Production, purification, and characterization of lipase from thermophilic and alkaliphilic Bacillus coagulans BTS-3

A thermophilic isolate Bacillus coagulans BTS-3 produced an extracellular alkaline lipase, the production of which was substantially enhanced when the type of carbon source, nitrogen source, and the initial pH of culture medium were consecutively optimized. Lipase activity 1.16 U/ml of culture medium was obtained in 48 h at 55 degrees C and pH 8.5 with refined mustard oil as carbon source and a combination of peptone and yeast extract (1:1) as nitrogen sources. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE-Sepharose column chromatography. Its molecular weight was 31 kDa on SDS-PAGE. The enzyme showed maximum activity at 55 degrees C and pH 8.5, and was stable between pH 8.0 and 10.5 and at temperatures up to 70 degrees C. The enzyme was found to be inhibited by Al3+, Co2+, Mn2+, and Zn2+ ions while K+, Fe3+, Hg2+, and Mg2+ ions enhanced the enzyme activity; Na+ ions have no effect on enzyme activity. The purified lipase showed a variable specificity/hydrolytic activity towards various 4-nitrophenyl esters.     

Isolation and purification of an extracellular protease from a new strain of Bacillus subtilis, viz.NCIM 2711

A new extracellular protease having a prospective application in the food industry was isolated from Bacillus sUbtilis NCIM 2711 by (NH4)2SO4 precipitation from the cell broth. It was purified using DEAE-Cellulose and CM-Sephadex C-50 ion-exchange chromatography. With casein as a substrate, the proteolytic activity of the purified protease was found to be optimal at pH 7.0 and temperature 55 degrees C with Km 1.06 mg/ml. The enzyme was stable over a pH range 6.5-8.0 at 30 degrees C for 1 hr in presence of CaCl2 x 2H2O. At 55 degrees C, the enzyme retained 60% activity up to 15 min in presence of CaCl2 x 2H2O. EDTA and o-phenanthroline (OP) completely inhibited the enzyme activity while DFP, PMSF and iodoacetamide were ineffective. The enzyme was completely inhibited by Hg2+ and partially by Cd2+, Cu2+, Ni2+, Pb2+ and Fe2+. The OP inhibited enzyme could be reactivated by Zn2+ and Co2+ up to 75% and 69% respectively. It is a neutral metalloprotease showing a single band of 43 kDa on SDS-PAGE.     

Purification and thermal characterization of a novel peroxidase from a local chick pea cultivar

A novel peroxidase isolated from a local chick pea (Cicer arietinum L.) cultivar (Balksar 2000) was purified by means of ammonium sulfate precipitation, DEAE-cellulose chromatography and two runs on gel filtration. The purified enzyme has a specific activity of 2045 U/mg with 17 % activity recovery. The molecular mass of the enzyme was estimated to be 39 kDa by SDS-polyacrylamide gel electrophoresis. Optimum pH and temperature of the enzyme were 5.5 and 45 degrees C respectively. The thermal denaturation of local chick pea peroxidase was studied in aqueous solution at temperatures ranging from 45 degrees C to 65 degrees C. The temperature of 50% inactivation of the enzyme was found to be 68 degrees C. The enthalpy (DeltaH*) and free energy (DeltaG*) of thermal denaturation of chick pea peroxidase were 101.4 and 103.4 k J/mol respectively at 65 degrees C.Metals like Zn2+, Mn2+, Hg2+, Co2+ and Al3+ slightly inhibited the peroxidase activity while Ca2+, Mg2+ and Ba2+ have no effect on enzyme activity. The high specific activity and thermal stability make chick pea peroxidase an alternative to horseradish peroxidase (HRP) in various applications.     

