嗜碱菌(Bacillus sp.) ZBAW6的木聚糖酶的分离纯化及其性质

通过硫酸铵分级沉淀,阴离子交换层析,凝胶过滤3步从嗜碱菌Bacillus sp. ZBAW6纯化了木聚糖酶。结果表明该酶分子量为45kD。N末端序列为DPFAAAVAPL。在pH5.5～10.5范围内均具有较高酶活性和稳定性;最适反应温度为65℃,酶活力基本不变。该酶作用于Beechxylan的Km为0.11mg/mL,Vmax为 23.89μmol/(min·mg)。 Hg2+对该酶有强的抑制作用。     

极端嗜盐古生菌(Natrinema sp.)R6-5胞外嗜盐蛋白酶的纯化和性质研究

采用bacitracin-Sepharose 4B亲和层析的方法得到凝胶电泳均一的来自极端嗜盐古生菌(Natrinema sp.)R6-5的胞外嗜盐蛋白酶。经SDS-PAGE分析该酶亚基分子量为62kDa。PMSF对它的活性完全抑制,表明它是一种丝氨酸蛋白酶,该酶反应的最适NaCl浓度为3mol/L,最适温度为45℃,最适pH值为8.0。在高盐条件下能维持高活性并十分稳定,具有重要的潜在应用价值。     

假单胞菌碱性木聚糖酶的纯化及性质

假单胞菌(Pseudomonas)G62可产生两种胞外木聚糖酶,即XynA和XynB。经过硫酸铵沉淀、阴离子和阳离子交换层析、分子筛色谱,最终得到 两种电泳纯酶。XynA的分子量及等电点分别为42kD和91,XynB的分子量和等电点分别是 20kD和88。经薄层色谱分析证明,两酶以不同的方式水解木聚糖,但都不产生木糖,即 两酶都为内切酶,它们的最适作用温度均为50℃。XynA的最适作用pH为7.0～9.8,而XynB的为7.0～7.5。在65℃时的半寿期XynA为6 min,XynB为140 min。XynA的Km和Vmax分别是5.56 mg·ml-1和543μmol·min-1·mg-1,XynB的Km和Vmax分别是7.72 mg·ml-1和819μmol·min-1·mg-1。两酶受Cu2+、Fe3+、Pb2+、Zn2+和Hg2+强烈抑制。化学修饰的初步结果表明,两酶的活性位点氨基酸均含有色氨酸和羧基氨基酸。     

嗜碱芽包杆菌N6—27碱性纤维素酶的纯化及性质

The alkaline cellulase produced by alkalophilic Bacillus sp. N6-27 was purified to electrophoresis homogeneity by (NH4)2SO4 fractionation, Sepharose CL-4B hydrophobic interaction chromatography, Bio-gel P-150 chromatography. The molecular weight and pI determined by SDS-PAGE and by PAGE-IEF were 94,000 and 4.2, respectively. The optimum temperature and pH for the enzymatic catalysis were 55 degrees C and 8.5, respectively. The enzyme activity was stable under 50 degrees C and in the pH range of 6-11. The substrate was carboxymethylcellulose (CMC). The enzyme activity was strongly inhibited by Fe2+, Cu2+ and Hg2+.     

黑曲霉木聚糖酶的纯化与性质

由凝胶电泳酶谱检测到黑曲霉１４９发本酵液中存在两型木聚糖酶,依次为Ｘ－Ⅰ和Ｘ－Ⅱ。通过硫酸铵分级沉淀及ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ５０柱层析分别将Ｘ－Ⅰ、Ｘ－Ⅱ纯化到凝胶电泳均一。由ＳＤＳ－凝胶电泳和浓度梯度凝胶电泳测得Ｘ－Ⅰ和Ｘ－Ⅱ的分子量分别为３７ｋＤａ,２４ｋＤａ和２３ｋＤａ,Ｘ－Ⅰ具有亚基。二者的含糖量分别为２７．６％和７．３％。Ｘ－Ⅰ和Ｘ－Ⅱ最适返应温度分别为５０℃和５５℃,ｐＨ为４．     

