Purification and characterization of cis-aconitic acid decarboxylase from Aspergillus terreus TN484-M1

cis-Aconitic acid decarboxylase (CAD) was assumed to be a key enzyme in the production of itaconic acid by comparing the activity of CAD from Aspergillus terreus TN484-M1 with that of CAD from the low-itaconate yielding strain Aspergillus terreus CM85J. The constitutive CAD was purified to homogeneity from A. terreus TN484-M1 by ammonium sulfate fractionation, and column chromatography on DEAE-toyopearl, Butyl-toyopearl, and Sephacryl S200HR, and then characterized. A molecular mass of 55 kDa for the native enzyme was determined by SDS-PAGE. The enzymic activity was optimal at a pH of 6.2 and temperature of 45 degrees C. The K(m) value for cis-aconitic acid was determined as 2.45 mM (pH 6.2, 37 degrees C). The enzyme was completely inactivated by Hg+, Cu2+, Zn2+, p-chloromercuribenzoate, and 5,5'-dithio-bis(2-nitrobenzoate).     

Purification and characterization of an alkaline manganese peroxidase from Aspergillus terreus LD-1

We report that Aspergillus terreus LD-1 produces an extracellular ligninolytic enzyme, manganese peroxidase (MnP), that reacts under alkaline conditions. This MnP was purified 13.1-fold from the culture supernatant to elicit a single band upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of this MnP was estimated as either 43 kDa by SDS-PAGE or 44 kDa by gel permeation chromatography, suggesting a monomeric structure. The optimum pH and temperature of this MnP are 12.5 and 37 degrees C, respectively. This MnP is stable in the pH range 11.0 to 12.5 and also up to 40 degrees C. The K(m) values of this MnP for hydrogen peroxide, 2,6-dimethoxyphenol (2,6-DMP) and Mn2+ were 320 microM, 20 microM and 33 microM at pH 12.5, respectively. The activity of the MnP is completely inhibited by Hg2+, Pb2+, Ag+ and lactate. On the other hand, the MnP is activated by oxalate, maleate and fumarate. Maleate at 5 mM increased the MnP activity 5-fold. EDTA at 1 mM inhibited the MnP activity completely, but this inhibition was not observed in the presence of 1 mM Fe2+.     

Purification and characterization of an extracellular protease from alkaliphilic and thermophilic Bacillus sp. PS719

An alkaline protease was purified to apparent homogeneity from culture supernatants of Bacillus sp. PS719, a novel alkaliphilic, thermophilic bacterium isolated from a thermal spring soil sample, by ammonium sulfate precipitation followed by DEAE-cellulose and alpha-casein agarose column chromatographies. The purified enzyme migrated as a single protein band of 42 kDa during both denaturing and nondenaturing gel electrophoresis, suggesting that it consists of a single polypeptide chain. Its isoelectric point was approximately 4.8. The protease exhibited maximum activity towards azocasein at pH 9.0 and at 75 degrees C. The enzyme activity was stimulated by Ca2+, but was inhibited in the presence of Fe2+ or Cu2+. The enzyme was stable in the pH range 8.0 to 10.0 and up to 80 degrees C in the absence of Ca2+. Since phenylmethylsulfonyl fluoride (PMSF) and 3,4-dichloroisocoumarin (DCI) in addition to N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) completely inhibited the activity, this enzyme appears to be a trypsin-like serine protease. Among the various oligopeptidyl-p-nitroanilides tested, the protease showed a preference for cleavage at arginine residues on the carboxylic side of the scissile bond of the substrate, liberating p-nitroaniline from N-carbobenzoxy (CBZ)-L-arginine-p-nitroanilide with the K(m) and V(max) values of 0.6 mM and 1.0 micromol.min(-1).mg protein(-1), respectively.     

