Purification and characterization of an extracellular alpha-neoagarooligosaccharide hydrolase from Bacillus sp. MK03

An agarolytic bacterium was isolated from soil in Gifu prefecture, Japan, and identified as Bacillus sp. strain MK03. The strain secreted neoagarooligosaccharide hydroluse into the culture medium. The enzyme was purified 49.7-fold from the culture fluid by ammonium sulfate precipitation and anion-exchange and gel-filtration column chromatographic methods. The purified enzyme appeared as a single band on polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate (SDS)-PAGE. Estimations of the molecular mass by gel filtration and SDS-PAGE gave values of 320 kDa and 42 kDa, respectively, indicating that the enzyme is octametric. The enzyme cleaved the alpha-1,3 linkage in neoagarobiose to produce 3,6-anhydro-L-galactose and D-galactose. It also selectively cleaved the alpha-1,3 linkage at the nonreducing end in neoagarotetraose or neoagarohexaose to give 3,6-anhydro-L-galactose and agarotriose or agaropentaose. The optimum temperature and pH for the enzyme were 30 degrees C and 6.1, respectively. The N-terminal amino acid sequence showed no homology to sequences of other known neoagarooligosaccharide hydrolases and agarases.     

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 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.     

Purification and characterization of extracellular 1,2-alpha-L-fucosidase from Bacillus cereus

Bacillus cereus isolated from a soil sample, inductively produced alpha-L-fucosidase in culture medium containing porcine gastric mucin (PGM). The production of the enzyme was also weakly induced by L-fucose and D-arabinose, but not by other sugars including glucose. The enzyme was purified 61-fold with an overall recovery of 1.8% from the culture fluid supplemented with PGM by ammonium sulfate precipitation, acetone fractionation, and subsequent column chromatography. The purified enzyme was found homogeneous by SDS-PAGE and its molecular mass was estimated to be approximately 196,000 kDa. Its optimum pH was 7.0 and it was stable in the pH range of 5.0 to 9.0. The enzyme hydrolyzed the alpha-(1-->2)-L-fucosidic linkage in oligosaccharides such as Fucalpha1-2Galbeta1-4Glc (2'-fucosyllactose), Fucalpha1-2Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose I), and the glycoprotein PGM. The enzyme was inactive on p-nitrophenyl alpha-L-fucoside, the alpha-(1-->3)-L-fucosidic linkages in Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose III) and orosomucoid, the alpha-(1-->4)-L-fucosidic linkage in Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3Galbeta1-4Glc (lacto-N-fucopentaose II), and the alpha-(1-->6)-L-fucosidic linkage in thyroglobulin.     

Purification and characterization of extracellular medium-chain-length polyhydroxyalkanoate depolymerase from Pseudomonas sp. RY-1

A novel bacterial strain capable of growing in a medium containing a medium-chain-length polyhydroxyalkanoate (MCL-PHA) as the sole carbon source was isolated from a soil sample. The isolate, which was identified as Pseudomonas sp. RY-1, secreted MCL-PHA depolymerase into the culture fluid only when it was cultivated in a medium containing a MCL-PHA, such as polyhydroxyoctanoate (PHO) or polyhydroxynonanoate (PHN). The extracellular MCL-PHA depolymerase of this organism was purified to electrophoretic homogeneity. The enzyme was a tetramer with identical subunits and a total molecular mass of 115 kDa. The isoelectric point of this enzyme was estimated to be 5.9 by isoelectric focusing. The maximal activity was observed at pH 8.5 and 35 degrees C. The enzyme was insensitive to phenylmethylsulfonyl fluoride and dithiothreitol, unlike other short-chain-length (SCL) PHA depolymerases. The K(m) values for PHO and PHN were 0.86 and 1.47 mg/ml, respectively. The enzyme could not hydrolyze SCL-PHAs and p-nitrophenyl esters of fatty acids.     

