木聚糖酶XYNB分子中折叠股B1和B2间的疏水作用对酶热稳定性的影响

对来源于Streptomycesolivaceoviridis的高比活木聚糖酶XYNB进行同源建模,并结合嗜热木聚糖酶氮末端芳香族氨基酸疏水作用的结构分析,设计了XYNB的T11Y定点突变,观察XYNB分子中折叠股B1和B2的疏水作用对酶的热稳定性的影响。将突变酶XYNB′在毕赤酵母中表达,表达的XYNB′经纯化后与原酶XYNB(同样经毕赤酵母表达后纯化)进行酶学性质比较,结果表明,XYNB′的耐热性比XYNB有明显的提高,但最适温度与原酶一样为60℃。另外,XYNB′的最适pH、Km值及比活性均有一定的改变。实验证实了木聚糖酶XYNB的氮端芳香族氨基酸之间的疏水相互作用与其热稳定性相关,为进一步的结构与功能研究提供了优良的基因材料。     

极端耐热木聚糖酶基因在大肠杆菌和毕赤酵母中的高效表达

以海栖热袍菌 (Thermotoga maritima) MSB8菌株基因组DNA为模板,通过PCR扩增出木聚糖酶（XylanaseB）基因, 将此基因克隆至大肠杆菌表达载体pET_28a（+）和毕赤酵母表达载体pPIC9K,并分别转化大肠杆菌 BL21和毕赤酵母GS115。该木聚糖酶在大肠杆菌细胞中表达量高, 但不能分泌; 而在毕赤酵母细胞的表达产物可分泌至胞外。酶学性质分析表明,此酶分子量约为40kD,其最适反应温度为90℃, 最适反应pH值为6.65,且在碱性条件下稳定,具有重要的工业应用前景。     

常压室温等离子体快速诱变绿色糖单孢菌筛选 木聚糖酶高产菌株及其酶学性质研究

[目的]利用常压室温等离子体快速诱变绿色糖单孢菌,筛选耐热耐碱木聚糖酶高产菌株,并对其进行酶学性质分析,确保其适用于生物制浆漂白工艺.[方法]采用刚果红平板水解圈法结合摇瓶发酵胞外酶测定法进行菌株筛选,并通过DNS木聚糖酶活性测定等方法对来源于不同突变株的木聚糖酶进行酶学性质分析对比.[结果]筛选出遗传稳定性良好的两株木聚糖酶高产菌株AT24和AT22-2,以麦草浆为诱导底物的粗酶液中,突变株AT24及AT22-2所产的木聚糖酶活性分别为512.74、552.70U/mL,分别为原始菌株S.v的16和17倍的.来源于突变株AT22-2的木聚糖酶的最适反应pH为9.5,最适反应温度为90℃,在50℃-90℃温度范围内具有良好的热稳定性,在100℃条件下处理30 min剩余酶活仍为68％;突变株AT24所产木聚糖酶的最适反应温度为60℃,最适pH为10.0,在60℃-80℃的高温环境下,突变株AT24所产的木聚糖酶具有良好的热稳定性.[结论]突变株AT22-2所产具有耐碱耐高温性质的木聚糖酶,在应用领域尤其在纸浆造纸行业具有较大的潜在应用价值.     

适冷海洋细菌交替假单胞菌（Pseudoalteromonas sp.）MB-1内切葡聚糖酶基因的克隆和表达

从黄海深海海底淤泥中筛选出一株产纤维素酶的适冷革兰氏阴性杆菌MB1,克隆和分析了MB1的16S rDNA序列（GenBank接受号：AY551321）,经鉴定为交替假单胞菌（Pseudoalt eromonas）,命名为Pseudoalteromonas sp.MB1。克隆了该菌适冷内切葡聚糖酶基因celA（GenBank接受号：AY551322）,并在大肠杆菌（Escherichia coli）BL21中进行了表达。重组E.coli菌体破碎后,获取上清液,其中融合蛋白GSTCelA浓度约为78.5mg/L。分析了融合酶GSTCelA的性质,其最适反应温度为35℃,最适反应pH值为72,为中性适冷酶。实验结果为交替假单胞菌低温纤维素酶的基础理论和应用研究奠定了基础。     

焦曲霉产木聚糖酶的研究*

从1144株真菌中筛选到一株产木聚糖酶活力较高的焦曲霉(Aspergillus ustus)。该菌株适宜的产酶条件为在4%麸皮液中添加0.5%葡萄糖,0.4%硝酸钠及0.1%氯化钠,30℃振荡培养6d,木聚糖酶活力可达2176 u/mL。酶反应的最适温度为55℃,最适pH为5.5,在pH5～8酶活力稳定。45℃保温1h,酶活力剩余35%。酶水解桦木木聚糖的Km值为4.3×10^-3g/mL,V_max值为4.9mg/(mL·min)。酶解产物以木三糖和木四糖为主,表明该酶是一种典型的内切糖苷酶。     