Purification and characterization of N-Acetylglucosamine 6-phosphate deacetylase from Thermus caldophilus

N-Acetylglucosamine 6-phosphate deacetylase [EC 3.5.1.25] was purified and biochemically characterized from an extreme thermophile, Thermus caldophilus GK24. The optimum temperature and pH of the enzyme were 80 degrees C and 7.5, respectively. The enzyme is a tetramer composed of identical 45 kDa subunits. The N-terminal amino acid sequence of the purified enzyme was determined to be MSVDLKTLHRRHVLTP. It hydrolyzed GlcNAc-6-P, but not GlcNAc-1-P or chitin oligosaccharides. The deacetylase activity was completely inhibited by the addition of 1 mM Cu2+, but moderately activated by that of 1 mM Mn2+ and Co2+. Within 2 h of reaction, 2 mM GlcNAc-6-P was completely hydrolyzed to GlcN-6-P and acetate by the action of the deacetylase.     

醋酸菌中乙醛脱氢酶的分离纯化及酶学性质

以实验室筛选得到的醋酸菌(Acetobacter pomorum)为实验菌株发酵产酶,通过细胞破壁,采用(NH₄)₂SO₄沉淀、透析、DEAE-Sepharose 离子交换层析及 Superdex G-75凝胶过滤层析分离纯化得到乙醛脱氢酶的酶液,并考察其酶学性质。该酶分子质量为221.60 kDa,单个亚基分子质量约为54.41 kDa,为四聚体结构;纯酶液比活力20.25 U/mg,纯化倍数为10.16倍,乙醛脱氢酶(aldehyde dehydrogenase,ALDH)的回收率为6.53%。酶学性质研究表明,ALDH促进乙醛分解的最适温度为50 ℃,40~50 ℃相对酶活力稳定性好;该酶的最适pH为7.0,当pH在5.5~7.5内酶活力表现稳定;金属离子对酶活性的影响实验表明,Na+、K+、Zn2+、Ba2+对该酶酶有不同程度抑制作用,而Mg2+、Ca2+、Al3+、Li+、Cu2+具有促进作用;ALDH的最适底物为乙醛,相对偏好直链醛类。ALDH活性较大,为后期表达和深入研究其生物学功能提供理论和数据支持。     

金龟子绿僵菌(Metarhizium anisopliae Ma83)几丁质酶的纯化及性质

由金龟子绿僵茵Ma83菌株产生的几丁质酶经硫酸铵盐盐析,Sephadex G-100柱层析,DEAE-纤维素柱层析分离纯化后,得到SDS-PAGE均一样品.酶的最适反应温度为50℃,半失活温度为65℃;酶的最适反应pH值为5.0,酶在pH4.0～7.0范围内较稳定.Ag+、Co2+、K+、Mg2+对Ma83几丁质酶有激活作用,而Hg2+、Zn2+、Pb2+对几丁质酶活力有抑制作用.经计算Ma83菌株几丁质酶对胶体几丁质的Km值为1.05 mg/mL.     

Purification and characterization of a beta-glucosidase with beta-xylosidase activity from Aspergillus sojae

A beta-glucosidase (EC 3.2.1.21) was purified as an electrophoretically homogeneous protein from a solid culture of Aspergillus sojae. The molecular mass of the purified enzyme was estimated to be 250 kDa by gel filtration chromatography and 118 kDa by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). The isoelectric point of the enzyme was 3.80. The maximum velocity of rho-nitrophenyl beta-d-glucopyranoside degradation by the beta-glucosidase was attained at 60 degrees C and at pH 5.0. The purified enzyme was stable from pH 6.0 to 8.0, and up to 50 degrees C. The activity of the enzyme was significantly inhibited by Hg2+ and Cu2+, and stimulated by Mn2+ and Fe3+. The purified enzyme hydrolyzed beta-D-xylopyranosides as well as beta-D-glucopyranosides; the Km and Vmax values on rho-nitrophenyl beta-D-glucopyranoside were 0.14 mM and 16.7 micromol/min/mg protein, and on rho-nitrophenyl beta-D-xylopyranoside 0.51 mM and 12.2 micromol/min/mg protein, respectively.     

