嗜热拟青霉固体发酵产木聚糖酶的纯化和性质

研究一株新的嗜热拟青霉J18的固体发酵产木聚糖酶的纯化和性质。固体发酵的粗酶液经硫酸铵沉淀、凝胶过滤层析和离子交换层析得到了一种分子量约为26 kDa的电泳纯木聚糖酶,酶活力回收率为33.5%,纯化了5.27倍。该木聚糖酶具有很好的温度和pH稳定性,在pH7.0~pH 9.0下,60℃处理24 h,酶活力能保存80%以上。该酶水解玉米芯木聚糖生成以木二糖、木三糖和木四糖为主的低聚木糖,薄层层析分析表明不含木糖,适合生产低聚木糖。     

耐冷皮壳正青霉一种木聚糖酶的纯化与性质研究

研究了耐冷皮壳正青霉Eupenicillium crustaceum一种木聚糖酶的纯化和酶学性质。采用硫酸铵沉淀和阴离子交换层析的方法,从耐冷皮壳正青霉液体发酵液中分离纯化出一种亚基分子量35kDa的木聚糖酶。酶学性质研究表明,酶的最适pH值为5.5,在pH4.5-6.5范围内具有较高的催化活性。最适温度为50℃,20℃下酶活为最高酶活的40%。Ag+和Fe2+大幅度提高木聚糖酶的酶活,而Mn2+和Hg2+强烈抑制木聚糖酶的活性。同时,该木聚糖酶具有严格的底物特异性。     

产碱性木聚糖酶菌株的筛选及酶学性质

从青海盐碱湖土壤中筛选到25株产碱性木聚糖酶的菌株,其中编号为QH14的菌株产酶量达648.79U/mL,纯化后比活可达1148.56 U/mg。16 SrDNA鉴定表明菌株QH14属于短小芽孢杆菌,命名为Bacillus sp.QH14。从该菌株的基因组中克隆获得了碱性木聚糖酶编码基因XynQH14,并在大肠杆菌E.coliBL21(DE3)中获得重组表达。通过Ni-NTA亲和层析分离纯化后的重组QH14木聚糖酶比活达700.47 U/mg。该碱性木聚糖酶的酶促反应最适温度为60℃,最适pH为9.2;55℃处理1h仍保持50%的活力;在pH7.0~11条件下37℃处理酶液24 h后均保持80%以上的活力,且在pH11缓冲溶液中50℃处理24 h仍保持31.02%的酶活,显示了该碱性木聚糖酶较好的热稳定性和碱稳定,提示该碱性木聚糖酶在制浆造纸、纺织等行业的应用潜力。     

产碱性木聚糖酶菌株的筛选及酶学性质

从青海盐碱湖土壤中筛选到25株产碱性木聚糖酶的菌株, 其中编号为QH14的菌株产酶量达648.79 U/mL, 纯化后比活可达1148.56 U/mg。16 SrDNA鉴定表明菌株QH14属于短小芽孢杆菌, 命名为Bacillus sp. QH14。从该菌株的基因组中克隆获得了碱性木聚糖酶编码基因XynQH14, 并在大肠杆菌E.coliBL21(DE3)中获得重组表达。通过Ni-NTA亲和层析分离纯化后的重组QH14木聚糖酶比活达700.47 U/mg。该碱性木聚糖酶的酶促反应最适温度为60℃, 最适pH为9.2; 55℃处理1h仍保持50%的活力; 在pH7.0~11条件下37℃处理酶液24 h后均保持80%以上的活力, 且在pH11缓冲溶液中50℃处理24 h仍保持31.02%的酶活, 显示了该碱性木聚糖酶较好的热稳定性和碱稳定, 提示该碱性木聚糖酶在制浆造纸、纺织等行业的应用潜力。     

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。     

酶法水解超级稻秸秆木聚糖制备低聚木糖的工艺研究

本文以超级稻秸秆为原料,碱溶液抽提法得到粗木聚糖,然后酶水解,制备低聚木糖。对酶解条件进行了优化,并采用高效液相色谱法对木聚糖酶解液主要组分进行了分析。结果表明,正交试验优化的最佳酶解条件为温度50℃、pH5. 0、加酶量4%,在该试验条件下低聚木糖得率为13. 5%。酶解液组分分析发现,其主要组分为木二糖和木三糖,只有少量的单糖,该结果有利于后续低聚木糖的纯化,符合低聚木糖制备的要求。     

