2-aminoethyl)ethyleneglycol- N, N, N′, N′-tetraacetic acid decreased the esterase activity, indicating that EST53 is dependent on the presence of Ca 2+ ion. In addition, its activity was inhibited by the addition of phenylmethylsulfonyl fluoride and diethyl pyrocarbonate,
indicating that it contains serine and histidine residues, which play key roles in the catalytic mechanism. EST53 shows a
relatively high degree of similarity to Burkholderia lipases that belong to family I.2 of the lipolytic enzymes, whereas the local sequence around the pentapeptide of EST53 is
most similar to those of Bacillus lipases belonging to family I.4. 相似文献
6.
EstA was purified from the supernatant by A. lwoffii 16C-1. Its molecular mass was determined to be 45 kDa, and the optimal activity occurred when the pH level was 8.0 at a temperature
of 37°C. The activation energies for the hydrolysis of p-nitrophenyl butyrate was determined to be 11.25 kcal/mol in the temperature range of 10–37°C. The enzyme was unstable at
temperatures higher than 50°C. The Michaelis constant ( K
m
) and V
max for p-nitrophenyl butyrate were 11 μ M and 131.6 μ M min −1 mg of protein-1, respectively. The enzyme was strongly inhibited by Hg 2−, Ca 2+, Mg 2+, Fe 2+, Cu 2+, Zn 2+, Mn 2+, Co 2+, ethylemediaminetetraacetic acid (EDTA), phenylmethylsulfonyl fluoride (PMSF), and diisopropyl fluorophosphate (DFP).
Received: 20 August 2001 / Accepted: 20 September 2001 相似文献
7.
BackgroundDienelactone hydrolases catalyze the hydrolysis of dienelactone to maleylacetate, which play a key role for the microbial degradation of chloroaromatics via chlorocatechols. Here, a thermostable dienelactone hydrolase from thermoacidophilic archaeon Sulfolobus solfataricus P1 was the first purified and characterized and then expressed in Escherichia coli.MethodsThe enzyme was purified by using several column chromatographys and characterized by determining the enzyme activity using p-nitrophenyl caprylate and dienelactones. In addition, the amino acids related to the catalytic mechanism were examined by site-directed mutagenesis using the identified gene. ResultsThe enzyme, approximately 29 kDa monomeric, showed the maximal activity at 74 °C and pH 5.0, respectively. The enzyme displayed remarkable thermostability: it retained approximately 50% of its activity after 50 h of incubation at 90 °C, and showed high stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme displayed substrate specificities toward trans-dienelactone, not cis-isomer, and also carboxylesterase activity toward p-nitrophenyl esters ranging from butyrate (C 4) to laurate (C 12). The kcat/ Km ratios for trans-dienelactone and p-nitrophenyl caprylate (C 8), the best substrate, were 92.5 and 54.7 s −1 μM −1, respectively. ConclusionsThe enzyme is a typical dienelactone hydrolase belonging to α/β hydrolase family and containing a catalytic triad composed of Cys151, Asp198, and His229 in the active site. General significanceThe enzyme is the first characterized archaeal dienelactone hydrolase. 相似文献
8.
Soil metagenome constitutes a reservoir for discovering novel enzymes from the unculturable microbial diversity. From three
plant rhizosphere metagenomic libraries comprising a total of 142,900 members of recombinant plasmids, we obtained 14 recombinant
fosmids that exhibited lipolytic activity. A selected recombinant plasmid, pFLP-2, which showed maximum lipolytic activity,
was further analyzed. DNA sequence analysis of the subclone in pUC119, pELP-2, revealed an open reading frame of 1,191 bp
encoding a 397-amino-acid protein. Purified EstD2 exhibited maximum enzymatic activity towards p-nitrophenyl butyrate, indicating that it is an esterase. Purified EstD2 showed optimal activity at 35 °C and at pH 8.0. The
K
m and K
cat values were determined to be 79.4 μM and 120.5/s, respectively. The esterase exhibited an increase in enzymatic activity
in the presence of 15% butanol and 15% methanol. Phylogenetic analysis revealed that the lipolytic protein EstD2 may be a
member of a novel family of lipolytic enzymes. Several hypothetical protein homologs of EstD2 were found in the database.
A hypothetical protein from Phenylobacterium zucineum HLK1, a close homolog of EstD2, displayed lipolytic activity when the corresponding gene was expressed in Escherichia coli. Our results suggest that the other hypothetical protein homologs of EstD2 might also be members of this novel family. 相似文献
9.
An esterase from Pseudomonas putida JD1 (PPE) was successfully cloned, actively expressed in Escherichia coli, and characterized. It was discovered that PPE is more active towards short-chain esters, hydrolyzed δ-valerolactone, and
ε-caprolactone and was most active at 37°C and pH 8. After purification to homogeneity by Ni–NTA-assisted affinity chromatography,
the kinetic parameters K
M and k
cat were determined for p-nitrophenyl acetate and butyrate, respectively, showing better catalytic efficiency for hydrolysis of the acetate residue.
