里氏木霉GXC木聚糖酶的研究*

研究了里氏木霉GXC产木聚糖酶的条件和酶学性质。结果表明,适宜产酶碳源为乳糖、甘露糖、棉子糖、木聚糖和麸皮,氮源为牛肉膏和酵母膏;产酶的最适初始pH为4.0,30℃培养60h。对以麸皮为碳源的培养液进行纯化的酶特性研究表明,木聚糖酶的最适反应温度为50℃,pH为5.5,该酶在pH5.0(7.0和40℃以下相对稳定。Fe3+和Mn2+对木聚糖酶有较大的促进作用,Cu～2+、Fe～2+和Ca～2+ 具有抑制作用。     

里氏木霉GXC木聚糖酶的研究

研究了里氏木霉GXC产木聚糖酶的条件和酶学性质。结果表明,适宜产酶碳源为乳糖、甘露糖、棉子糖、木聚糖和麸皮,氮源为牛肉膏和酵母膏;产酶的最适初始pH为4.0,30℃培养60h。对以麸皮为碳源的培养液进行纯化的酶特性研究表明,木聚糖酶的最适反应温度为50℃,pH为5.5,该酶在pH5.0(7.0和40℃以下相对稳定。Fe3+和Mn2+对木聚糖酶有较大的促进作用,Cu～2+、Fe～2+和Ca～2+ 具有抑制作用。     

嗜酸青霉酸性木聚糖酶产生特性及部分酶学性质

从煤矿酸性废水中分离到一株产木聚糖酶青霉,通过酸性液体培养研究了菌体生长对pH的响应及木聚糖酶的产生特征,并测定了木聚糖酶的部分应用性质.结果表明:实验菌株嗜酸,菌丝生长最适pH为2.0,孢子萌发生长适宜pH为3.0～4.0;木聚糖诱导菌体在生长稳定期大量产生木聚糖酶,蛋白胨是菌体产酶的适宜氮源;菌株所产木聚糖酶属于酸性木聚糖酶,反应最适pH 3.5、最适温度50 ℃～55 ℃,pH 2.0时酶活达到最高活力的72%,在最适反应条件下保温60 min,残余酶活接近70%,适用于较强酸性的高温加工环境.     

M-26碱性木聚糖酶的分离纯化及酶学性质研究

从Bacillus pumilus M-26发酵液中分离纯化碱性木聚糖酶,进行酶学性质研究,同时制备工业用碱性木聚糖酶制剂。首先将M-26发酵液进行硫酸铵盐析,制备工业用碱性木聚糖酶干品;然后进行sephadexG-25层析脱盐和cellulose DE-52层析得以纯化。硫酸铵的饱和度50%,酶制剂的酶活可达9 000 IU/g,收率为85%;分离纯化使酶的比活为126.32 IU/mg蛋白,纯化倍数为19.89,酶的回收率12.83%;分子量约为20 ku;M-26碱性木聚糖酶的最适温度和pH分别是55℃和pH 8.0,具有一定的耐碱性;该酶无纤维素酶活性,Fe2＋对其有激活作用;Mn2＋、Zn2＋、Fe3＋、Cu2＋对其具有抑制作用。短小芽胞杆菌M-26碱性木聚糖酶具有纸浆生物漂白应用前景。     

点青霉葡萄糖氧化酶基因的克隆及其酶学性质研究北大核心CSCD

旨在克隆点青霉菌(Penicillium notatum)中的葡萄糖氧化酶基因(GOD),在毕赤酵母(Phchia pastoris)中异源表达,纯化并研究其酶学性质。利用PCR技术从点青霉No.8312菌株的基因组DNA中克隆得到GOD基因,将该基因克隆到穿梭载体p MD-AOX上并在毕赤酵母X33中表达,对纯化后的葡萄糖氧化酶的酶学性质进行分析。结果显示,X33-GOD可高表达具有活性的GOD,在30℃、pH6.5的条件下,其培养液上清GOD酶活可达496 U/mL,比活123.0 U/mg;重组表达的葡萄糖氧化酶最适温度为40-45℃,最适pH为6.0,酶的稳定性研究表明,该酶在pH3.5-7.0区间和温度低于50℃下稳定。1 mmol/L Zn^(2+)对其有激活作用;Ag^+对该酶活性有较大抑制作用。构建出GOD的高产毕赤酵母工程菌株,与点青霉GOD相比,具有更高的发酵酶活和比活。     