嗜热和嗜碱木聚糖酶研究进展

木聚糖酶是降解半纤维素主要成分木聚糖的关键酶,广泛应用在食品、饲料、制浆造纸、生物脱胶等行业。特别是在造纸工业中,木聚糖酶显示出巨大的应用潜力,已成为国内外研究的热点。纸浆漂白工艺中需要酶在高温碱性条件下发挥作用。目前,主要通过筛选野生型木聚糖酶资源和对现有中性中温木聚糖酶分子改造的方法获得嗜热碱木聚糖酶。文中就嗜热嗜碱木聚糖酶的筛选、嗜热嗜碱机制研究及分子改造进展进行了综述,并对其前景进行了展望。     

嗜碱细菌Cellulomonas bogoriensis 69B4T木聚糖酶的表达纯化及性质研究

【目的】对嗜碱细菌Cellulomonas bogoriensis 69B4~T产碱性木聚糖酶进行研究,克隆来源于该菌株的木聚糖酶基因,并对其进行异源表达、纯化及酶学性质的表征,为后续研究碱性木聚糖酶的耐碱机制及应用奠定基础。【方法】采用单因素分析法对菌株产碱性木聚糖酶情况进行研究;通过基因组分析,锚定5个内切木聚糖酶基因,利用同源扩增的方法进行克隆,并在大肠杆菌中重组表达,利用亲和层析对重组酶进行纯化,以木聚糖为底物表征木聚糖酶的酶学性质。【结果】来源于C. bogoriensis 69B4~T的5种木聚糖酶Xyn370、Xyn393、Xyn425、Xyn466和Xyn486均在大肠杆菌内实现了异源表达,并经亲和层析获得纯酶组分,其最适反应温度分别为60、50、40、40、60°C,在50°C范围内保温2h,残余酶活均在90%以上;最适反应p H分别为7.0、8.0、8.0、8.0、9.0,在p H5.0–9.0时具有较好的稳定性;5种重组木聚糖酶对部分金属离子和高浓度盐表现出较好的耐受性,对榉木木聚糖的酶活性最高,均为内切型木聚糖酶。【结论】本研究表达纯化的5种重组木聚糖酶具有耐盐碱的优良特性,且对温度、某些金属离子和化学试剂耐受,为研究木聚糖酶的耐碱机制及工业应用提供了酶源。     

黑曲霉木聚糖酶的纯化与性质

由凝胶电泳酶谱检测到黑曲霉１４９发酵液中存在两型木聚糖酶,依次为Ｘ－Ⅰ和Ｘ－Ⅱ．通过硫酸铵分级沉淀及ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ５０柱层析分别将Ｘ－Ⅰ、Ｘ－Ⅱ纯化到凝胶电泳均一。由ＳＤＳ一凝胶电泳和浓度梯度凝胶电泳测得Ｘ－Ⅰ和Ｘ－Ⅱ的分子量分别为３７ｋＤａ,７６ｋＤａ,２４ｋＤａ和２３ｋＤａ,Ｘ－Ⅰ具有亚基。二者的含糖量分别为２７６％和７．３％。Ｘ－Ⅰ和Ｘ－Ⅱ最适反应温度分别为５０℃和５５℃,ｐＨ为４６和５．２。在ｐＨ４．６～９．２和ｐＨ４．０～１０．０之间Ｘ－Ⅰ、Ｘ－Ⅱ活力稳定。５０℃保温２４ｈ,Ｘ－Ⅰ活力仍为１００％,而Ｘ－Ⅱ的活力已降为２．８％。ＨｇＣｌ２和ＡｇＮＯ３显著抑制Ｘ－Ⅰ、Ｘ－Ⅱ的活力。Ｘ－Ⅰ与Ｘ－Ⅱ水解不同来源的木聚糖,其产物有所不同。     