Investigation of the protein sequence revealed not only the classical catalytic triad for carboxylesterases, additionally
the interesting GGG(A)X-motif, which is associated to activity towards tertiary alcohols, was found. Indeed, enzymatic activity
was shown for a set of different tertiary alcohols with enantioselectivities up to E = 20, suggesting PPE to be a promising biocatalyst. In addition, PPE also hydrolyzed 4-hydroxyphenyl acetate, the product
of a Baeyer–Villiger monooxygenase-catalyzed oxidation of 4-hydroxyacetophenone with a specific activity of 34.36 U/mg suggesting
a physiological role in P. putida JD1. 相似文献
10.
The genome sequence of Streptomyces coelicolor A3(2) contains 51 putative lipase and esterase genes mostly of unknown function. The gene estB (locus SCO 6966) was expressed as a His-tagged protein in E. coli. Esterase B was active at low temperatures exerting its maximum activity at 30°C and retaining more than 25% of its activity
at 4°C. The optimum pH was 8–8.5. The enzyme was active against short synthetic p-nitrophenylesters (C 2–C 10) with maximum activity towards the acetate ester (C 2). The esterase was tested on 13 series of racemic esters of potential interest for the synthesis of chiral pharmaceutical
compounds. 4 of the series were substrates and a modest degree of enantioselectivity was observed (enantiomeric ratios of
1.1–1.9). 相似文献
11.
Dienelactone hydrolase, an α/β hydrolase enzyme, catalyzes the hydrolysis of dienelactone to maleylacetate, an intermediate for the Krebs cycle. Genome sequencing of the psychrophilic yeast, Glaciozyma antarctica predicted a putative open reading frame (ORF) for dienelactone hydrolase (GaDlh) with 52% sequence similarity to that from Coniophora puteana. Phylogenetic tree analysis showed that GaDlh is closely related to other reported dienelactone hydrolases, and distantly related to other α/β hydrolases. Structural prediction using MODELLER 9.14 showed that GaDlh has the same α/β hydrolase fold as other dienelactone hydrolases and esterase/lipase enzymes, with a catalytic triad consisting of Cys–His–Asp and a G–x–C–x–G–G motif. Based on the predicted structure, GaDlh exhibits several characteristics of cold-adapted proteins such as glycine clustering in the binding pocket, reduced protein core hydrophobicity, and the absence of proline residues in loops. The putative ORF was amplified, cloned, and overexpressed in an Escherichia coli expression system. The recombinant protein was overexpressed as soluble proteins and was purified via Ni–NTA affinity chromatography. Biochemical characterization of GaDlh revealed that it has an optimal temperature at 10 °C and that it retained almost 90% of its residual activity when incubated for 90 min at 10 °C. The optimal pH was at pH 8.0 and it was stable between pH 5–9 when incubated for 60 min (more than 50% residual activity). Its Km value was 256 μM and its catalytic efficiency was 81.7 s−1. To our knowledge, this is the first report describing a novel cold-active dienelactone hydrolase-like protein. 相似文献
13.
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 p I 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. 相似文献
14.
Extracellular cholesterol esterase of Burkholderia cepacia strain ST-200 was purified from the culture supernatant. Its molecular mass was 37 kDa. The enzyme was stable at pH 5.5–12 and active at pH 5.5–6, showing optimal activity at pH 7.0 at 45°C. Relative to the commercially available cholesterol esterases, the purified enzyme was highly stable in the presence of various water-miscible organic solvents. The enzyme preferentially hydrolyzed long-chain fatty acid esters of cholesterol, except for that of cholesteryl palmitate. The enzyme exhibited lipolytic activity toward various p-nitrophenyl esters. The hydrolysis rate of p-nitrophenyl caprylate was enhanced 3.5- to 7.2-fold in the presence of 5–20% (vol/vol) water-miscible organic solvents relative to that in the absence of organic solvents. The structural gene encoding the cholesterol esterase was cloned and sequenced. The primary translation product was predicted to be 365 amino acid residues. The mature product is composed of 325 amino acid residues. The amino acid sequence of the product showed the highest similarity to the lipase LipA (87%) from B. cepacia DSM3959. 相似文献
15.
A C–C hydrolase gene ( bphD
LA-4
) from strain Dyella ginsengisoli LA-4 was cloned and expressed in Escherichia coli BL21 (DE3). BphD LA-4 together with another hydrolase MfphA LA-4, which derived from the same strain, possessed esterase activities. p-Nitrophenyl butyrate was the best substrate for both enzymes. BphD LA-4 had high catalytic efficiency to p-nitrophenyl benzoate, whereas MfphA LA-4 had no activity. Homology modeling and docking studies demonstrated that the proper hydrogen bond interaction was important
for the reactivity of specific substrate. 相似文献
16.