青霉L1来源具有生产木寡糖应用潜力的高比活GH11木聚糖酶

木聚糖酶是一种备受关注的糖苷水解酶,能够应用于酿造、饲料、制药、生物能源等多个领域,但是目前大部分木聚糖酶在低于30℃的环境中活力较低。为了获得在较低温度下具有高活力的木聚糖酶,从青霉L1(Penicilliumsp.L1)中克隆到一条GH11木聚糖酶基因XYN11A,并在毕赤酵母GS115中进行异源表达。经过纯化和酶学性质测定,该酶的最适p H和最适温度分别为3.5-4.0和55℃,能够在酸性和中性缓冲液(p H 1.0-7.0)中以及40℃下保持稳定,同时对所有已测试的金属离子和化合物都有一定的抗性。值得注意的是,该酶具有GH11家族中比较高的比活力6 700 U/mg,另外,该酶在较低温度20-40℃亦可展现出较高的酶活力(24%-58%)。经过16 h的榉木木聚糖水解实验,该木聚糖酶的水解产物主要是木二糖、木三糖和木四糖,几乎不产生单体木糖。因该酶同时具有产寡糖、较低温度下活力高以及嗜酸性等3种特性,XYN11A在食品和饲料工业中具有巨大的应用潜力。     

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。     

嗜热木聚糖酶基因在毕赤酵母中的表达及其酶学性质

木聚糖酶Umxyn10A,属于GH10家族,包含一段跨膜区和GH10家族催化功能域,将跨膜区去掉后,剩余部分重命名为Umxyn10AQ。按毕赤酵母密码子偏好性将Umxyn10AQ序列进行密码子优化,与毕赤酵母表达载体p PIC9K相连。重组质粒p PIC9K-Umxyn10AQ经SalⅠ单酶切线性化后转至毕赤酵母(Pichia pastoris)GS115。经G418筛选得到重组毕赤酵母菌株GS115/Umxyn10AQ,每12 h添加1%甲醇诱导剂。DNS法测定重组木聚糖酶re Umxyn10AQ酶活,最高酶活达到15 U/m L,SDS-PAGE分析表明,re Umxyn10AQ相对分子质量为45.0 k D。重组木聚糖酶re Umxyn10AQ的最适p H为8.0,最适反应温度85℃,Co2+对该酶活有显著促进作用,提高了近20%,水解产物为木糖(14%)、木二糖(86%)。结果表明,木聚糖酶Umxyn10AQ在毕赤酵母中成功表达并且分泌到胞外,相较于大肠杆菌表达产物Umxyn10A,最适p H提高了1.5个单位、最适温度提高了10个单位,p H耐受性和温度耐受性都有所改善,并且主要水解产物仍为木二糖。     

Enrichment of new alkane oxidizing bacterial strains for human drug metabolite production

An extracellular xylanase produced by Streptomyces matensis DW67 was purified from the culture supernatant by ammonium sulfate precipitation, ion exchange and gel filtration chromatography and characterized. The xylanase was purified to 14.5-fold to homogeneity with a recovery yield of 14.1%. The purified xylanase appeared as a single protein band on SDS-PAGE with a molecular mass of 21.2 kDa. However, it had a very low apparent molecular mass of 3.3 kDa as determined by gel filtration chromatography. The N-terminal sequence of first 15 amino acid residues was determined as ATTITTNQTGYDGMY. The optimal temperature and pH for purified xylanase was 65 °C and pH 7.0, respectively. The enzyme was stable within the pH range of 4.5–8.0 and was up to 55 °C. The xylanase showed specific activity towards different xylans and no activity towards other substrates tested. Hydrolysis of birchwood xylan by the xylanase yielded xylobiose and xylotriose as principal products. The enzyme hardly hydrolyzed xylobiose and xylotriose, but it could hydrolyze xylotetraose and xylopentaose to produce mainly xylobiose and xylotriose through transglycosylation. These unique properties of the purified xylanase make this enzyme attractive for biotechnological applications, such as bioblenching in paper and pulp industries, production of xylooligosaccharides. This is the first report of the xylanase from S. matensis.     