A_2菌产蛋白水解酶的分离纯化

利用A2菌种液态摇瓶发酵产蛋白水解酶,后采用硫酸铵分级沉淀、Sephadex G-75凝胶过滤层析、DEAE离子交换层析对粗酶液进行分离纯化后,蛋白酶比活力达到521.47U/mL,纯化倍数是粗酶液的3.52倍,回收率为8.71%。通过SDS-PAGE凝胶电泳得到单一条带,并测得其相对分子质量约为30kDa。     

Purification and properties of an extracellular inulinase from Rhizopus sp. strain TN-96

A filamentous fungus, Rhizopus sp. strain TN-96, was isolated from rhizosphere soil samples. An extracellular inulinase was purified from the culture filtrate of strain TN-96 grown on inulin by DEAE-Cellulofine A-500 and Sephacryl S-200 HP chromatographies. The enzyme was homogeneous as judged by SDS-polyacrylamide gel electrophoresis, with an apparent M(r) of 83 kDa. The purified enzyme had specific activities of 17 U/mg toward inulin (I) and 0.32 U/mg toward sucrose (S) (I/S ratio, 53). Inulinase activity was optimal at pH 5.5 and 40 degrees C. The inulinase exhibited an apparent K(m) value of 9.0 mM for inulin. The enzyme also hydrolyzed raffinose, but not bacterial levan.     

Bacillus sp.LS 壳聚糖酶的分离纯化及性质研究

以 Bacillus sp IS 产生的壳聚糖酶粗酶液出发,经 (NH4)2SO4盐析、DEAE-Sephadex A25 阴离子交换、Sephadex G-100 凝胶过滤分离纯化后,SDS-PAGE 显示为一条带,测得其分子量为 30.9k u.同时对其酶学性质进行了初步研究,发现该酶在低于 40℃、pH6.0～7.5 范围内较稳定.最适反应温度和pH值分别为55℃和 5.0;Zn2 、Ag 、Ca2 、Co2 、Mn2 对该酶有明显的促进作用,而 Fe3 、Hg2 对该酶有强烈的抑制作用;该酶的米氏常数为2.50 mg/mL,最大反应速度为 4.19μmol/mL·min.     

Purification and characterization of extracellular caseinolytic enzyme of Micrococcus sp. MCC-315 isolated from cheddar cheese

Micrococcus sp. MCC-315, an organism isolated from Cheddar cheese, produced an extracellular calcium metalloenzyme. This protease was purified to homogeneity from culture supernatant by precipitation with ammonium sulfate (50 to 70% saturation) and gel filtration through Sephadex G-100, resulting in about 82 times increase of specific activity and 53% recovery of the enzyme. The protease exhibited a pH optimum at 10.6 for both whole casein and beta-casein. It had optimum activity for whole casein in the presence and absence of calcium++ at 60 and 50 degrees C, respectively, and at 37 to 40 degrees C for beta-casein with or without calcium++. The enzyme was stable at 45 degrees C but lost activity at higher temperatures. It was inhibited by heavy metal ions but calcium++, cobalt++, manganese++, strontium++, and iron++ had a slight stimulatory effect. The enzyme was inhibited completely and irreversibly by metal chelating agents. Calcium ions were required for maintenance of an active conformation of the enzyme. The enzyme had molecular weight of 28,900 and Michaelis constants 6.66 and 5.00 mg/ml for whole casein and beta-casein. Amino acid analysis of the hydrolyzed enzyme revealed the absence of sulfhydryl groups as was indicated also by lack of inhibition by thiol reagents.     

Bacillus sp.AA5所产壳聚糖酶的纯化及性质研究

纯化Bacillussp.AA5所产的壳聚糖酶,经SDS-聚丙烯酰胺凝胶电泳测得该酶的分子量大约为38.1kDa。同时对其酶学性质进行了初步研究,发现该壳聚糖酶在4℃下10d内稳定性良好,酶的最适温度和pH值分别为43℃和7.0,Ca2 、Mg2 、Ba2 对它的酶活有不同程度的促进作用。     

Purification and characterization of an l-amino acid oxidase from Pseudomonas sp. AIU 813