绿色糖单孢菌木聚糖酶的分离纯化与酶学性质研究

木聚糖酶是一种重要的具有工业应用前景的木聚糖降解酶,在充分利用自然资源、保护生态环境等方面具有十分重要的意义.通过硫酸铵分级沉淀及Sephadex G-100凝胶柱层析方法从一株中度耐热耐碱放线菌—绿色糖单孢菌的胞外酶中纯化得到单一的木聚糖酶,相对分子质量为51 kD,酶纯度提高了13.01倍.纯酶最适反应温度为60℃,最适反应pH值为7.0;75℃以下该酶具有良好的热稳定性,在pH 7～10范围内具有较强的耐受力.金属离子Ca2+、Fe2+、Zn2+对该酶具有明显促进作用,Cu2+、Mn2+、Al3+和SDS具有抑制作用而K+,Na+,Mg2+没有明显的作用.该酶为大分子质量的糖基化蛋白,含糖量为21.96%.     

嗜碱芽孢杆菌N6-27碱性纤维素酶的纯化及性质

The alkaline cellulase produced by alkalophilic Bacillus sp. N6-27 was purified to electrophoresis homogeneity by (NH4)2SO4 fractionation, Sepharose CL-4B hydrophobic interaction chromatography, Bio-gel P-150 chromatography. The molecular weight and pI determined by SDS-PAGE and by PAGE-IEF were 94000 and 4.2,respectively. The optimum temperature and PH for the enzymatic catalysis were 55℃ and 8.5, respectively. The enzyme activity was stable under 50℃ and in the PH range of 6-11. The substrate was carboxy…     

短小芽孢杆菌A-30耐碱性木聚糖酶的纯化及性质研究

木聚糖广泛存在于自然界 ,通常占高等植物干重的 1 5%～ 30 % ,由木糖经β- 1 ,4-糖苷键连接起来形成主链 ,并由阿拉伯糖、乙酰基甘露糖、葡萄糖醛酸等复杂侧链共同组成 .在众多可降解木聚糖的酶中 ,β-内切木聚糖酶 ( E.C3.2 .1 .8,β- 1 ,4- xylanxylanohydrolase)起主要作用 .在纺织、制浆造纸、饲料及食品等工业中具有潜在的应用价值 .近年来 ,欧美等国已将其应用在造纸制浆工业 ,降低了漂白时氯的用量 ,改善了纸张性能 ,并且减少了环境污染 .对木聚糖酶的研究成为生物技术领域研究的热点之一 .国内外对来源于不同菌种的木聚糖酶的分离…     

Thermostable alkaline cellulase from an alkaliphilic isolate, Bacillus sp. KSM-S237

Thermostable alkaline cellulase (endo-1,4-β-glucanase, EC 3.2.1.4) activity was detected in the culture medium of a strictly alkaliphilic strain of Bacillus, designated KSM-S237. This novel enzyme was purified to homogeneity by a two-step column-chromatographic procedure with high yield. The N-terminal amino acid sequence of the purified enzyme was Glu-Gly-Asn-Thr-Arg-Glu-Asp-Asn-Phe-Lys-His-Leu-Leu-Gly-Asn-Asp-Asn-Val-Lys-Arg. The enzyme had a molecular mass of approximately 86 kDa and an isoelectric point of pH 3.8. The enzyme had a pH optimum of 8.6–9.0 and displayed maximum activity at 45°C. The alkaline enzyme was stable up to 50°C and more than 30% of the original activity was detectable after heating at 100°C and at pH 9.0 for 10 min. The enzyme hydrolyzed carboxymethylcellulose, lichenan (β-1,3;1,4-linkage), and p-nitrophenyl derivatives of cellotriose and cellotetraose. Crystalline forms of cellulose (Avicel and filter paper), H₃PO₄-swollen cellulose, NaOH-swollen cellulose, curdlan (β-1,3-linkage), laminarin (β-1,3;1,6-linkage), and xylan were barely hydrolyzed at all. Received: April 28, 1997 / Accepted: May 24, 1997     