Expression of recombinant Bacillus licheniformis xylanase A in Pichia pastoris and xylooligosaccharides released from xylans by it

The mature peptide of Bacillus licheniformis xylanase A (BlxA) was successfully expressed in Pichia pastoris under the control of AOX1 promoter. After 96-h 0.25% methanol induction, the activity of recombinant B. licheniformis xylanase A (reBlxA) in culture supernatant was 122.9 U/mg. Enzymatic properties assays showed that the optimum temperature and pH for reBlxA were 60 degrees C and pH 6.0, respectively. When treated at 70 degrees C, pH 6.0 for 2 min, the residual activities of the reBlxA were 76%. Over 80% of reBlxA activity was retained after treatment of the enzyme by preincubation over a pH range of 5.0-9.0 for 1h at 25 degrees C. High performance liquid chromatography (HPLC) analysis revealed that xylotriose (X3) was the main hydrolysis product released from birchwood xylan and wheat bran insoluble xylan by reBlxA. The mode of action studies showed that reBlxA was an endo-acting xylanase and xylobiose (X2), xylotriose, xylotetraose (X4), xylopentaose (X5), and xylohexaose (X6) could be hydrolyzed by it. This is the first report on the expression of reBlxA in yeast and on determining and quantifying the hydrolysis products released from xylans by reBlxA.     

Purification and characterization of a moderately thermostable xylanase from Bacillus sp. strain SPS-0

A Bacillus spp. strain SPS-0, isolated from a hot spring in Portugal, produced an extracellular xylanase upon growth on wheat bran arabinoxylan. The enzyme was purified to homogeneity by ammonium sulfate precipitation, anion exchange, gel filtration, and affinity chromatography. The optimum temperature and pH for activity was 75 degrees C and 6.0. Xylanase was stable up to 70 degrees C for 4 h at pH 6.0 in the presence of xylane. Xylanase was completely inhibited by the Hg(2+) ions. beta-Mercaptoethanol, dithiothreitol, and Mn(2+) stimulated the xylanase activity. The products of birchwood xylan hydrolysis were xylose, xylobiose, xylotriose, and xylotetraose. Kinetic experiments at 60 degrees C and pH 6.0 gave V(max) and K(m)values of 2420 nkat/mg and 0.7 mg/ml.     

海枣曲霉木聚糖酶降解寡聚木糖的特性

利用滤纸层析或ＡｃｒｙｌｅｘＰ－２凝胶过滤从落叶松木聚糖硫酸水解液中分离纯化子木二糖至木五糖。采用硅胶薄层层析分析底物和产物的方法研究了海枣霉木聚糖酶降解寡聚木糖的特点。此酶作用于寡糖的最适ＰＨ为５．０,终产物为Ｘ和Ｘ２。酶作用于Ｘ３、Ｘ４及Ｘ５的相对初速度分别为１、３４和４００,Ｘ２几乎不被酶解,推断该酶的底物结合部位至少具有５个亚位点,在高底物浓度,低酶量,远离最适ＰＨ以及在反应初期都能检测到     

Purification and characterization of a xylobiose- and xylose-producing endo-xylanase from Aspergillus niger

A xylanase from a commercial Aspergillus niger pentoglycanase was purified to homogeneity by column chromatography on Ultrogel AcA 54, SP-Sephadex, Sephadex G-50, and SP-Sephadex. The enzyme hydrolyzed xylotriose slowly to xylose and xylobiose, and xylotetraose and higher xylo-oligosaccharides rapidly to mixtures of smaller xylo-oligosaccharides, with xylobiose and xylose being the preponderant final products. The anomeric configuration of the products was inverted, in contrast to the behavior of most other carbohydrases that initially produce mixtures of oligosaccharides. This enzyme is a glycoprotein having an amino acid composition high in acidic residues. Its molecular weight is 20,800 and its isoelectric point is at pH 6.7. Optimal pH values for activity and stability are between 4 and 6 and, in a 20-min assay, maximal activity is attained at 55°.     

Purification and characterization of two xylanases from Trichoderma longibrachiatum.

Two endoxylanases were purified from the culture medium of Trichoderma longibrachiatum. Both enzymes were highly basic, and lacked activity on carboxymethyl-cellulose. An enzyme of 21.5 kDa (xylanase A) had a specific activity of 510 U/mg protein, a Km of 0.15 mg soluble xylan/ml, possessed transglycosidase activity and generated xylobiose and xylotriose as the major endproducts from xylan or xylose oligomers. A larger enzyme of 33 kDa (xylanase B) had a specific activity of 131 U/mg protein, a Km of 0.19 mg soluble xylan/ml, lacked detectable transglycosidase activity and generated xylobiose and xylose as major endproducts from xylan and xylose oligomers. Xylotriose was the smallest oligomer attacked by both enzymes. In addition, xylotriose inhibited hydrolysis of xylopentanose by both enzymes, while xylobiose appeared to inhibit xylanase B, but not xylanase A.     

