Molecular cloning, and characterization of a modular acetyl xylan esterase from the edible straw mushroom Volvariella volvacea

A new Volvariella volvacea gene encoding an acetyl xylan esterase (designated as Vvaxe1) was cloned and expressed in Pichia pastoris. The cDNA contained an ORF of 1047 bp encoding 349 amino acids with a calculated mass of 39 990 Da. VvAXE1 is a modular enzyme consisting of an N-terminal signal peptide, a catalytic domain, and a cellulose-binding domain. The amino acid sequence of the enzyme exhibited a high degree of similarity to cinnamoyl esterase B from Penicillium funiculosum, and acetyl xylan esterases from Aspergillus oryzae, Penicillium purpurogenum, and Aspergillus ficuum. Recombinant acetyl xylan esterase released acetate from several acetylated substrates including beta-d-xylose tetraacetate and acetylated xylan. No activity was detectable on p-nitrophenyl acetate. Enzyme-catalyzed hydrolysis of 4-methylumbelliferyl acetate was maximal at pH 8.0 and 60 degrees C, and reciprocal plots revealed an apparent K(m) value of 307.7 microM and a V(max) value of 24 733 IU micromol(-1) protein. ReAXE1 also exhibited a capacity to bind to Avicel and H(3)PO(4) acid-swollen cellulose.     

Purification and properties of an acetylxylan esterase from Fibrobacter succinogenes S85.

An acetylxylan esterase (EC 3.1.1.6) was purified to apparent homogeneity from the nonsedimentable extracellular culture fluid of Fibrobacter succinogenes S85 grown on cellulose. This enzyme had an apparent molecular mass of 55 kDa and an isoelectric point of 4.0. The temperature and pH optima were 45 degrees C and 7.0, respectively. The apparent Km and Vmax were 2.7 mM and 9,100 U/mg, respectively, for the hydrolysis of alpha-naphthyl acetate. The enzyme cleaved acetyl residues from birchwood acetylxylan but did not hydrolyze carboxymethylcellulose, larchwood xylan, ferulic acid-arabinose-xylose polymer, p-nitrophenyl-alpha-L-arab-inofuranoside, or longer-chain naphthyl fatty acid esters. The esterase enzyme may play a role in enhancing hemicellulose degradation by F. succinogenes, thereby allowing it greater access to cellulose present in forage cell walls.     

Acetyl esterase production by Termitomyces clypeatus

Production of acetyl esterase by Termitomyces clypeatus was stimulated by xylan, cellulose, arabinose and arabinose-containing polysaccharides in the growth medium. The culture filtrate was equally active with p-nitrophenyl acetate and acetyl xylan. Acetyl xylan was completely deacetylated by the enzyme. Activity was optimum at pH 6.5 and at 50¡C. The Km values for p-nitrophenyl acetate and acetyl xylan were 0.83 mM and 0.38% (w/v) with Vm of 48 and 55 mmole acetate produced/min.mg protein, respectively.     

Purification and properties of an acetylxylan esterase from Fibrobacter succinogenes S85

An acetylxylan esterase (EC 3.1.1.6) was purified to apparent homogeneity from the nonsedimentable extracellular culture fluid of Fibrobacter succinogenes S85 grown on cellulose. This enzyme had an apparent molecular mass of 55 kDa and an isoelectric point of 4.0. The temperature and pH optima were 45 degrees C and 7.0, respectively. The apparent Km and Vmax were 2.7 mM and 9,100 U/mg, respectively, for the hydrolysis of alpha-naphthyl acetate. The enzyme cleaved acetyl residues from birchwood acetylxylan but did not hydrolyze carboxymethylcellulose, larchwood xylan, ferulic acid-arabinose-xylose polymer, p-nitrophenyl-alpha-L-arab-inofuranoside, or longer-chain naphthyl fatty acid esters. The esterase enzyme may play a role in enhancing hemicellulose degradation by F. succinogenes, thereby allowing it greater access to cellulose present in forage cell walls.     