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)。     

木霉T6木聚糖酶制剂研究

本文研究了木霉Ｔ６（Ｔｒｉｃｈｏｄｅｒｍａ ｓｐ．）产木聚糖酶固态发酵过程和木聚糖酶制剂制备,结果显示,固体曲培养４ｄ时酶活力最高,固体曲最适液固浸提比为７：１。木聚糖酶在６０～６５％硫酸铵饱和度下盐析效果最好。冷冻干燥和４０℃烘干酶粉得率分别为６８．３％和４５．７％。酶最适反应ＰＨ为４．５,最适反应温度５０℃,在不同温度下１小时后的半失活温度为４７．７℃。     

棘孢曲霉SM-L22木聚糖酶系主要组分的纯化与性质

经超滤浓缩、分子筛色谱、阴离子和阳离子交换层析 ,由棘孢曲霉发酵液最终分离得到 4个电泳纯的木聚糖酶主要组分Xy 1、Xy 2、Xy 3和Xy 4。通过SDS 聚丙烯酰胺凝胶电泳测得各组分的分子量分别是 92 1 3、32 4 0、4 2 4 0和 2 7 0 3kD。实验证明这些酶组分均属于酸性木聚糖酶 ,Xy 1、Xy 2、Xy 3和Xy 4的最适反应pH分别为 5 0、4 0、4 6和 3～ 3 5。各酶组分在酸性条件下较稳定 ,碱性条件下酶活丧失较快。Xy 1及Xy 2的最适反应温度在75℃ ,在 5 0℃以下比较稳定 ;Xy 3及Xy 4最适反应温度为 5 5℃ ,在 4 0℃以下比较稳定。通过对各酶组分米氏常数的测定可知 ,Xy 1及Xy 2对底物桦木木聚糖的Km 值分别为 0 36 %和 0 2 6 % ,Xy 3及Xy 4的Km 值为 2 4 6 %和1 3 9%。 4种组分的Vmax 分别为 4 0 1 μmol min mg、8 81 μmol min mg、81 97μmol min mg、4 71 μmol min mg。Cu2 、Ag 对各组分都有较强的抑制作用 ,Mg2 、Ba2 、Ca2 能促进Xy 3的木聚糖酶活 ,Ca2 也可大幅度促进Xy 4的木聚糖酶活性。     

Purification and characterization of a neutral endoxylanase from Aspergillus nidulans

Abstract A neutral endoxylanase from a culture filtrate of Aspergillus nidulans grown on oat spelt xylan was purified to apparent homogeneity. The purified enzyme showed a single band on SDS-PAGE with a molecular mass of 22,000 and had an isoelectric point of 6.4. The enzyme was a non-debranching endoxylanase highly specific for xylans and completely free from cellulolytic activity. The xylanase showed an optimum activity at pH 5.5 and 62°C and had a K m of 4.2 mg oat spelt xylan per ml and a V max of 710 μmol min⁻¹ (mg protein)⁻¹.     

Purification and characterization of a novel neutral β-glucanase and an alkaline β-glucanase from an alkaliphilic Bacillus isolate

A relatively high β-glucanase-producing strain Bacillus sp. III-3 was isolated from soda lakes in Neimenggu, China. This alkaliphilic strain was found to produce two thermostable β-glucanases including a novel neutral β-glucanase III-3-A and an alkaline β-glucanase III-3-B. The β-glucanases were purified to homogeneity from the culture supernatant with a two-step column chromatographic procedure. III-3-A and III-3-B had molecular mass of approximately 45 and 85 kDa, respectively. Mass spectrometry analysis indicated that III-3-A was probably different from the β-glucanases reported, whereas III-3-B showed high homology with those of family A5 alkaline β-glucanases from alkaliphilic bacilli. The optimum pH of III-3-A was about 7.0, while that of III-3-B was 8.0–10.0. Both enzymes exhibited maximum activity at 45 °C and were stable up to 50 °C. Ca²⁺ ion stimulated the activity of III-3-A but enhanced the thermostability of III-3-B. The two enzymes were resistant to most metal ions and reagents examined.     