Purification and properties of a phytase from Candida krusei WZ-001

A phytase from Candida krusei WZ-001 isolated from soil was purified to electrophoretic homogeneity by ion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. The phytase is composed of two different subunits with molecular masses of 116 kDa and 31 kDa on SDS-PAGE (or 120 kDa and 30 kDa on gel chromatography), with the larger subunit having a glycosylation rate of around 35%. The phytase has an optimum pH of 4.6, an optimum temperature of 40 degrees C and a pI value of 5.5. The phytase activity was stimulated by 2-mercapto-ethanol and dithiothreitol (DTT), and inhibited by Zn2+, Mg2+, iodoacetate, pI value of 5.5. The phytase activity was stimulated by 2-mercapto-ethanol ethanol and dithiothreitol (DTT), and inhibited by Zn2+, Mg2+, iodoacetate, p-chroloromercuribenzoate (pCMB) and phenylmethylsulfonyl fluoride (PMSF). The phytase displayed a broad substrate specificity and the K(m) for phytate was 0.03 mM. Phytate was sequentially hydrolyzed by the phytase. Furthermore, 1D and 2D NMR analyses and bioassay of myoinositol indicated that the end hydrolysis product of phytate was myoinositol 2-monophosphate.     

Purification and characterization of a novel beta-agarase from an alkalophilic bacterium, Alteromonas sp. E-1

A novel beta-agarase (EC 3.2.1.81) was purified from an agar-degrading alkalophilic bacterium, Alteromonas sp. E-1 isolated from the soil. This enzyme was obtained from a cell-free extract after sonication and purified 40.9-fold through treatment with streptomycin, ammonium sulfate fractionation and successive chromatography on anion-exchange and gel filtration columns. The molecular weight was estimated to be 82 kDa by SDS-polyacrylamide gel electrophoresis and 180 kDa by Superdex 200 gel filtration. The enzyme was inhibited by Mn2+, Cu2+, Fe2+, Zn2+ and Hg2+, and activated by K+, Na+ and EDTA, and its optimum pH and temperature for agarose degradation were 7.5 and 40 degrees C, respectively. This beta-agarase hydrolyzed agarose with rapid reduction of viscosity, and neoagarobiose [O-3,6-anhydro-alpha-L-galactopyranosyl(1-->3)-D-galactose] was detected from the early stage of the reaction. Neoagarobiose as the final product was selectively released from agarose, neoagarohexaose and neoagarotetraose by the reaction with this beta-agarase. This observation was different from that of other beta-agarases which produced mixtures of neoagarobiose and neoagarotetraose as the final hydrolysis products. The N-terminal amino acid sequence of this beta-agarase shows no homology to those of other beta-agarases that were so far reported.     

Partial purification and characterization of an extracellular proteinase from Aeromonas hydrophila strain A4

An extracellular metalloproteinase from Aeromonas hydrophila strain A4, isolated from milk, was purified by a factor of 300 by chromatography on DEAE-cellulose and Sephadex G-150. The enzyme had a mol. wt of 43,000 and contained 2 g atom Ca/mol. It was active over a pH range 4.8-9.5 and had optimum activity on casein at pH 7.0 with Km = 0.17 mM. It was strongly inactivated by metal chelators and the apoenzyme was fully reactivated with Ca2+, Mn2+ or Co2+. Heavy metal ions such as Ag+, Hg2+, Fe2+, Zn2+, Cd2+, Ni2+ and Cu2+ totally or partly inactivated the enzymic activity at 5 mM concentration. The enzyme was not inactivated by diisopropylfluorophosphate, soyabean trypsin inhibitor or sulphydryl group reagents. It was optimally active at 45 degrees C; above 50 degrees C activity declined rapidly, but significant activity persisted at 4 degrees C. It was heat labile in phosphate or Tris-maleate buffer but exogenous Ca2+ afforded protection.     