Purification and characterization of a heat-stable esterase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius.

A heat-stable esterase has been purified 1080-fold to electrophoretic homogeneity from Sulfolobus acidocaldarius, a thermoacidophilic archaebacterium; 20% of the starting activity is recovered. The purified enzyme shows a specific activity of 158 units/mg, based on the hydrolysis of p-nitrophenyl acetate. The esterase hydrolyses short-chain p-nitrophenyl esters, aliphatic esters and triacylglycerols. It is strongly inhibited by paraoxon and phenylmethanesulphonyl fluoride, but only weakly by eserine. From sedimentation-equilibrium data and molecular sieving in polyacrylamide gels, the Mr of the esterase is estimated to be 117000-128000. SDS/polyacrylamide-gel electrophoresis reveals a single band of protein, of Mr 32000. The purified esterase crystallizes in the presence of poly(ethylene glycol) in short rods. The enzyme is inactivated only on prolonged storage at temperature above 90 degrees C.     

Purification and characterisation of an ester hydrolase from a strain of Arthrobacter species: its application in asymmetrisation of 2-benzyl-1,3-propanediol acylates

An ester hydrolase (ABL) has been isolated from a strain of Arthrobacter species (RRLJ-1/95) maintained in the culture collection of this laboratory. The purified enzyme has a specific activity of 1700 U/mg protein and is found to be composed of a single subunit (Mr 32,000), exhibiting both lipase and esterase activities shown by hydrolysis of triglycerides and p-nitrophenyl acetate respectively. Potential application of the enzyme concerns the asymmetrisation of prochiral 2-benzyl-1,3-propanediol esters besides enantioselective hydrolysis of alkyl esters of unsubstituted and substituted 1-phenyl ethanols.     

利用结构多样性的对硝基苯酚羧酸酯和脂肪醇乙酸酯评估7个枯草芽胞杆菌酯水解酶的指纹图谱

基于GenBank公布的枯草芽胞杆菌168基因组序列,克隆表达了30个预测的酯水解酶基因。结果发现:其中7个酶对对硝基苯酚酯表现出明显的酯水解活力。它们在α/β水解酶家族中分属5个不同的亚家族。通过显色底物和pH指示剂进行的高通量筛选,分别绘制了这7个酶的底物指纹谱。考察了酶催化手性酯水解反应的对映选择性,结果表明:对硝基苄基酯酶PnbA和S-脱乙酰化酶Cah对手性醇的乙酸酯具有较广的底物谱,而PnbA和羧酸酯酶Nap分别对DL-薄荷醇乙酸酯和2-氯-1-苯乙醇乙酸酯/2-萘乙醇乙酸酯有极好的对映选择性(E>200)。此外,发现酯酶YitV催化2-氯-1-苯乙醇乙酸酯水解的反应遵循反-Kazlauskas规则。     

Purification and characterization of an endo-1,4-beta-glucanase from Neisseria sicca SB that hydrolyzes beta-1,4 linkages in cellulose acetate

An enzyme catalyzing hydrolysis of beta-1,4 bonds in cellulose acetate was purified 18.3-fold to electrophoretic homogeneity from a culture supernatant of Neisseria sicca SB, which can assimilate cellulose acetate as the sole carbon and energy source. The molecular mass of the enzyme was 41 kDa and the isoelectric point was 4.8. The pH and temperature optima of the enzyme were 6.0-7.0 and 60 degrees C. The enzyme catalyzed hydrolysis of water-soluble cellulose acetate (degree of substitution, 0.88) and carboxymethyl cellulose. The Km and Vmax for water-soluble cellulose acetate and carboxymethyl cellulose were 0.242% and 2.24 micromol/min/mg, and 2.28% and 12.8 micromol/min/mg, respectively. It is estimated that the enzyme is a kind of endo-1,4-beta-glucanase (EC 3.2.1.4) from the substrate specificity and hydrolysis products of cellooligosaccharides. The enzyme and cellulose acetate esterase from Neisseria sicca SB degraded water-insoluble cellulose acetate by synergistic action.     