定点突变提高里氏木霉木聚糖酶 (XYN II) 的稳定性

通过定点突变的方法,在来源于里氏木霉Trichderma reesei的木聚糖酶XYN II的N-末端两个β折叠片层间添加二硫键,以提高木聚糖酶的稳定性。原酶XYN-OU和突变酶XYN-HA12 (T2C、T28C和S156F) 分别在毕赤酵母中分泌表达,突变酶与原酶纯化后进行酶学性质比较。结果表明：突变酶最适反应温度由50℃提高到60℃;在70℃的半衰期由1 min提高到14 min;最适反应pH为5.0,与原酶保持一致,但是在50℃、30 min条件下的pH稳定范围由4.0~9.0扩展到3.0~10.0。对木聚糖酶分子改良的结果反映出在β片层间添加二硫键可以有效改善酶在较高温度下三维结构的刚性,提高热稳定性。     

Bacillus pumilus WL-11木聚糖酶A的纯化、鉴定及其底物降解方式

对一株Bacilluspumilus WL_11木聚糖酶的纯化、酶学性质及其底物降解模式进行了研究。经过硫酸铵盐析、CM_Sephadex及SephadexG_75层析分离纯化,获得一种纯化的WL_11木聚糖酶A ,其分子量为26.0kD ,pI值9.5 ,以燕麦木聚糖为底物时的表观Km 值为16.6mg mL ,Vmax值为12.63μmol (min·mg)。木聚糖酶A的pH稳定范围为6 0至10 4 ,最适作用pH范围则在7.2至8.0之间,是耐碱性木聚糖酶;最适作用温度为45℃～55℃,在37℃、45℃以下时该酶热稳定性均较好;50℃保温时,该酶活力的半衰期大约为2h ,在超过50℃的环境下,该酶的热稳定较差,55℃和60℃时的酶活半衰期分别为35min和15min。WL_11木聚糖酶A对来源于燕麦、桦木和榉木的可溶性木聚糖的酶解结果发现,木聚糖酶A对几种不同来源的木聚糖的降解过程并不一致。采用HPLC法分析上述底物的降解产物生成过程发现木聚糖酶A为内切型木聚糖酶,不同底物的降解产物中都无单糖的积累,且三糖的积累量都较高;与禾本科的燕麦木聚糖底物降解不同的是,木聚糖酶A对硬木木聚糖降解形成的五糖的继续降解能力较强。采用TLC法分析了WL-11粗木聚糖酶降解燕麦木聚糖的过程,结果表明燕麦木聚糖能够被WL-11粗木聚糖酶降解生成系列木寡糖,未检出木糖,这说明WL-11主要合成内切型木聚糖酶A,同时发酵液中不含木糖苷酶,适合用来酶法制备低聚木糖。     

棒曲霉22产木聚糖酶的研究

从105株霉菌和酵母菌中筛选到一株木聚糖酶活力较高的棒曲霉(Aspergillua clavatus)菌株22。该菌株适宜的产酶培养基为(g/1):蔗渣半纤维素30,NH₄NO₃ 5,酵母膏5,麸皮10,吐温801和少量无机盐,初始pH5.5。最适的孢子接种量为4.9×10⁶个/ml。在上述培养基中28℃振荡培养72h.木聚糖酶活力可达335.9u/ml。酶反应的最适温度为50℃;最适pH为4.8,在pH 6-11酶活性稳定。     

里氏木霉内切-β-甘露聚糖酶基因在毕赤酵母中的表达

采用PCR方法从里氏木霉(Trichoderma reesei)基因组中获得含有两个内含子的内切-β-甘露聚糖酶全长基因,末端重叠延伸PCR去除内含子后,将其插入到巴斯德毕赤酵母(Picher pastoris)表达载体pPIC9K中,位于α-因子信号肽序列的下游,并与之同框,获得重组质粒pM242。重组质粒线性化后用电击法转化毕赤酵母菌株GS115。经大量筛选,获得高效分泌表达内切甘露聚糖酶的毕赤酵母工程菌株Gpmf25。摇瓶发酵结果表明,培养基中甘露聚糖酶的活力可达12.5IU/mL。重组酶最适pH和最适反应温度分别为5.0和80℃,在pH5.0～6.0时酶活稳定,在pH5.4时70℃保温30min酶活维持50％以上。     

Purification and characterization of the overexpressed Aspergillus oryzae xylanase, XynF1

The Aspergillus oryzae xynF1 gene coding for a xylanase, XynF1, was successfully overexpressed under the strong A. oryzae TEF1 gene promoter. The high-XynF1-producing transformant secreted about 180 mg/l of XynF1 in the glucose-containing medium. The overexpressed XynF1 was purified by only one chromatographic step. The purified XynF1 had a molecular mass of 35.0 kDa, a pH optimum of 5.0, and a temperature optimum of 60 degrees C.     