里氏木霉GXC木聚糖酶的研究

研究了里氏木霉GXC产木聚糖酶的条件和酶学性质。结果表明,适宜产酶碳源为乳糖、甘露糖、棉子糖、木聚糖和麸皮,氮源为牛肉膏和酵母膏;产酶的最适初始pH为4．0,30℃培养60h。对以麸皮为碳源的培养液进行纯化的酶特性研究表明,木聚糖酶的最适反应温度为50℃,pH为5．5,该酶在pH5．0－7．0和40℃以下相对稳定。Fe^2 和Mn^2 对木聚糖酶有较大的促进作用,Cu^2 、Fe^2 具有抑制作用。     

点青霉葡萄糖氧化酶基因的克隆及其酶学性质研究

旨在克隆点青霉菌(Penicillium notatum)中的葡萄糖氧化酶基因(GOD),在毕赤酵母(Phchia pastoris)中异源表达,纯化并研究其酶学性质。利用PCR技术从点青霉No.8312菌株的基因组DNA中克隆得到GOD基因,将该基因克隆到穿梭载体p MD-AOX上并在毕赤酵母X33中表达,对纯化后的葡萄糖氧化酶的酶学性质进行分析。结果显示,X33-GOD可高表达具有活性的GOD,在30℃、pH6.5的条件下,其培养液上清GOD酶活可达496 U/mL,比活123.0 U/mg;重组表达的葡萄糖氧化酶最适温度为40-45℃,最适pH为6.0,酶的稳定性研究表明,该酶在pH3.5-7.0区间和温度低于50℃下稳定。1 mmol/L Zn~(2+)对其有激活作用;Ag~+对该酶活性有较大抑制作用。构建出GOD的高产毕赤酵母工程菌株,与点青霉GOD相比,具有更高的发酵酶活和比活。     

绵毛嗜热丝孢菌木聚糖酶的纯化与性质

研究了绵毛嗜热丝孢菌Thermomyces lanuginosus W205胞外木聚糖酶的纯化与性质。粗酶液经硫酸铵沉淀和Q-Sepharose FF离子交换层析即可得到电泳纯木聚糖酶,回收率为46.6%,比酶活为1396.9U/mg。该酶的最适pH和最适温度分别为pH7.0和75℃,pH稳定范围为5.5-10.8,70℃处理30min残存酶活在70%以上。薄层层析结果显示该酶水解桦木木聚糖的主要产物是木二糖和木三糖,并且能够通过转糖苷作用将木三糖转化为木二糖。该木聚糖酶易于纯化并且具有较宽的pH稳定性及良好的热稳定性,具有较大的潜在工业应用价值。     

嗜冷嗜酸β-1,4-木聚糖酶及其钙离子依赖型碳水化合物结合模块

【目的】β-1,4-木聚糖酶是木聚糖降解的关键酶之一,嗜冷嗜酸木聚糖酶在功能性低聚木糖的制备中具有重要作用,但相关报道较少。【方法】从太平洋火色杆菌(Flammeovirga pacifica)菌株WPAGA1基因组发掘到一条新型的木聚糖酶序列,经基因合成、质粒构建和表达,并对其进行分离纯化及酶学性质研究。【结果】该木聚糖酶(Xyl4513)具有2个保守结构域,一个属于糖苷水解酶11家族(glycoside hydrolase family 11,GH11)催化模块(Xyl4513-T),另一个属于碳水化合物结合模块(carbohydrate-binding module,CBM)60家族(CBM4513),这是一种非常罕见的GH11家族木聚糖酶含有CBM的现象。纯化后的Xyl4513最适反应温度和pH值分别为30℃、3.0,这一特性说明Xyl4513为嗜冷嗜酸β-1,4-木聚糖酶;而截短的木聚糖酶Xyl4513-T最适反应温度和pH值分别为20℃、4.0,且催化效率(kcat/Km)较前者下降了20%,说明CBM4513对酶稳定性和催化效率有较大影响。Ca^(2+)、Mg2+和Ni2+对酶催化活性均有明显促进作用,其中Ca^(2+)效果更为明显。仅当含有Ca^(2+)时,CBM4513才对β-1,4-木聚糖具有特异性结合能力,属于Ca^(2+)依赖型CBM,其最大结合量为9.13μmol/g。【结论】本文获得了一种新型的嗜冷嗜酸木聚糖酶和相应的Ca^(2+)依赖型CBM,进一步丰富了它们的基因和蛋白资源。     