Purification of isocitrate lyase from Saccharomyces cerevisiae

Isocitrate lyase purified to homogeneity from Saccharomyces cerevisiae was composed of four identical subunits with a molecular mass of 75 kDa. The enzyme was most active at pH 7.0 in the presence of 5 mM-Mg2+. The Km value for threo-Ds-isocitrate was 1.4 mM. Isocitrate lyase was shown to be thermostable at 50 degrees C for 60 min at a high salt concentration, but rapidly lost activity at -20 degrees C or by dialysis.     

鸭卵清溶菌酶的分离纯化及性质

通过硫酸铵的分级沉淀、CM-Sepharose阳离子交换层析、Superdex-200凝胶过滤层析等步骤,从鸭卵清中获得电泳纯的溶菌酶,该酶的比活力达到33687.26U/mg,纯化倍数为109.44,回收率为28.00%。测得该酶分子质量约为14.82kD,对溶壁微球菌的最适反应温度为50℃,最适pH值为7,且在50℃以下及pH5～9有较好的稳定性,同时在最适条件下测得其Km值为0.0864mg/mL。Fe2+、Mg2+、Mn2+等金属离子对该酶有较强的抑制作用,而Zn2+、Cu2+、Co2+对该酶有一定的激活作用。     

香菜叶多酚氧化酶的分离纯化与部分酶学性质研究

新鲜香菜叶经匀浆、缓冲液提取、硫酸铵分级沉淀、DEAE-Sepharose离子交换层析、Superdex-200凝胶过滤层析,获得电泳纯的多酚氧化酶。该酶比活力达到5 622.95 U/mg,酶活回收率为3.90%,纯化倍数为126.08;全酶分子质量为111.10 kD,亚基分子质量为55.60 kD;最适温度为37 ℃,最适pH值为6.5;在25～45 ℃及pH 6.0～7.0范围内有较好的稳定性;在最适条件下测得其Km值为4.04×10－2 mol/L;甲醇、乙醇、异丙醇、氯仿及柠檬酸、抗坏血酸、Ca2＋、Hg2＋、Ba2＋对其有抑制作用,Co2＋、Pb2＋对其具有一定的激活作用。     

Ammonia assimilation in Klebsiella pneumoniae F-5-2 that can utilize ammonium and nitrate ions simultaneously: purification and characterization of glutamate dehydrogenase and glutamine synthetase

The two ammonia-assimilating enzymes glutamate dehydrogenase (GDH; EC 1.4.1.4) and glutamine synthetase (GS; EC 6.3.1.2) were synthesized steadily during the cell growth of Klebsiella pneumoniae F-5-2 that can utilize NH4+ and NO3- simultaneously under aerobic conditions. The enzymes were purified to homogeneity from cell extracts and characterized. The molecular mass of the purified GDH was 300 kDa with six identical 52-kDa subunits. GDH showed its maximal activity (aminating) at pH 8.0 and was stable between pHs 5.5 and 11.5. The enzyme was NADP-specific and strongly inhibited by Ag+. It catalyzed the amination of 2-ketovalerate, 2-ketoadipate, and 2-ketobutyrate, in addition to 2-ketoglutarate. The purified GS has a molecular mass of 470 kDa with eight identical 60-kDa subunits. GS showed its maximal activity at pH 8.0 and was stable between pHs 6.0 and 7.0. The enzyme was strongly inhibited by Fe3+, Hg2+, and Cu2+.     

Purification and characterization of a serine carboxypeptidase from Kluyveromyces marxianus

We purified a carboxypeptidase (CPY) from the yeast of Kluyveromyces marxianus. This enzyme was purified 170 times from a soluble extract of 100000 x g. Purification consisted in a fractionated precipitation with ammonium sulfate and two chromatographic steps consisting of anion exchange chromatography and hydrophobic interactions chromatography. The native enzyme depicted a molecular mass of 67 kDa by gel filtration. This serine carboxypeptidase depicted an optimal pH of 8.5 and was stable at a pH ranging from 6.0 to 9.0, its optimal temperature was of 45 degrees C and was unstable at temperatures above 55 degrees C; Michaelis constant and Vmax for N-benzoyl-l-tyrosine-p-nitroanilide were of 29 microM and 612 microM/min mg of protein, respectively. The enzyme was strongly inhibited by phenylmethylsufonyl fluoride (PMSF) and, to a lesser degree, by trans-epoxysuccinyl-l-leucylamido-(4-guanidine)-butane. This study indicated that K. marxianus carboxypeptidase could be an alternative to other animal-source carboxypeptidases in the industry.     