Molecular cloning, characterization, and expression analysis of the xynF3 gene from Aspergillus oryzae

The gene encoding xylanase F3 (xynF3) was isolated from a genomic library of Aspergillus oryzae KBN616, used for making shoyu koji. The structural part of xynF3 was found to be 1468 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynF3 was interrupted by ten short introns and encoded 323 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynF3 had a signal peptide of 22 amino acids. The predicted amino acid sequence of XynF3 has strong similarity to other family 10 xylanases from fungi. The xynF3 gene was successfully overexpressed in A. oryzae and the XynF3 was purified. The molecular mass of XynF3 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 32,000. This was almost the same as the molecular mass of 32,437 calculated from the deduced amino acid sequence. The purified XynF3 showed an optimum activity at pH 5.0 and 58 degrees C. It had a Km of 6.5 mg/ml and a Vmax of 435 micromol x min(-1) x mg(-1) when birch wood xylan was used as a substrate. Expression of the xynF3 gene was analyzed using an Escherichia coli beta-glucuronidase gene as a reporter. The result indicated that xynF3 is expressed in the medium containing wheat bran as a carbon source.     

米曲霉耐热木聚糖酶纯化及酶学特性研究

对米曲霉原始发酵液中耐热木聚糖酶进行纯化和酶学特性研究,利用甘蔗渣为碳源培养米曲霉,通过超滤和阴离子交换柱两步纯化得到木聚糖酶XynH1,分子量35．402kDa,利用飞行时间质谱和SDS—PAGE分析,推断XynH1为XylanaseXynF1,分子量为35．402kDa。XynH1属于糖苷水解酶家族10,酶活为442．2IU／nag,最适pH和温度分别为pH6．0和65℃,80℃以下及pH4．0～10．5范围内较稳定。     

Characterization of a xylanase from the newly isolated thermophilic Thermomyces lanuginosus CAU44 and its application in bread making

AIMS: A xylanase from the newly isolated thermophilic fungus, Thermomyces lanuginosus CAU44, was characterized and evaluated for its suitability in bread making. METHODS AND RESULTS: Xylanase was purified 3.5-fold to homogeneity with a recovery yield of 32.8%. It appeared as a single protein band on SDS-PAGE gel with a molecular mass of c. 25.6 kDa. The purified xylanase had an optimum pH of 6.2, and it was stable over pH 5.6-10.3. The optimal temperature of xylanase was 75 degrees C and it was stable up to 65 degrees C at pH 6.2. Study was further carried out to investigate the effect of the purified xylanase on the properties of wheat bread and its staling during storage. CONCLUSIONS: The purified xylanase from T. lanuginosus CAU44 was stable up to 65 degrees C and had a broad pH range. The presence of thermostable xylanase during bread making led to an improvement of the specific bread volume and better crumb texture. Besides, addition of xylanase provided an anti-staling effect. SIGNIFICANCE AND IMPACT OF THE STUDY: The xylanase from the newly isolated Thermomyces lanuginosus CAU44 shows great promise as a processing aid in the bread-making industry.     

Characterization of a cellulase-free, neutral xylanase from Thermomyces lanuginosus CBS 288.54 and its biobleaching effect on wheat straw pulp

A xylanase purified from the thermophilic fungus Thermomyces lanuginosus CBS 288.54 was characterized and its potential application in wheat straw pulp biobleaching was evaluated. Xylanase was purified 33.6-fold to homogeneity with a recovery yield of 21.5%. It appeared as a single protein band on SDS-PAGE gel with a molecular mass of approx. 26.2 kDa. The purified xylanase had a neutral optimum pH ranging from pH 7.0 to pH 7.5, and it was also stable over pH 6.5-10.0. The optimal temperature of the xylanase was 70-75 degrees C and it was stable up to 65 degrees C. The purified xylanase was found to be not glycosylated. The xylanase was highly specific towards xylan, but did not exhibit other enzyme activity. Apparent Km values of the xylanase for birchwood, beechwood, soluble oat-spelt and insoluble oat-spelt xylans were 4.0, 4.7, 2.0 and 23.4 mg ml-1, respectively. The potential application of the xylanase was further evaluated in biobleaching of wheat straw pulp. The brightness of bleached pulps from the xylanase pretreated wheat straw pulp was 1.8-7.79% ISO higher than that of the control, and showed slightly lower tensile index and breaking length than the control. Although chlorine consumption was reduced by 28.3% during bleaching, the xylanase pretreated pulp (15 U g-1 pulp) still maintained its brightness at the control level. Besides, pretreatment of pulp with the xylanase was also effective at an alkaline pH as high as pH 10.0.     