Purification and characterization of a thermophilic alkaline xylanase from thermoalkaliphilic Bacillus sp. strain TAR-1

Thermoalkaliphilic Bacillus sp. strain TAR-1 isolated from soil produced an extracellular xylanase. The enzyme (xylanase R) was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography. The molecular mass of xylanase R was 40 kDa and the isoelectric point was 4.1. The enzyme was most active over the range of pH 5.0 to 10.0 at 50°C. The optimum temperatures for activity were 75°C at pH 7.0 and 70°C at pH 9.0. Xylanase R was stable up to 65°C at pH 9.0 for 30 min in the presence of xylan. Mercury(ll) ion at 1 mM concentration abolished all the xylanase activity. The predominant products of xylan-hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that xylanase R was an endo-acting enzyme. Xylanase R had a K_m of 0.82 mg/ml and a V_max of 280 μmol min⁻¹ mg⁻¹ for xylan at 50°C and pH 9.0.     

Characterization of a novel, ultra-large xylanolytic complex (xylanosome) from Streptomyces olivaceoviridis E-86

A novel, ultra-large xylanolytic complex (xylanosome) from Streptomyces olivaceoviridis E-86 was purified to homogeneity by ammonium sulfate precipitation and Sephacryl S-300 gel filtration chromatography. The purified xylanosome appeared as a single protein band on the non-denaturing (native) polyacrylamide gel electrophoresis (PAGE) gel with a molecular mass of approximately 1200 kDa. The optimal temperature and pH for xylanase activity was 60 °C and pH 6.0, respectively. The xylanase activity was stable within pH 4.1–10.3. It was stable up to 60 °C at pH 6.0. The xylanosome was highly specific towards oat-spelt xylan, and showed low activity towards corncob powder, but exhibited very low activity towards lichenan, CMC and p-nitrophenyl derivatives. Apparent K_m values of the xylansosome for birchwood, beechwood, soluble oat-spelt and insoluble oat-spelt xylans were 2.5, 3.6, 1.7 and 4.9 mg ml⁻¹, respectively. The main hydrolysis products of birchwood xylan were xylotriose, xylobiose and xylose. Analysis of the products from wheat arabinoxylan degradation by xylanosome confirmed that the enzyme had endoxylanase and debranching activities, with xylotriose, xylobiose, xylose and arabinose as the main degradation products. These unique properties of the purified xylanosome from Streptomyces olivaceoviridis E-86 make this enzymatic complex attractive for biotechnological applications.     

Cloning of a Bacillus sp. BP-23 gene encoding a xylanase with high activity against aryl xylosides

Abstract The xynB gene encoding a xylanase from the recently isolated Bacillus sp. strain BP-23 has been cloned and expressed in Escherichia coli . The enzyme produced in this host shows a molecular size of 41 kDa and a pI of 4.5. The pH and temperature at which the highest activity was found were 5.5 and 50°C respectively. Crude xylanase B showed activity on xylan, aryl xylosides, xylotetraose and xylotriose, while xylobiose was not hydrolyzed by the enzyme. Xylanase B showed high specific activity on aryl xylosides, probably as a result of the transxylosidase activity detected.     

Cloning, sequencing, and expression of a xylanase gene from the extreme thermophile Dictyoglomus thermophilum Rt46B.1 and activity of the enzyme on fiber-bound substrate.

A genomic library of the Dictyoglomus sp. strain Rt46B.1 was constructed in the phage vector lambda ZapII and screened for xylanase activity. A plaque expressing xylanase activity, designated B6-77, was isolated and shown to contain a genomic insert of 5.3 kb. Subcloning revealed that the xylanase activity was restricted to a internal 1,507-bp PstI-HindIII fragment which was subsequently sequenced and shown to contain a single complete open reading frame coding for a single-domain xylanase, XynA, with a putative length of 352 amino acids. Homology comparisons show that XynA is related to the family F group of xylanases. The temperature and pH optima of the recombinant enzyme were determined to be 85 degrees C and pH 6.5, respectively. However, the enzyme was active across a broad pH range, with over 50% activity between pH 5.5 and 9.5. XynA was shown to be a true endo-acting xylanase, being capable of hydrolyzing xylan to xylotriose and xylobiose, but it could not hydrolyze xylobiose to monomeric xylose. XynA was also shown to hydrolyze xylan present in Pinus radiata kraft pulp, indicating that it may be of use as an aid in pulp bleaching. The equivalent xylanase gene was also isolated from the related bacterium Dictyoglomus thermophilum, and DNA sequencing showed these genes to be identical, which, together with the 16S small-subunit rRNA gene sequencing data, indicates that Rt46B.1 and D. thermophilum are very closely related.