毛壳霉内切菊粉酶的纯化与性质

毛壳霉 (Chaetomiumsp .)C34发酵液经硫酸铵分级沉淀、DEAE 纤维素 11离子交换层析、Q SepharoseFastFlow离子交换层析、SephacrylS 2 0 0凝胶过滤、PhenolSepharoseTM HP疏水层析 ,得到电泳纯的内切菊粉酶组分 ,纯化倍数为 30 8倍 ,活力回收率为 7 7%。用SDS PAGE测得该酶亚基的分子量为 6 6kD。菊粉酶的最适pH为 6 0 ,最适温度为 5 0～ 5 5℃。菊粉酶在 5 0℃以下 ,pH5 0～ 8 0时较稳定。Cu2 完全抑制酶的活性 ,Mn2 、Zn2 、Fe2 、EDTA以及NBS(N bromosuccinimide ,N 溴代丁二酰亚胺 )对该酶有很强的抑制作用。该酶对菊粉有较强底物专一性 ,产物主要为低聚果糖 ,也可作用于蔗糖 ,I S值为 2 0。以菊粉为底物时 ,Km 为 0 199mmol L ,Vmax为 115 μmol (mg·min)。     

Purification and characterization of intestinal alkaline phosphatase from harp seal

1. Alkaline phosphatase (EC 3.1.3.1.) from harp seal (Phagophilus groenlandicus) has been purified by concanavalin A-Sepharose chromatography to homogeneity with a specific activity of 1200-1500 units/mg of protein. 2. The mol. wt of the enzyme and its subunits were estimated as 260,000 and 70,000, respectively. By chromatofocusing the isoelectric point of this enzyme is 5.5. 3. With p-nitrophenylphosphate, pH-optimum and KM for the enzyme are 9.8 and 0.9 mM, respectively. 4. The enzyme was strongly inhibited by Sn4+, Fe3+ and Zn2+, whereas Mg2+ and Mn2+ were effective activators of the enzyme. Seal alkaline phosphatase was slightly inhibited by high concentrations of Ca2+ and Cr3+. 5. The enzyme activity reached a maximum at 55-60 degrees C. It was shown that the heat stability of seal and calf intestinal alkaline phosphatases were equal at 37 and 56 degrees C.     

重组超耐热酸性α-淀粉酶的分离纯化及其性质研究

基因工程菌所产生的重组超耐热酸性α-淀粉酶,通过超滤浓缩、脱盐和聚丙烯酰胺垂直板凝胶电泳进行纯化,得到电泳纯的超耐热酸性α-淀粉酶,纯化倍数为11.7,活力回收率为29.8%。用SDSPAGE测得该酶的分子量为55kD,酶的等电点pI(室温)为5.0,以可溶性淀粉为底物的Km值为1.12gL,用硫酸酚法测得其含糖量为15.4%。该酶的最适反应温度为95℃,最适反应pH值为4.5。在pH4.0～7.0室温放置48h酶活没有变化,110℃保温1h残留60%活力。Cr3 、Fe2 、Cu2 抑制酶的活性,Ca2 对酶活无影响。EDTA和DTT对酶的活性无影响。     

疏绵状嗜热丝孢菌热稳定几丁质酶的纯化及其性质研究

采用硫酸铵沉淀、DEAE SepharoseFastFlow阴离子层析、Phenyl Sepharose疏水层析等步骤获得了凝胶电泳均一的疏绵状嗜热丝孢菌 (Thermomyceslanuginosus)几丁质酶。经SDS PAGE和凝胶过滤层析测得纯酶蛋白的分子量在 4 8～ 4 9 .8kD之间。该酶反应的最适温度和最适pH分别为 5 5℃和 4 5 ,在pH4 5条件下 ,该酶在 5 0℃以下稳定 ;6 5℃的半衰期为 2 5min ;70℃保温 2 0min后 ,仍保留 2 4 %的酶活性。其N 端氨基酸序列为AQGYLSVQYFVNWAI。金属离子对几丁质酶的活性影响较大 ,Ca2 、Na 、K 、Ba2 对酶有激活作用 ;Ag 、Fe2 、Cu2 、Hg2 对酶有显著的抑制作用 ;以胶体几丁质为底物的Km 和Vmax值分别为 9 .5 6mg mL和 2 2 . 12 μmol min。抗菌活性显示 ,该酶对供试病原菌有不同程度的抑制作用。     