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.     

Purification and characterization of xylanase from a thermophilic Streptomyces sp. K37

Extracellular xylanase (EC 3.2.1.8) from Streptomyces sp. K37 was purified 33.53 by ultrafiltration and cation exchange chromatography followed by gel filtration chromatography. The optimum pH and temperature for purified xylanase were found to be pH 6.0 and 60 degrees C. The Km and V(max) values of the purified xylanase were 15.4 mg ml(-1) and 0.67 micromole reducing sugar min(-1) ml(-1). High performance liquid chromatography (HPLC) gel filtration of the purified xylanase eluted xylanase activity as a peak corresponding to the molecular weight of about 24.3 kDa while the molecular weight determined by SDS-PAGE was found to be 26.4 kDa. The purified xylanase of Streptomyces sp. K37 was found to be endoxylanase and non arabinose liberating enzyme and was highly glycosylated (73.97%).     

木聚糖酶XYNB的N46D突变、表达及酶学性质变化

对来源于Streptomyces olivaceoviridis的高比活木聚糖酶XYNB进行同源建模和同源序列比较,发现第11族木聚糖酶的催化结构域在β折叠股A3和B3之间存的一个保守的氨基酸位点,该位点与木聚糖酶的pH特性有关.据此设计了XYNB的N46D定点突变.将突变酶XYNBN46D在毕赤酵母中表达,表达的XYNBN46D经纯化后与原酶XYNB(同样经毕赤酵母表达后纯化)进行酶学性质比较,结果表明, XYNBN46D的最适pH值由5.2下降到4.2,pH稳定性也向酸性pH偏移,同时,热稳定性和最适温度也有一定的提高, 但酶的比活性显著下降.结果证实,木聚糖酶XYNB的第46位Asn与其最适pH值相关.对导致酶学性质改变的可能因素进行了分析,结果为进一步的结构与功能研究提供了资料.     

An alkali-tolerant xylanase produced by the newly isolated alkaliphilic <Emphasis Type="Italic">Bacillus pumilus</Emphasis> from paper mill effluent

An alkaline active xylanase, XynBYG, was purified from an alkaliphilic Bacillus pumilus BYG, which was newly isolated from paper mill effluent. It had an optimum pH of 8.0–9.0, and showed good stability after incubated at pH 9.0 for 120 min. The optimum temperature for the activity was 50°C, and the enzyme retained below 55% of its original activity for 30 min at 55°C. The gene coding for XynBYG consists of 687 bp and encodes 229 amino acids. Similarity analysis indicated that XynBYG belong to family 11 glycosyl hydrolases. Site-directed mutagenesis was performed to replace five sites (Tyr/Ser) to Arg/Glu and the results demonstrated that the optimum temperature of the mutant Y7 (S39R-T146E) increased 5°C and the half-life of inactivation (T1/2) at 60 and 65°C was 1 h and 25 min, respectively. Thus, it provides a potential xylanase that can meet the harsh conditions in the industrial applications.     

Obtaining a mutant of Bacillus amyloliquefaciens xylanase A with improved catalytic activity by directed evolution

This study aimed to obtain xylanase exhibiting improved enzyme properties to satisfy the requirements for industrial applications. The baxA gene encoding Bacillus amyloliquefaciens xylanase A was mutated by error-prone touchdown PCR. The mutant, pCbaxA50, was screened from the mutant library by using the 96-well plate high-throughput screening method. Sequence alignment revealed the identical mutation point S138T in xylanase (reBaxA50) produced by the pCbaxA50. The specific activity of the purified reBaxA50 was 9.38 U/mg, which was 3.5 times higher than that of its parent expressed in Escherichia coli BL21 (DE3), named reBaxA. The optimum temperature of reBaxA and reBaxA50 were 55 °C and 50 °C, respectively. The optimum pH of reBaxA and reBaxA50 were pH 6 and pH 5, respectively. Moreover, reBaxA50 was more stable than reBaxA under thermal and extreme pH treatment. The half-life at 60 °C and apparent melting temperature of reBaxA50 were 9.74 min and 89.15 °C, respectively. High-performance liquid chromatography showed that reBaxA50 released xylooligosaccharides from oat spelt, birchwood, and beechwood xylans, with xylotriose as the major product; beechwood xylan was also the most thoroughly hydrolyzed. This study demonstrated that the S138T mutation possibly improved the catalytic activity and thermostability of reBaxA50.