In this work, a metagenomic library was generated from peat-swamp forest soil obtained from Narathiwat Province, Thailand. From a fosmid library of approximately 15,000 clones, six independent clones were found to possess lipolytic activity at acidic pH. Analysis of pyrosequencing data revealed six ORFs, which exhibited 34–71% protein similarity to known lipases/esterases. A fosmid clone, designated LP8, which demonstrated the highest level of lipolytic activity under acidic conditions and demonstrated extracellular activity, was subsequently subcloned and sequenced. The full-length lipase/esterase gene, estPS2, was identified. Its deduced amino acid was closely related to a lipolytic enzyme of an uncultured bacterium, and contained the highly conserved motif of a hormone-sensitive family IV lipase. The EstPS2 enzyme exhibited highest activity toward p-nitrophenyl butyrate (C 4) at 37 °C at pH 5, indicating that it was an esterase with activity and secretion characteristics suitable for commercial development. 相似文献
17.
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. 相似文献
18.
A new lipase gene designated as SlLipA was isolated from Serratia liquefaciens S33 DB-1 by the genomic-walking method. The cloned gene contained an open reading frame (ORF) of 1,845 bp encoding 615 amino
acids with a conserved GXSXG motif. Genome sequence analysis showed that an aldo/keto reductase gene closed to the SlLipA gene. The lipase gene was cloned into the expression vector pPICZαA and successfully integrated into the heterologous host,
methylotrophic yeast Pichia pastoris GS115. Five transformants could be expressed as secreted recombinant proteins with the high activity on Triglyceride–Agarose
plate and as candidates to produce the recombinant enzyme. A C-terminal His tag was used for its purification. The lipase
activity of different transformants against substrate para-nitrophenyl laurate ( p-NPL) varied from 14 to 16 U ml −1. For the substrates para-nitrophenyl caprate ( p-NPC), p-NPL, para-nitrophenyl myristate ( p-NPM), para-nitrophenyl palmitate ( p-NPP), and para-nitrophenyl stearate ( p-NPS), the specific activity was shown to be preferred to long acyl chain length of p-NPS. 相似文献
19.
Two extracellular tannin acyl hydrolases (TAH I and TAH II) produced by an Antarctic filamentous fungus Verticillium sp. P9 were purified to homogeneity (7.9- and 10.5-fold with a yield of 1.6 and 0.9%, respectively) and characterized. TAH
I and TAH II are multimeric (each consisting of approximately 40 and 46 kDa sub-units) glycoproteins containing 11 and 26%
carbohydrates, respectively, and their molecular mass is approximately 155 kDa. TAH I and TAH II are optimally active at pH
of 5.5 and 25 and 20°C, respectively. Both the enzymes were activated by Mg 2+and Br − ions and 0.5–2.0 M urea and inhibited by other metal ions (Zn 2+, Cu 2+, K +, Cd 2+, Ag +, Fe 3+, Mn 2+, Co 2+, Hg 2+, Pb 2+ and Sn 2+), anions, Tween 20, Tween 60, Tween 80, Triton X-100, sodium dodecyl sulphate, β-mercaptoethanol, α-glutathione and 4-chloromercuribenzoate.
Both tannases more efficiently hydrolyzed tannic acid than methyl gallate. E
a of these reactions and temperature dependence (at 0–30°C) of k
cat, k
cat/ K
m, Δ G*, Δ H* and Δ S* for both the enzymes and substrates were determined. The k
cat and k
cat/ K
m values (for both the substrates) were considerably higher for the combined preparation of TAH I and TAH II. 相似文献
20.
A clone which conferred lipolytic activity at low temperature was identified from a fosmid library constructed from a South
China Sea marine sediment sample. The gene responsible, estF, consisted of 1,080 bp that encoded 359 amino acid residues, with a typical N-terminal signal peptide of 28 amino acid residues.
A phylogenetic analysis of amino acid sequence with other lipolytic enzymes revealed that EstF and seven closely related putative
lipolytic enzymes comprised a unique clade in the phylogenetic tree. Moreover, these hypothetic esterases showed unique conservative
sites in the amino acid sequence. The recombinant EstF was overexpressed and purified, and its biochemical properties were
partially characterized. The optimal substrate for EstF to hydrolyze among a panel of p-nitrophenyl esters (C2 to C16) was p-nitrophenyl butyrate (C4), with a K
m of 0.46 mM. Activity quickly decreased with substrates containing an acyl chain length longer than 10 carbons. We found that
EstF was active in the temperature range of 0–60°C, showed the best activity at 50°C, but was unstable at 60°C. It exhibited
a high level of activity in the pH range of 7.0–10.0 showing the highest activity at pH 9.0. 相似文献
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