Purification and Characterization of an Esterase Involved in Poly(vinyl alcohol) Degradation by Pseudomonas vesicularis PD

An esterase catalyzing the hydrolysis of acetyl ester moieties in poly(vinyl alcohol) was purified 400-fold to electrophoretic homogeneity from the cytoplasmic fraction of Pseudomonas vesicularis PD, which was capable of assimilating poly(vinyl alcohol) as the sole carbon and energy source. The purified enzyme was a homodimeric protein with a molecular mass of 80 kDa and the isoelectric point was 6.8. The pH and temperature optima of the enzyme were 8.0 and 45°C. The enzyme catalyzed the hydrolysis of side chains of poly(vinyl alcohol), short-chain p-nitrophenyl esters, 2-naphthyl acetate, and phenyl acetate, and was slightly active toward aliphatic esters. The enzyme was also active toward the enzymatic degradation products, acetoxy hydroxy fatty acids, of poly(vinyl alcohol). The K _m and V _max of poly(vinyl alcohol) (degree of polymerization, 500; saponification degree, 86.5-89.0 mol%) and p-nitrophenyl acetate were 0.381% (10.6 mM as acetyl content in the polymer) and 2.56 μM, and 6.52 and 12.6 μmol/min/mg, respectively. The enzyme was strongly inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate at a concentration of 5 mM, which indicated that the enzyme was a serine esterase. The pathway for the metabolism of poly(vinyl alcohol) is also discussed.     

Isolation and characterization of sialate 9(4)-O-acetylesterase from influenza C virus

An esterase was isolated from influenza C virus with a specific activity from 1.7-5 U/mg protein, and its substrate specificity was tested with various naturally occurring O-acylated sialic acids, synthetic carbohydrate acetates, and other esters. The enzyme hydrolyses only acetic acid esters at significant rates. The non-natural substrates 4-methyl-umbelliferyl acetate, 4-nitrophenyl acetate, and alpha-naphthyl acetate are cleaved at highest hydrolysis rates, followed by the natural substrate N-acetyl-9-O-acetylneuraminic acid. The esterase also acts on N-glycoloyl-9-O-acetylneuraminic acid and, much slower, on N-acetyl-4-O-acetylneuraminic acid; N-acetyl-7-O-acetylneuraminic acid is not hydrolysed. 2-Deoxy-2,3-didehydro-N-acetyl-9-O-acetylneuraminic acid is also a substrate for this enzyme, however, 6-O-acetylated N-acetylmannosamine and glucose are not. Esterification of the carboxyl function of sialic acids strongly reduces or prevents esterase action on O-acetyl groups. The carboxyl ester is not hydrolysed. The relative cleavage rates also depend on the type of the non-sialic acid part of the molecule. N-Acetyl-9-O-acetylneuraminic acid as component of sialyllactose and rat serum glycoprotein shows hydrolysis rates close to the free form of this sugar, while acetyl ester groups of bovine submandibular gland mucin and rat erythrocytes are hydrolysed at slower rates. Gangliosides and 4-O-acetylated glycoproteins are no substrates for the purified enzyme. A slow hydrolysis is observed by incubation of 9-O-acetylated GD1a with intact influenza C viruses. As other natural acetyl esters (acetyl-CoA and acetylthiocholine iodide) are not hydrolysed, the enzyme can be classified as sialate 9(4)-O-acetylesterase (EC 3.1.1.53).     

Coenzyme A- and NADH-dependent esterase activity of methylmalonate semialdehyde dehydrogenase.