Purification and characterization of a novel cyclohexylamine oxidase from the cyclohexylamine-degrading Brevibacterium oxydans IH-35A

Cyclohexylamine oxidase (CHAO) from a cell extract of Brevibacterium grown on cyclohexylamine was purified 50.2-fold, to electrophoretic homogeneity, by serial chromatographies. The molecular mass of the native enzyme was estimated to be approximately 50 kDa by gel filtration and SDS-PAGE. The optimum pH was 7.4 and the stable pH range was 6.0 to 7.0. The enzyme was thermostable up to 30 degrees C. The enzyme was found to be highly specific for the deamination of alicyclic monoamines such as cyclopentylamine, cycloheptylamine, and N-methylcyclohexylamine and aliphatic monoamines, such as sec-butylamine. The apparent K(m) value for cyclohexylamine was 1.23 mM. The enzyme was inhibited by flavin enzyme inhibitors such as quinine and quinacrine. The N-terminal 27 amino acid residues were determined as Gly-Ser-Val-Thr-Pro-Asp-Pro-Asp-Val-Asp-Val-Ile-Ile-His-Gly-Ala-Gly-Ile-Ser-Gly-Ser-Ala-Ala-Ala-Lys-Ala-Leu-, revealing homology to conventional flavin-containing amine oxidases (EC 1.4.3.4).     

Purification and characterization of an extracellular trypsin-like protease of Fusarium oxysporum var. lini

An alkaline serineprotease, capable of hydrolyzing Nalpha-benzoyl- dl arginine p-nitroanilide, was secreted by Fusarium oxysporum var. lini grown in the presence of gelatin as the sole nitrogen and carbon source. The protease was purified 65-fold to electrophoretic homogenity from the culture supernatant in a three-step procedure comprising QSepharose chromatography, affinity chromatography, and FPLC on a MonoQ column. SDS-PAGE analysis of the purified protein indicated an estimated molecular mass of 41 kDa. The protease had optimum activity at a reaction temperature of 45 degrees C and showed a rapid decrease of activity at 48 degrees C. The optimum pH was around 8.0. Characterization of the protease showed that Ca2+ and Mg2+ cations increased the activity, which was not inhibited by EDTA or 1,10-phenanthroline. The enzyme activity on Nalpha-benzoyl-DL arginine p-nitroanilide was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, p-aminobenzamidine dihydrochloride, aprotinin, 3-4 dichloroisocoumarin, and N-tosyl-L-lysine chloromethyl ketone. The enzyme is also inhibited by substrate concentrations higher than 2.5 x 10(-4)M. The protease had a Michaelis-Menten constant of 0.16 mM and a V(max) of 0.60 mumol released product.min(-1).mg(-1) enzyme when assayed in a non-inhibiting substrate concentration. The activity on Nalpha-benzoyl- dl arginine p-nitroanilide was competitively inhibited by p-aminobenzamidine dihydrochoride. A K(i) value of 0.04 mM was obtained.     

Purification and properties of hydrogen sulfide oxidase from Bacillus sp. BN53-1

A hydrogen sulfide oxidase was purified to homogeneity from the heterotroph Bacillus sp. BN53-1 isolated from pig feces compost. The enzyme was found to be a monomer with a M(r) value of approximately 37 kDa. It required FAD for its activity, which was not replaced by FMN. The optimum reaction pH and temperature were 7.5 and 40 degrees C, respectively. The enzyme was stable between pH 6.0 and 7.0 and up to 30 degrees C. Its activity was stimulated by Ca2+ and Mn2+ and inhibited by Al3+, dithiothreitol, and 2-mercaptoethanol. The main product was elemental sulfur, and H2O2 was not detected. The N-terminal sequence of the enzyme showed similarity to other FAD-requiring enzymes.     