An l-amino acid oxidase was found from a newly isolated strain, Pseudomonas sp. AIU 813. This enzyme was remarkably induced by incubation with l-lysine as a nitrogen source, and efficiently purified using an affinity chromatography with l-lysine as ligand. The enzyme oxidized l-lysine, l-ornithine and l-arginine, but not other l-amino acids and d-amino acids. The oxidase activity for l-lysine was detected in a wide pH range, and its optimal was pH 7.0. In contrast, the oxidase activity for l-ornithine and l-arginine was not shown in acidic region from pH 6.5, and optimal pH for both substrates was 9.0. The enzyme was a flavoprotein and composed of two identical subunits with molecular mass of 54.5?kDa. The N-terminal amino acid sequence was similar to that of putative flavin-containing amine oxidase and putative tryptophan 2-monooxygenase, but not to that of l-amino acid oxidases.     

Purification and characterization of an extracellular lipase from Mucor hiemalis f. corticola isolated from soil

We have screened 39 microfungi isolates originated from soil in terms of lipolytic activity. Out of all screened, a novel strain of Mucor hiemalis f. corticola was determined to have the highest lipase activity. The extracellular lipase was produced in response to 2% glucose and 2.1% peptone. The lipase was purified 12.63-folds with a final yield of 27.7% through following purification steps; ammonium sulfate precipitation, dialysis, gel filtration column chromatography and ion exchange chromatography, respectively. MALDI-TOF MS analysis revealed 31% amino-acid identity to a known lipase from Rhizomucor miehei species. The molecular weight of the lipase was determined as 46kDa using SDS-PAGE and analytical gel filtration. Optimal pH and temperature of the lipase were determined as 7.0 and 40°C, respectively. The enzyme activity was observed to be stable at the pH range of 7.0-9.0. Thermostability assays demonstrated that the lipase was stable up to 50°C for 60min. The lipase was more stable in ethanol and methanol than other organic solvents tested. Furthermore, the activity of the lipase was slightly enhanced by SDS and PMSF. In the presence of p-NPP as substrate, K(m) and V(max) values of the lipase were calculated by Hanes-Woolf plot as 1.327mM and 91.11μmol/min, respectively.     

Purification and characterization of extracellular cysteine protease inhibitor, ECPI-2, from Chlorella sp

An extracellular cysteine protease inhibitor (ECPI-2) was purified to homogeneity from the culture filtrate of Chlorella sp. 4533 by the combination of various column chromatographies. The molecular mass of the inhibitor was estimated to be 340 kDa by SDS-PAGE. The inhibitor was extremely heat-stable under acidic or neutral condition. ECPI-2 exhibited an inhibitory activity against the proteolytic activity of papain, ficin, or chymopapain, but not against stem bromelain or cathepsin B. The inhibitor showed no inhibitory activity against trypsin, alpha-chymotrypsin or thermolysin. ECPI-2 contains 33.6% carbohydrate residues by weight and inhibits papain at a molar ratio of 1:2. The proteolysis of the inhibitor by trypsin or alpha-chymotrypsin was apparent, but the inhibitory activity of ECPI-2 was unaffected by these enzymes. The alpha-chymotrypsin hydrolysis product from ECPI-2 was further separated into six fractions by gel filtration. From these results, it is suggested that ECPI-2 has several reactive sites for papain.     

芽孢杆菌B15胞外蛋白酶和淀粉酶的酶学性质研究

为探索芽孢杆菌B15胞外蛋白酶和淀粉酶在饲用酶制剂方面的应用,试验对其产酶情况及胞外酶的酶学性质进行了研究。结果显示,该菌株产蛋白酶和淀粉酶的最高酶活性分别为(181.9±3.5)U/mL和(50.2±1.6)U/mL。蛋白酶和淀粉酶的最适作用温度范围分别为40℃～50℃和40℃～70℃,最适作用pH值为7和5～7。蛋白酶和淀粉酶经40℃～50℃热处理,以及在pH值为5～9的缓冲液中较稳定。蛋白酶被EDTA所抑制,属金属蛋白酶,金属离子(10mmol/L)Mg2+和K+对其有激活作用,Fe2+有抑制作用,而Fe2+对淀粉酶有一定的激活作用。     