Isolation and characterization of a novel phytase fromPenicillium simplicissimum

Eighty-three isolates from different soil samples exhibited the potential for producing active extracellular phytase. The most active fungal isolate with phytase activity was identified as Penicillium simplicissimum. In shaking culture with enrichment medium, the highest extracellular phytase activity of the producing strain was 3.8 U/mL. The crude enzyme filtrate was purified to homogeneity using ultrafiltration. IEC and gel filtration chromatography. The molar mass of the purified enzyme was estimated to be 65 kDa on SDS-PAGE. The saccharide identification with periodic acid-Schiff reagent (PAS) and activity recognition by 1-naphthyl phosphate was all positive. The isoelectric point of the enzyme, as deduced by isoelectric focusing, was pH 5.8, the optimum pH and temperature being pH 4.0 and 55 degrees C, respectively. The purified enzyme revealed broad substrate specificity and was strongly inhibited by Fe2+, Fe3+ and Zn2+; however, no inhibition was found by EDTA and PMSF. Phytase activity was inhibited when 2 mmol/L of dodecasodium phytate was added and the Km for it was determined to be 813 mmol/L.     

Purification and some properties of beta-N-acetyl-D-glucosaminidase from viscera of green crab (Scylla serrata)

Beta-N-acetyl-D-glucosaminidase was purified from viscera of green crab (Scylla serrata) by extraction with 0.01 M Tris-HCl buffer (pH 7.5) containing 0.2 M NaCl, ammonium sulfate fractionation, and then chromatography on Sephadex G-100 and DEAE-cellulose (DE-32). The purified enzyme showed a single band on polyacrylamide gel electrophoresis, and the specific activity was determined to be 7990 U/mg. The molecular weight of the whole enzyme was determined to be 132.0 kD, and the enzyme is composed of two identical subunits with molecular mass of 65.8 kD. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-NAG) were found to be at pH 5.6 and at 50 degrees C, respectively. The study of its stability showed that the enzyme is stable in the pH range from 4.6 to 8.6 and at temperatures below 45 degrees C. The kinetic behavior of the enzyme in the hydrolysis of pNP-NAG followed Michaelis-Menten kinetics with Km of 0.424 +/- 0.012 mM and Vmax of 17.65 +/- 0.32 micromol/min at pH 5.8 and 37 degrees C, and the activation energy was determined to be 61.32 kJ/mol. The effects of some metal ions on the enzyme were surveyed, and the results show that Na+ and K+ have no effects on the enzyme activity; Mg2+ and Ca2+ slightly activate the enzyme, while Ba2+, Zn2+, Mn2+, Hg2+, Pb2+, Cu2+, and Al3+ inhibit the enzyme to different extents.     

Optimization of culture conditions and properties of immobilized sulfide oxidase from Arthrobacter species

Arthrobacter species strain FR-3, isolated from sediments of a swamp, produced a novel serine-type sulfide oxidase. The production of sulfide oxidase was maximal at pH 7.5 and 30 degrees C. Among various carbon and nitrogen sources tested, glucose and yeast extract were found to be the most effective substrates for the secretion of sulfide oxidase. The sulfide oxidase was purified to homogeneity and the molecular weight of the purified enzyme was 43 kDa when estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified sulfide oxidase can be effectively immobilized in DEAE (diethylaminoethyl)-cellulose matrix with a yield of 66%. The purified free and immobilized enzyme had optimum activity at pH 7.5 and 6.0, respectively. Immobilization increases the stability of the enzyme with respect to temperature. The half-life of the immobilized enzyme was 30 min at 45 degrees C, longer than that of the free enzyme (10 min). The purified free sulfide oxidase activity was completely inhibited by 1 mM Co2+ and Zn2+ and sulfhydryl group reagents (para-chloromercuribenzoic acid and iodoacetic acid). Catalytic activity was not affected by 1 mM Ca2+, Mg2+, Na+ and metal-chelating agent (EDTA).     

Production and characterization of N-acyl-D-glutamate amidohydrolase from Pseudomonas sp. strain 5f-1.