Purification and characterization of sea squirt alpha-N-acetylgalactosaminidase

Sea squirt alpha-N-acetylgalactosaminidase was purified to homogeneity. Its molecular weight was estimated to be approximately 160,000 by gel filtration and 40,000 by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing condition. The chromatographic and electrophoretic behaviors indicated that the enzyme was composed of four subunits. The optimum pH of the enzyme reaction was about 4.0 at 37 degrees C, while the enzyme was stable in the range of pH 5.0 to 6.0 during 4 h preincubation at 37 degrees C. Although the enzyme (0.1 unit) was stable at 0 degrees C for 30 min in the presence of 7.5 mM metal ions (Al3+, Ba2+, Ca2+, K+, Mn2+, Pb2+, Sr2+, and Zn2+), almost 40% of the enzyme activity was lost in the presence of Cu2+, Hg2+, monoiodoacetic acid, and EDTA. The enzyme hydrolyzed aryl N-acetyl-alpha-D-galactosaminide as well as GalNAcalpha1(-->4GalNAcalpha1-->)n 4GalNAc-p-aminobenzoic acid ethyl ester (ABEE) (n = 1-4), but GalNAcalpha1-->4GalNAc-ABEE only scarcely. Furthermore, an allergenic pentasaccharitol ABEE derivative, GalNAcalpha1-->2Fucalpha1-->3(GalNAcbeta1-->4) GlcNAcbeta1-->2(3-acetoamido-3-deoxy)L-threose-ABEE, the minimum structural unit for the sea squirt allergenicity was hydrolyzed to 95 mol% for 72 h incubation with the enzyme. The enzyme could be utilized as a powerful tool for the structural analyses of the carbohydrate epitopes of the sea squirt allergen molecules.     

Purification and characterization of an enzyme that has dihydroxyacetone-reducing activity from methanol-grown Hansenula ofunaensis

An intracellular enzyme having reduction activity towards dihydroxyacetone (DHA), and that was induced by DHA, was purified and characterized from a methanol-grown yeast, Hansenula ofunaensis. After harvesting cells grown in a 1% methanol medium until the early stationary phase, the enzyme was purified through ammonium sulfate fractination and a series of ion-exchange, hydrophobic, and gel-filtration column chromatographies. SDS-PAGE and HPLC showed the enzyme to be a homo dimer composed of two identical subunits, each with a molecular mass of 38 kDa. The optimum pHs for DHA reduction and glycerol oxidation were 6.0 and 7.0, respectively. The optimum temperature for enzyme activity was 55 degrees C. The enzyme reduced several other compounds, including acetaldehyde, acetol, 2-butanone and 3-methyl-2-butanone, more effectively than it did DHA, while its oxidation activity was higher towards ethanol, 2-propanol, 1,2-propanediol, 2,3-butanediol and 1,3-butanediol than towards glycerol. The K(m) values for DHA in reduction and glycerol in oxidation were 430 mM and 4 M, respectively. The K(m) values for DHA in reduction and glycerol in oxidation were 430 mM and 4 M, respectively. The purified enzyme had high K(m) values for glycerol and DHA and low K(m) values for 2-butanol and butanone, although physiologically it had a role in DHA metabolism. There were similarities between the purified enzyme and sec-alcohol dehydrogenases reported previously in their behavior towards inhibitors and metal ions, as well as in their K(m) values for 2-butanol and 2-butanone, but differences in their subunit molecular masses and activities for ethanol. At pH 9.8, the oxidative activity of the purified enzyme for l-2-butanol was about eleven times higher than that for d-2-butanol.