Methylmalonate semialdehyde dehydrogenase purified to homogeneity from rat liver possesses, in addition to its coupled aldehyde dehydrogenase and CoA ester synthetic activity, the ability to hydrolyze p-nitrophenyl acetate. The following observations suggest that this activity is an active site phenomenon: (a) p-nitrophenyl acetate hydrolysis was inhibited by malonate semialdehyde, substrate for the dehydrogenase reaction; (b) p-nitrophenyl acetate was a strong competitive inhibitor of the dehydrogenase activity; (c) NAD+ and NADH activated the esterase activity; (d) coenzyme A, acceptor of acyl groups in the dehydrogenase reaction, accelerated the esterase activity; and (e) the product of the esterase reaction proceeding in the presence of coenzyme A was acetyl-CoA. These findings suggest that an S-acyl enzyme (thioester intermediate) is likely common to both the esterase reaction and the aldehyde dehydrogenase/CoA ester synthetic reaction.     

Purification and characterization of cholesterol esterase from porcine pancreas.

A protein catalyzing the hydrolysis of cholesterol esters and p-nitrophenyl acetate has been purified 200-fold from porcine pancreas. The enzyme is homogenous as judged by polyacrylamide gel electrophoresis and exhibits a molecular weight of 80 000 as determined by sodium dodecyl sulfate electrophoresis and gel filtration. Activity toward p-nitrophenylacetate exhibits a broad pH optimum and is influenced by a group with a pKa of 5.5--6.0. The enzyme is completely inhibited by diisopropylfluorophosphate at concentrations as low as 10(-5) M, suggesting that it is a serine esterase. Partial inhibition was observed with p-chloromercuribenzoate.     

Human mitochondrial aldehyde dehydrogenase substrate specificity: comparison of esterase with dehydrogenase reaction.

Substrate specificity of human mitochondrial low Km aldehyde dehydrogenase (EC 1.2.1.3) E2 isozyme has been investigated employing p-nitrophenyl esters of acyl groups of two to six carbon atoms and comparing with that of aldehydes of one to eight carbon atoms. The esterase reaction was studied under three conditions: in the absence of coenzyme, in the presence of NAD (1 mM), and in the presence of NADH (160 microM). The maximal velocity of the esterase reaction with p-nitrophenyl acetate and propionate as substrates in the presence of NAD was 3.9-4.7 times faster than that of the dehydrogenase reaction. Under all other conditions the velocities of dehydrogenase and esterase reactions were similar; the lowest kcat was for p-nitrophenyl butyrate in the presence of NAD. Stimulation of esterase activity by coenzymes was confined to esters of short acyl chain length; with longer acyl chain lengths or increased bulkiness (p-nitrophenyl guanidinobenzoate) no effect or even inhibition was observed. Comparison of kinetic constants for esters demonstrates that p-nitrophenyl butyrate is the worst substrate of all esters tested, suggesting that the active site topography is uniquely unfavorable for p-nitrophenyl butyrate. This fact is, however, not reflected in kinetic constants for butyraldehyde, which is a good substrate. The substrate specificity profile as determined by comparison of kcat/Km ratios was found to be quite different for aldehydes and esters. For aldehydes kcat/Km ratios increased with the increase of chain length; with esters under all three conditions, a V-shaped curve was produced with a minimum at p-nitrophenyl butyrate.     

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.     

Purification and properties of an acetyl specific carboxylesterase from Nocardia mediterranei

Summary An acetyl specific carboxylesterase has been purified from Nocardia mediterranei. The purified enzyme is homogeneous as shown by SDS polyacrylamide gel electrophoresis. The esterase has a molecular weight of 68,000 and is composed of two identical subunits. The enzyme exhibits optimal activity at pH 7.5 and at 35°C and is stable below 40°C. The enzyme activity is inhibited by several sulfhydryl reagents. The esterase hydrolyzes preferentially acetyl esters. Propionyl esters are cleaved very slowly whereas butyryl esters are no substrates at all. In addition, the esterase shows a pronounced regiospecificity. On the other hand the enantiospecificity is rather low as demonstrated by the hydrolysis of prochiral and racemic substrates.     