Purification and characterization of an epsilon-poly-L-lysine-degrading enzyme from the epsilon-poly-L-lysine-tolerant Chryseobacterium sp. OJ7

The epsilon-poly-L-lysine-degrading enzyme of the epsilon-poly-L-lysine-tolerant Chryseobacterium sp. OJ7 was purified and characterized. The bacterium excreted the enzyme into the culture filtrate. The purified enzyme has a molecular mass of approximately 38.4 kDa and consists of two identical subunits with a molecular mass of 19.5 kDa. The enzyme catalyzed the endo-type degradation of epsilon-poly-L-lysine. A correlation between the epsilon-poly-L-lysine tolerance of the bacterium and the high epsilon-poly-L-lysine-degrading activity was suggested.     

苏云金芽孢杆菌精氨酸酶的纯化及酶学性质研究

从L-精氨酸诱导的苏云金芽孢杆菌SK20.001中分离纯化出SDS-PAGE电泳纯的精氨酸酶[EC3.5.3.1]。纯化酶的比活力为589.2U/mg,纯化过程酶的回收率为22.4%。酶经过聚丙烯酰胺凝胶电泳得到亚基的相对分子量约31000u。该酶的最适pH为9.8,最适温度为40℃,并且Mn2+显示出对酶有最强的激活作用。通过Lineweaver-Burk双倒数作图得到的精氨酸酶Km为15.2mmol/L。      

一株芽孢杆菌胞外多糖的抗氧化性研究

在连云港潮间带筛选到一株芽孢杆菌。经液体发酵后分离纯化得其胞外多糖(EPS)。用分光光度法研究了该菌胞外多糖在不同体系中对DPPH自由基、羟自由基、超氧阴离子的清除作用。结果表明,该菌胞外多糖能够有效地清除这3种自由基。所以该菌胞外多糖是一种很好的天然抗氧化剂。     

枯草芽孢杆菌纤溶酶的纯化及性质研究

以从牛肉酱中筛选的1株高产纤溶酶的枯草芽孢杆菌为材料,进行发酵产酶。其发酵液经过硫酸铵盐析、Sephadex G-100凝胶过滤、C M SepharoseCL-6B离子交换层析和电泳制备,得到电泳纯的枯草芽孢杆菌纤溶酶;其分子质量为32.5ku;pI10.9～11.8;具有直接溶解纤维蛋白和激活纤溶酶原的双重作用;胰蛋白酶对此酶无降解作用,对其纤溶活性无影响;该酶在pH4.0～13.0稳定,对温度的适应范围较广。     

Purification and characterization of polyphenol oxidase from Ferula sp.

Polyphenol oxidase (PPO) of several Ferula sp. was extracted and purified through (NH₄)₂SO₄ precipitation, dialysis, and gel filtration chromatography. Leaf and stem extracts were used for the determination of enzyme properties. Optimum conditions, for pH, temperature, and ionic strength were determined. The best substrates of PPO were catechol for leaf and (−) epicatechin for stem samples. Optimum pH and temperature were determined. K_M and V_max values were 2.34 × 10⁻³ M and 8541 EU/ml for catechol, and 2.89 × 10⁻³ M and 5308 EU/ml for (−) epicatechin. The most effective inhibitor was sodium diethyl dithiocarbamate for leaf samples and sodium metabisulphite for stem samples. Both inhibitors indicated competitive reactions. PPO showed irreversible denaturation after 40 min at 60 °C.     

Purification and characterization of arylmalonate decarboxylase from Achromobacter sp. KU1311

We have isolated, purified and characterized arylmalonate decarboxylase (AMDase; EC 4.1.1.76). This is an unique enzyme that gives optically pure arylpropionates from the corresponding arylmalonates. Recently, we have screened similar enzyme producers from soil samples and succeeded in isolating Achromobacter sp. KU1311. The gene encoding the enzyme was cloned and sequenced. The AMDase gene consists of 720 nucleotides, which specifies a 240 amino acid protein with a relative molecular mass of 24,735. This enzyme was purified and its characteristics were compared with those of the hitherto known enzyme from Alcaligenes bronchisepticus KU1201.