以类弹性蛋白多肽为标签的表达质粒构建及其用于木聚糖酶的非色谱纯化

【目的】旨在构建一个能以非色谱纯化目标蛋白的表达质粒,使用自行设计的类弹性蛋白多肽(ELPs)作为非色谱纯化标签,以纯化目标蛋白。该ELPs长度短,对盐非常敏感。【方法】从头设计了木聚糖酶,将其通过一段无规则卷曲同ELPs相连,合成了编码上述序列的基因,并构建重组表达载体pET-22b-SoxB-M2-S-ELP,转化至大肠杆菌BLR(DE3)中诱导表达,采用可逆相变循环经高速离心纯化木聚糖酶,并考察纯酶的酶学性质。【结果】成功构建了表达载体并表达,在pH=7.0时0.5 mol/L碳酸钠可使ELPs的相变温度降至22℃。在上述条件下,对木聚糖酶进行了非色谱纯化,其纯化倍数为3.2,回收率为21.2%,纯度为64.3%。经测定,未连接ELPs的酶、粗酶及纯化酶学性质基本一致,其最适温度为60℃,最适pH为6.0,最适反应时间为30 min,粗酶70℃保温1 h相对酶活仍有50%,为嗜热木聚糖酶,与预期相符。【结论】ELPs作为非色谱纯化标签纯化重组木聚糖酶具有操作简单、易于放大、成本较低的优势,故所构建的重组质粒可望通用于分离多种重组蛋白,具有较广泛的用途。     

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%.     

产木聚糖酶白地霉培养特性及部分纯化的酶学特性

本文对白地霉Ref1的培养特性、产酶条件和酶学特性进行了初步研究。结果表明:该菌为低温型菌株,其最佳生长条件为pH6、20℃和酵母膏作为氮源;最佳产酶条件为pH3-7、15℃及以酵母膏氮源;条件优化后产酶可达118.7U/mL,可溶蛋白含量可达到60μg/mL,酶溶液的比活可达到1250U/mg蛋白质;该木聚糖酶的最适反应温度和pH分别为50℃和5,金属离子Mg2+、Na+和8mmol/L的Fe2+、Cu2+、Zn2+等对木聚糖酶的活性有抑制作用,而Ca2+、4mmol/L的Fe2+、Cu2+、Zn2+和8mmol/L的Mn2+等对该酶反应则有促进作用;该木聚糖酶在保温2h后在15-40℃范围内能保持80%以上的酶活性,在50℃时能保持68%的酶活性;用lineweaver-Burk作图法(双倒数作图法)求得该酶的最大反应速度Vmax和Km值分别为163.38mmol/mg/min和0.75mg/mL。     

Properties and application of a partially purified alkaline xylanase from an alkalophilic fungus Aspergillus nidulans KK-99

An alkalophilic Aspergillus nidulans KK-99 produced an alkaline, thermostable xylanase (40 IU/ml) in a basal medium supplemented with wheat bran (2% w/v) and KNO3 (at 0.15% N) pH 10.0 and 37 degrees C. The partially purified xylanase was optimally active at pH 8.0 and 55 degrees C. The xylanase was stable in a broad pH range of 4.0-9.5 for 1 h at 55 degrees C, retaining more than 80% of its activity. The enzyme exhibited greater binding affinity for xylan from hardwood than from softwood. The xylanase activity was stimulated (+25%) by Na+ and Fe2+ and was strongly inhibited (maximum by 70%) by Tween-20, 40, 60, SDS, acetic anhydride, phenylmethane sulphonyl fluoride, Triton-X-100. The xylanase dose of 1.0 IU/g dry weight pulp gave optimum bleach boosting of Kraft pulp at pH 8.0 and temperature 55 degrees C for 3 h reaction time.     