N-Acyl-D-glutamate amidohydrolase from Pseudomonas sp. strain 5f-1 was inducibly produced by D isomers of N-acetylglutamate, glutamate, aspartate, and asparagine. The enzyme has been purified to homogeneity by DEAE-cellulose, (NH4)2SO4 fractionation, and chromatofocusing followed by gel filtration on a Sephadex G-100 column. The enzyme was a monomer with molecular weight of 55,000. The enzyme activity was optimal at pH 6.5 to 7.5 and 45 degrees C. The isoelectric point and the pH stability were 8.8 and 9.0, respectively. N-Formyl, N-acetyl, N-butyryl, N-propionyl, N-chloroacetyl derivatives of D-glutamate and glycyl-D-glutamate were substrates for the enzyme. At pH 6.5 in 100 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer at 30 degrees C, a Km of 6.67 mM and a Vmax of 662 mumol/min/mg of protein for N-acetyl-D-glutamate were obtained. None of the metal ions stimulated the enzyme activity. Na+, K+, Mg2+, and Ba2+ acted as stabilizers. Hg2+, Cu2+, Zn2+, Fe3+, and EDTA were strongly inhibitory.     

Xylanase production by Burkholderia sp. DMAX strain under solid state fermentation using distillery spent wash

Xylanase production by a newly isolated strain of Burkholderia sp. was studied under solid state fermentation using anaerobically treated distillery spent wash. Response surface methodology (RSM) involving Box-Behnken design was employed for optimizing xylanase production. The interactions between distillery effluent concentration, initial pH, moisture ratio and inoculum size were investigated and modeled. Under optimized conditions, xylanase production was found to be in the range of 5200-5600 U/g. The partially purified enzyme recovered after ammonium sulphate fractionation showed maximum activity at 50 degrees C and pH 8.6. Kinetic parameters like Km and Vmax for xylan were found to be 12.75 mg/ml and 165 micromol/mg/min. In the presence of metal ions such as Ca2+, Co2+, Mn2+, Ba2+, Mg2+ and protein disulphide reducing agents such as beta-mercaptoethanol and dithiotheritol (DTT) the activity of enzyme increased, where as strong inhibition of enzyme activity was observed in the presence of Cu2+, Ag+, Fe2+ and SDS. The crude enzyme hydrolysed lignocellulosic substrate, wheat bran as well as industrial pulp.     

Production and characterization of N-acyl-D-glutamate amidohydrolase from Pseudomonas sp. strain 5f-1.

N-Acyl-D-glutamate amidohydrolase from Pseudomonas sp. strain 5f-1 was inducibly produced by D isomers of N-acetylglutamate, glutamate, aspartate, and asparagine. The enzyme has been purified to homogeneity by DEAE-cellulose, (NH4)2SO4 fractionation, and chromatofocusing followed by gel filtration on a Sephadex G-100 column. The enzyme was a monomer with molecular weight of 55,000. The enzyme activity was optimal at pH 6.5 to 7.5 and 45 degrees C. The isoelectric point and the pH stability were 8.8 and 9.0, respectively. N-Formyl, N-acetyl, N-butyryl, N-propionyl, N-chloroacetyl derivatives of D-glutamate and glycyl-D-glutamate were substrates for the enzyme. At pH 6.5 in 100 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer at 30 degrees C, a Km of 6.67 mM and a Vmax of 662 mumol/min/mg of protein for N-acetyl-D-glutamate were obtained. None of the metal ions stimulated the enzyme activity. Na+, K+, Mg2+, and Ba2+ acted as stabilizers. Hg2+, Cu2+, Zn2+, Fe3+, and EDTA were strongly inhibitory.     