Esterases from Bacillus subtilis and B. stearothermophilus share high sequence homology but differ substantially in their properties

A novel esterase from Bacillus subtilis (BsubE) was cloned, functionally expressed in Escherichia coli and biochemically characterized. BsubE shows high homology (74% identity, >95% homology) to an esterase from the thermophilic B. stearothermophilus (BsteE). Both enzymes were efficiently expressed in E. coli, using a L-rhamnose-expression system [11,500 units/l (BsteE), 3,400 units/l (BsubE)] and were purified by Ni-nitrilotriacetic acid chromatography, yielding specific activities of 70 units/mg (BsteE) and 40 units/mg (BsubE), as determined by the hydrolysis of p-nitrophenyl acetate. Despite the high homology, both esterases revealed remarkable differences in their properties. As expected, the esterase from the thermophilic organism showed significantly higher temperature stability. Whereas BsteE showed highest activity at 65-70 degrees C, BsubE was almost inactivated at 50 degrees C. Moreover, both enzymes showed quite different substrate patterns in the hydrolysis of various esters. Whilst the B. subtilis esterase accepted esters with a branched alcohol moiety well, the B. stearothermophilus esterase was more useful in the hydrolysis of substrates with a sterically demanding carboxylic acid group. BsteE showed excellent enantioselectivity ( E>100) in the kinetic resolution of menthyl acetate and even accepted the bulky menthyl benzoate as substrate ( E=19). In contrast, BsubE converted 1-phenethylacetate with higher selectivity ( E>150 vs E=8).     

Enzymatic production of ethanol from cellulose using soluble cellulose acetate as an intermediate

A two-stage process for the enzymatic conversion of cellulose to ethanol is proposed as an alternative to currently incomplete and relatively slow enzymatic conversion processes employing natural insoluble cellulose. This alternative approach is designed to promote faster and more complete conversion of cellulose to fermentable sugars through the use of a homogeneous enzymatic hydrolysis reaction. Cellulose is chemically dissolved in the first stage to form water-soluble cellulose acetate (WSCA). The WSCA is then converted to ethanol in a simultaneous saccharification-fermentation with Pestal-otiopsis westerdijkii enzymes (containing cellulolytic and acetyl esterase components) and yeast.Water-soluble cellulose acetate was successfully prepared from purified wood cellulose (Solka Floe) and chemical reagents. Enzyme pretreatment of WSCAto form metabolizable sugars was a necessary step in achieving practical conversion of WSCA to ethanol using yeast. The results showed that WSCA has a low enzyme requirement and a high convertibility to reducing sugars with enzymes from P. westerdijkii fungus. Pestalotiopsis westerdijkii enzymes were found to be superior to enzymes from Trichoderma viride in producing metabolizable glucose from WSCA. The yeast utilized 55-70% of the hydrolyzate sugars that were produced by P. westerrlijkii enzymes on WSCA and produced ethanol. The acetate that was liberated into solution by the action of acetyl esterase enzymes on WSCA was found to have a stimulatory effect on ethanol production in yeast. This is an important feature that can be used to advantage in manipulating the conversion to maximize the production of ethanol. Hence, the simultaneous saccharification-fermentation of WSCA to ethanol using P. westerdijkii enzymes and yeast has features that are highly desirable for developing an economical cellulose conversion process.     

Purification and characterization of the enantioselective esterase from Kluyveromyces marxianus CBS 1553