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…     

Production and biochemical characterization of xylanase from an alkalitolerant novel species Aspergillus niveus RS2

The novel fungus Aspergillus niveus RS2 isolated from rice straw showed relatively high xylanase production after 5 days of fermentation. Of the different xylan-containing agricultural by-products tested, rice husk was the best substrate; however, maximum xylanase production occurred when the organism was cultured on purified xylan. Yeast extract was found to be the best nitrogen source for xylanase production, followed by ammonium sulfate and peptone. The optimum pH for maximum enzyme production was 8 (18.2 U/ml); however, an appreciable level of activity was obtained at pH 7 (10.9 U/ml). Temperature and pH optima for xylanase were 50°C and 7.0, respectively; however the enzyme retained considerably high activity under high temperature (12.1 U/ml at 60°C) and high alkaline conditions (17.2 U/ml at pH 8 and 13.9 U/ml at pH 9). The enzyme was strongly inhibited by Hg²⁺, while Mn²⁺ was slight activator. The half-life of the enzyme was 48 min at 50°C. The enzyme was purified by 5.08-fold using carboxymethyl-sephadex chromatography. Zymogram analysis suggested the presence of a single candidate xylanase in the purified preparation. SDS-PAGE revealed a molecular weight of approximately 22.5 kDa. The enzyme had K _m and V _max values of 2.5 and 26 μmol/mg per minute, respectively.     

Purification and characterization of an alkaline xylanase from alkaliphilic Micrococcus sp AR-135

An xylanase producing alkaliphilic Micrococcus sp was isolated from an alkaline soda lake. Xylose and xylan induced enzyme production but no activity was detected when it was grown using other carbohydrate sources. The level of xylanase production was higher in the presence of xylose than in the presence of xylan. The enzyme was purified to homogeneity and its molecular weight was estimated to be 56 kD on SDS-PAGE. The optimum temperature and pH for xylanase activity were 55°C and 7.5–9.0, respectively. Sixty per cent of the maximum activity was displayed at pH 11. The enzyme was very stable in the pH range of 6.5–10 and up to a temperature of 40°C. Xylanase activity was inhibited by Cu²⁺ and Hg²⁺. Received 03 October 1997/ Accepted in revised form 03 February 1998     

Purification and characterization of low molecular weight alkali stable xylanase from Neosartorya spinosa UZ-2-11

Alkaliphilic xylanase from Neosartorya spinosa UZ-2-11 was purified using a three-step of purification scheme of ammonium sulphate precipitation followed by Sephadex G-100 gel filtration and DEAE-cellulose ion-exchange chromatography, and compared its properties with N. tatenoi KKU-CLB-3-2-4-1 of our previous report. The purified xylanase from N. spinosa UZ-2-11 exhibited maximum activity at pH 9.0 and 45 °C which was similar to endo-xylanase from N. tatenoi KKU-CLB-3-2-4-1. However, this enzyme was stable in a range of pH 6.0–11.0. It was also more stable at a high temperature of 50 °C where the activity was still up to 50% after heating for 120 min. The xylanase was purified 7.89-fold with 3.0% of yield to obtain a specific activity of 11.88 U/mg. The molecular weight of xylanase from this fungus was 27.68 kDa. The K_m and V_max values of the purified xylanase were 0.24 mg/mL and 15.85 μmol/min/mg, respectively. The xylanase activity was moderately inhibited by Hg²⁺ at a concentration of 10 mM, which was different to the case of N. tatenoi KKU-CLB-3-2-4-1 where Hg²⁺ was a strong inhibitor. In addition, the hydrolysed birchwood xylan was obtained mailnly xylobiose, xylotriose, xylotetraose and xylopentaose as end products, suggesting that it was an endo-xylanase.