A fibrinolytic metalloprotease from the fruiting bodies of an edible mushroom, Armillariella mellea

A fibrinolytic metalloprotease has been purified from the fruiting bodies of the edible honey mushroom (Armillariella mellea). The enzyme has a molecular weight of 18538.1508, as measured by MALDI-TOF mass spectrometry and includes Zn2+ ion as found by ICP/MS. The N-terminal amino acid sequence, XXYNGXTXSRQTTLV, do not match any known protein or open reading frame. It hydrolyzes fibrinogen as well as fibrin, but does not show any proteolytic activity for other blood proteins such as thrombin, human albumin, bovine albumin, human IgG, hemoglobin, or urokinase. This protease hydrolyzes both A alpha and B beta subunits of human fibrinogen with equal efficiency. The enzyme activity was strongly inhibited by EDTA and 1,10-phenanthroline, indicating that the enzyme is a metalloprotease. No inhibition was found with PMSF, E-64, pepstatin, and 2-mercaptoethanol. The activity of the purified enzyme was slightly increased by Mg2+, Zn2+, and Co2+, but the enzyme was totally inhibited by Hg2+. It has broad substrate specificity for synthetic peptides, and a pH optimum at 7, suggested that the purified enzyme was a neutral protease. It was thermally stable up to 60 degrees C and the maximum fibrinolytic activity was at 55 degrees C.     

Isolation and characterization of NADP+-linked isocitrate dehydrogenase of germinating pea seeds (Pisum sativum)

NADP+-linked isocitrate dehydrogenase (E.C.1.1.1.42) has been purified to homogeneity from germinating pea seeds. The enzyme is a tetrameric protein (mol wt, about 146,000) made up of apparently identical monomers (subunit mol wt, about 36,000). Thermal inactivation of purified enzyme at 45 degrees and 50 degrees C shows simple first order kinetics. The enzyme shows optimum activity at pH range 7.5-8. Effect of substrate [S] on enzyme activity at different pH (6.5-8) suggests that the proton behaves formally as an "uncompetitive inhibitor". A basic group of the enzyme (site) is protonated in this pH range in the presence of substrate only, with a pKa equal to 6.78. On successive dialysis against EDTA and phosphate buffer, pH 7.8 at 0 degrees C, yields an enzymatically inactive protein showing kinetics of thermal inactivation identical to the untreated (native) enzyme. Maximum enzyme activity is observed in presence of Mn2+ and Mg2+ ions (3.75 mM). Addition of Zn2+, Cd2+, Co2+ and Ca2+ ions brings about partial recovery. Other metal ions Fe2+, Cu2+ and Ni2+ are ineffective.     

Purification and characterization of extracellular phytase from Aspergillus niger ATCC 9142

Extracellular phytase produced by Aspergillus niger ATCC 9142 was purified to homogeneity by employing an initial ultrafiltration step, followed by chromatography using ion exchange, gel filtration and chromatofocusing steps. The purified enzyme was an 84 kDa, monomeric protein. It possessed a temperature optimum of 65 degrees C, and a pH optimum of 5.0. Km and Vmax values of 100 microM and 7 nmol/s, respectively, were recorded and these values fall well within the range of those previously reported for microbial phytases. Substrate specificity studies indicated that, while the enzyme could hydrolyse a range of non-phytate-based phosphorylated substrates, its preferred substrate was phytate. Phytase activity was moderately stimulated in the presence of Mg2+, Mn2+, Cu2+, Cd2+, Hg2+, Zn2+ and F- ions. Activity was not significantly affected by Fe2- or Fe3- and was moderately inhibited by Ca2+. The enzyme displayed higher thermostability at 80 degrees C than did two commercial phytase products. Initial characterisation of the purified enzyme suggested that it could be a potential candidate for use as an animal feed supplement.     

Purification and characterization of carboxymethyl cellulase from Bacillus sp. isolated from a paddy field

A microorganism hydrolyzing carboxymethyl cellulose was isolated from a paddy field and identified as Bacillus sp. Production of cellulase by this bacterium was found to be optimal at pH 6.5, 37 degrees C and 150 rpm of shaking. This cellulase was purified to homogeneity by the combination of ammonium sulphate precipitation, DEAE cellulose, and sephadex G-75 gel filtration chromatography. The cellulase was purified up to 14.5 fold and had a specific activity of 246 U/mg protein. The enzyme was a monomeric cellulase with a relative molecular mass of 58 kDa, as determined by SDS-PAGE. The enzyme exhibited its optimal activity at 50 degrees C and pH 6.0. The enzyme was stable in the pH range of 5.0 to 7.0 and its stability was maintained for 30 min at 50 degrees C and its activity got inhibited by Hg2+, Cu2+, Zn2+, Mg2+, Na2+, and Ca2+.     