An intracellular esterase from the yeast Kluyveromyces marxianus CBS 1553 with interesting enantioselective hydrolytic activity towards racemic esters of 1,2-O-isopropylidene glycerol (IPG) was purified and characterized. Optimal culture conditions for the obtainment of the enantioselective esterase on a 5 l-fermentation scale were investigated. Two esterase activities (EST1 and EST2) in the crude cell extract were identified by native PAGE with specific activity staining and separated from each other by anion-exchange chromatography. EST1 showed higher activity and enantioselectivity than EST2 in the resolution of racemic IPG acetate and was further purified by hydrophobic interaction chromatography and preparative electrophoresis (final specific activity approximately = 300 U mg(-1), showing a main protein band with a molecular mass of 29 kDa. EST1 showed optimal activity between pH 8.0 and 10.0 and was stable in the pH range 7.0-10.0. Moreover, it was rather thermostable and active up to 80 degrees C, and retained most of its activity in the presence of 15% (v/v) of various organic solvents. The enzyme showed similar Vmax in the hydrolysis of the acetate esters of IPG, whereas the Km value towards (S)-IPG acetate was significantly lower than the one towards the (R)-enantiomer (5.3 and 70 microM, respectively). Finally, comparison of EST1 activity in the presence of different glycerol esters and synthetic substrates with different chain lengths showed a strong preference of this biocatalyst for short-chain substrates.     

Purification and characterization of the tween-hydrolyzing esterase of Mycobacterium smegmatis.

An esterase hydrolyzing Tween 80 (polyoxyethylene sorbitan monooleate) was purified from sonicated cell lysates of Mycobacterium smegmatis ATCC 14468 by DEAE-cellulose, Sephadex G-150, phenyl Sepharose, and diethyl-(2-hydroxypropyl) aminoethyl column chromatography and by subsequent preparative polyacrylamide gel electrophoresis. The molecular weight was estimated to be 36,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 41,000 by gel filtration on a Sephadex G-150 column. The esterase contained a single polypeptide. The esterase was stable to heat treatment at 100 degrees C and to a wide range of pH. The temperature and pH optima for the hydrolysis of Tween 80 were 50 degrees C and 8.3, respectively. The esterase had a narrow substrate specificity; it exhibited a high activity only on compounds having both polyoxyethylene and fatty acyl moieties, such as Tweens. Monoacylglyceride was hydrolyzed more slowly by this esterase and this enzyme exhibited a nonspecific esterase activity on p-nitrophenyl acyl esters, especially those having short chain acyl moieties. The Km and Vmax were 19.2 mM and 1,670 mumol/min per mg of protein for Tween 20, 6.6 mM and 278 mumol/min per mg of protein for Tween 80, and 0.25 mM and 196 mumol/min per mg of protein for p-nitrophenyl acetate, respectively. Observations of the effects of various chemical modifications on the activity of the esterase indicated that tyrosine, histidine, arginine, and methionine (with tryptophan) residues may be active amino acids which play important roles in the expression of Tween 80-hydrolyzing activity of the enzyme.     

Isolation and characterization of a fatty acyl esterase from rat lung

In an effort to facilitate studies of the reaction involved in the removal of fatty acids from acyl proteins, we have synthesized an octanoic acid ester of doubly blocked serine, specifically octanoyl N-carbobenzoxy-L-serine-benzyl ester (octanoyl boc-serine), and used it as a substrate to guide the purification of an esterase from rat lung. The esterase was purified 228-fold by column chromatography on DE-52 cellulose, hydroxylapatite, octyl-Sepharose, and concanavalin A-Sepharose and by HPLC gel filtration. The final enzyme preparation ran as a single 77,000-Da band when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exhibited a single symmetrical peak (sedimentation coefficient, 4.5 S) when centrifuged through a sucrose density gradient (empirical Mr, 63,000). The esterase is an acidic protein, pI 4.1, and is very active against p-nitrophenyl esters comprised of C4-C14 fatty acids; the highest specific activity (26.5 mumol/min/mg) was obtained using p-nitrophenyl caprylate as substrate. The pH optimum of the lung esterase is near 8.0 and the activity on octanoyl boc-serine is maximum when 0.3% (w/v) Myrj-52 is included in the assay medium. The activity of the esterase is not dependent on calcium ions. The enzyme does not remove acyl groups from the G-protein of vesicular stomatitis virus or the proteolipid of bovine brain. The possible role of the esterase in the metabolism of acylated proteins is considered.