枯草芽孢杆菌ZC-7中性蛋白酶的分离纯化及酶学性质研究

枯草芽孢杆菌ZC-7的发酵液,经离心分离得到粗酶液,再经硫酸铵盐析、中空纤维膜除盐浓缩、DEAE-Sepharose Fast Flow离子交换层析、Sephadex G-75柱层析等步骤获得电泳纯的中性蛋白酶。SDS-PAGE测得其分子量大约为42KDa。以酪蛋白为底物时,该酶的Km为5×10-3,Vmax为2.5×104ug/min,酶的最适作用pH为7.0,最适反应温度为55℃,在pH6.5～8.0, 40℃以下较稳定,对1mol/L H2O2具有一定的耐受性。EDTA、异丙醇和乙醇对该酶有抑制作用,Ca2+、Mg2+和Li+离子对其具有保护作用。     

Purification of xylanase from alkaliphilic Bacillus sp. K-8 by using corn husk column

The objective of this work was to apply low cost materials, agricultural residues, to the purification of xylanase. The results showed that crude extracellular, cellulase-free xylanase of an alkaliphilic Bacillus sp. strain K-8 could be purified in a single step by affinity adsorption–desorption on a corn husk column using a high flow rate, under the conditions 25 mM acetate buffer, pH 4.0, 4 °C, which prevented the hydrolysis of xylan by xylanase. After adsorption, the xylanase was eluted from the enzyme–corn husk complex with 500 mM Urea. The enzyme was purified 5.3-fold to homogeneity from culture supernatant. The molecular weight of the purified enzyme was 24 kDa as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The specific activity and recovery yield after purification were 25.4 U/mg protein and 42.3%, respectively.     

Purification and characterization of alkaline soda-bleach stable protease from Bacillus sp. APP-07 isolated from Laundromat soil

The detergent-compatible alkaline protease was produced from the bacterial strain Bacillus sp. APP-07 isolated from Laundromat soil of Solapur, Maharashtra, India. The culture was grown in 1000?ml capacity baffled flask with a working volume of 100?ml and incubated at 55?°C for 33?h on a rotary shaker. After incubation, alkaline protease was partially purified by the sequential method of acetone precipitation followed by nominal molecular weight limit (NMWL) cut-off ultrafiltration using 50?K and 10?K filters. Finally, Sephadex G-100 gel filtration chromatographic purification was performed to obtain 3.12 fold purified alkaline protease enzyme with a 66.67% final yield. The purified enzyme showed 31907.269 units (U) of enzyme activity containing 8741.718?U/mg of specific enzyme activity. The molecular weight of the enzyme was confirmed about 33.0?kDa (kDa) by the SDS-PAGE analysis. The purified enzyme was stable at higher pH and temperature range, with an optimum pH 10.5 and temperature 55?°C. The enzyme showed excellent stability and compatibility in various detergents, surfactants, bleach, and oxidizing agents. The enzyme activity enhanced in the presence of Ca²⁺, Cu²⁺, and surfactants, whereas; the phenylmethylsulphonyl fluoride (PMSF) and Diisopropyl fluorophosphate (DFP) completely inhibit the enzymatic activity, which pointed out that the enzyme affiliated to serine-centered metalloproteases family.In conclusion, the remarkable tolerance and stability of the enzyme explored the promising candidature for the several potential applications in the laundry detergents. The sustainability of the enzyme might serve several possible applications in the laundry detergents, leather industries, and other harsh industrial processes.