Purification and characterization of a thermostable xylanase from Bacillus stearothermophilus T-6.

Bacillus stearothermophilus T-6 produces an extracellular xylanase that was shown to optimally bleach pulp at pH 9 and 65 degrees C. The enzyme was purified and concentrated in a single adsorption step onto a cation exchanger and is made of a single polypeptide with an apparent M(r) of 43,000 (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Xylanase T-6 is an endoxylanase that completely degrades xylan to xylose and xylobiose. The pIs of the purified protein were 9 and 7 under native and denaturing conditions, respectively. The optimum activity was at pH 6.5; however, 60% of the activity was still retained at pH 10. At 65 degrees C and pH 7, the enzyme was stable for more than 10 h; at 65 degrees C and pH 9, the half-life of the enzyme was approximately 6 h. Kinetic experiments at 55 degrees C gave Vmax and Km values of 288 U/mg and 1.63 mg/ml, respectively. The enzyme had no apparent requirement for cofactors, and its activity was strongly inhibited by Zn2+, Cd2+, and Hg2+. Xylan completely protected the protein from inactivation by N-bromosuccinimide. The N-terminal sequence of the first 45 amino acids of the enzyme showed high homology with the N-terminal region of xylanase A from the alkalophilic Bacillus sp. strain C-125.     

Characterization of a thermostable Bacillus stearothermophilus alpha-amylase

Liquefying-type Bacillus stearothermophilus alpha-amylase was characterized. The coding gene was cloned in Bacillus subtilis and the enzyme was produced in three different host organisms: B. stearothermophilus, B. subtilis, and Escherichia coli. Properties of the purified enzyme were similar irrespective of the host. Temperature optimum was at 70-80 degrees C and pH optimum at 5.0-6.0. The enzyme was stable for 1 h in the pH range 6.0-7.5 at 80 degrees C. The enzyme was stabilized by Ca2+, Na+, and bovine serum albumin. About 50% of the activity remained after heating at 70 degrees C for 5 days or 45 min at 90 degrees C. Metal ions Cd2+, Cu2+, Hg2+, Pb2+, and Zn2+ were inhibitory, whereas EDTA, ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, and Tendamistat were without effect. The enzyme was fully active after treatment in acetone or ethanol at 55 or 70 degrees C, respectively, for 30 min. Sodium dodecyl sulfate (1%) did not affect stability, whereas 6 M urea denatured totally at 70 degrees C. The Km value for soluble starch was 14 mg/ml. Mr is 59,000 and pI 8.8. The only difference between the enzymes produced in different hosts was in signal peptide processing.     

Cloning, expression, and characterization of 5-aminolevulinic acid synthase from Rhodopseudomonas palustris KUGB306

The hemA gene encoding 5-aminolevulinic acid synthase (ALAS) was cloned from the genomic DNA of photosynthetic bacterium Rhodopseudomonas palustris KUGB306. The deduced protein (ALAS) of this gene contained 409 amino acids. The hemA gene was subcloned into an expression vector pGEX-KG and the encoded protein was overexpressed as a fusion protein with glutathione-S-transferase (GST) in Escherichia coli BL21. The recombinant ALAS was purified and isolated free of the fusion partner (GST) by affinity purification on glutathione-Sepharose 4B resin and cleavage of the purified fusion protein by thrombin protease. The optimum pH and temperature of the recombinant ALAS was found to be at pH 7.5-8.0 and 35-40 degrees C, respectively. The Km value of the enzyme was 2.01 mM for glycine and 49.55 microM for succinyl-CoA. The enzyme activity was strongly inhibited by Pb2+, Fe2+, Co2+, Cu2+, and Zn2+ at 1 mM, but slightly affected by Mg2+ and K+. The recombinant ALAS required pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor led to complete loss of the activity. Ultraviolet-visible spectroscopy with the ALAS suggested the presence of an aldimine linkage between the enzyme and PLP.