西门塔尔牛瘤胃木聚糖酶基因的克隆及相关序列分析

目的克隆西门塔尔牛(Simmental)瘤胃木聚糖酶基因Rua11A,并进行相关序列分析。方法采用巢式PCR及单侧PCR技术从西门塔尔牛瘤胃微生物宏基因组DNA内直接克隆木聚糖酶基因Rua11A,并利用生物信息学软件对该基因序列及其编码氨基酸序列进行预测和分析。结果拼接后获得了1 278 bp的Rua11A编码区DNA序列,包括762 bp的木聚糖酶编码区、102 bp的连接序列编码区和411 bp的碳水化合物结合域(carbohydrate binding module,CBM)编码区,共编码425个氨基酸。氨基酸序列N-末端有1处富含疏水性氨基酸,不存在跨膜结构域;包含31个氨基酸的信号肽(signal peptide)及394个氨基酸的成熟肽(mature peptide),且理化性质稳定;预测的Rua11A空间结构具有11家族木聚糖酶的典型特征,属于CBM第四家族;含有11家族木聚糖酶高度保守的氨基酸片段及催化活性中心(catalytic active center)。结论成功克隆了西门塔尔牛瘤胃木聚糖酶基因Rua11A,并进行了相关序列分析,为Rua11A的异源高效表达及分子改造奠定了基础。     

糖苷水解酶第10家族真菌木聚糖酶保守区及进化关系的分析

目的对糖苷水解酶第10家族(glucoside hydrolase family 10,GHF10)真菌木聚糖酶保守区及进化关系进行分析。方法应用CLUSTALW2软件对41条GHF10真菌木聚糖酶氨基酸序列进行多序列比对分析,获得完全保守的氨基酸位点;应用Block Maker和Consensus软件对CLUSTALW2比对结果进一步分析,确定第10家族真菌木聚糖酶共有的保守区及保守氨基酸位点;应用Pymol软件对源于GenBank中登录的6种10家族木聚糖酶进行比对,并找到这41条序列中共有的β折叠和α螺旋;利用MEGA 4.0软件对CLUSTALW2比对结果构建系统进化树,并根据进化树进行分组。结果 41条序列长度虽然不等,但均包含E270和E405两个谷氨酸催化位点。通过序列比对,得到第10家族真菌木聚糖酶的5个高度保守区TPENSMK、RGHTLVWHSQLPSWV、WDVVEN、AKLYINDYYNL、TELDI以及20个完全保守的氨基酸位点,这些保守区和保守位点多数分布在木聚糖催化区域以及(β/α)8折叠桶的内壁上。根据木聚糖酶的亲缘关系,可将第10家族真菌木聚糖酶分为3个大组,其中第1和第3组的木聚糖酶分别有12和27种,且均来源于子囊菌门;第2组仅包含2种木聚糖酶序列,来源于Neocallimastigomycota和担子菌门。结论分析了GHF10真菌木聚糖酶的保守区及进化关系,为木聚糖酶的工程改造奠定了基础。     

半纤维素酶的分子生物学

对半纤维素酶，主要是木聚糖酶的分子生物学研究进展作了简要评述，包括酶的结构、酶分子的多形性、酶基因克隆与表达等。重点对各种细菌来源和真菌来源的木聚精酶的遗传学研究新进展进行了概括。     

黑曲霉木聚糖酶基因xynA的克隆和序列分析

通过PCR方法从黑曲霉（Aspergillus niger ty-1）中克隆获得木聚糖酶基因xynA。序列分析表明,克隆得到的黑曲霉木聚糖酶基因,xynA序列全长685bp,其中含有49bp的内含子,xyn A cDAN序列全长636bp,编码212个氨基酸,理论上编码22.5kDa的蛋白,含有17个氨基酸的信号肽序列,该蛋白质属于G/11族木聚糖酶。     

纤维素酶半纤维素酶的应用及分子相关性

对纤维素酶和半纤维素酶(主要是木聚糖酶)的应用、分子结构上的相关性及其分子酶工程的研究进展作了简要评述,重点对纤维素酶与木聚糖酶分子结构上的相关性进行了分析和概括。     

木聚糖酶热稳定性分子改造的研究进展

木聚糖酶广泛应用于食品加工、生物制药、酒的澄清及纸浆漂白等行业。热稳定性对于木聚糖酶在工业领域中的应用起着至关重要的作用。根据催化结构域的氨基酸组成不同,木聚糖酶主要分为F/10和G/11两大家族。本文主要就这两大家族木聚糖酶热稳定性分子改造的技术和方法作一综述,为进一步提高木聚糖酶的热稳定性提供参考。     

从明胶分子学的角度讨论明胶的功能性质

明胶不是一种化学上均一的实体,但它代表的是一类多肽,具有一定的分子大小、分子形状和电荷分布,并随其原料来源及制造方法的不同而有所不同。当它们使用不同原始材料或不同工艺时,一个明胶样品的功能性质未必可能是相同的。虽然明胶可以在用作为食品添加剂时具有许多功能,如乳化剂、稳定剂、增稠剂、打泡剂或保水剂,但最主要的作用则是作为凝冻剂。明胶独特的氨基酸组成和序列使明胶获得了高级凝冻剂的声誉,它在浓度低至1%或更低时仍能凝冻。这种凝胶是由富亚氨酸区域形成的,这一区域中的相对柔顺的多肽链在冷却时形成螺旋构象,这些螺旋体通过氢键部分地重现了胶原的三元螺旋结构而获得稳定。它们是凝胶的结合区,但不是所有的螺旋体都进入此结合区。此类区域的数量和大小影响着凝胶的性质,并与溶剂的性质和系统的受热史,以及多肽链的尺寸和氨基酸组成与序列有关。     

耐热木聚糖酶研究进展

木聚糖是自然界中含量第二丰富的多糖,是一种可再生资源,木聚糖酶能够降解木聚糖,被广泛应用于化工、食品、饲料、造纸、废物处理和制药等工业领域。大多数天然木聚糖酶热稳定性较差,利用基因工程和蛋白质工程手段对木聚糖酶进行改造,使木聚糖酶热稳定性提高,对工业生产有着重要的现实意义。在分析耐热木聚糖酶结构特点的基础上,对提高木聚糖酶热稳定性的技术手段的研究进展进行了综述,包括固定化、定向进化、半理性设计、理性设计、构建嵌合体,为拓宽耐热木聚糖酶的应用范围提供了帮助。     

木聚糖酶在制备低聚木糖中的应用进展

低聚木糖(Xylo-oligosaccharide,XOS)是一类由木糖分子连接而成的益生元化合物,具有促进双歧杆菌的生长、改善肠道菌群的组成结构、提高人体免疫力以及预防疾病等功能特性.酶法是较适合的XOS制备方法.简要阐述了木聚糖和XOS的结构,并对XOS降解酶系中木聚糖酶的结构、作用位点和酶系分类以及在制备低聚木糖...     

木聚糖酶原位协同水解预处理大米草的研究

用氨水与双氧水预处理大米草,与原料大米草一起进行球磨,用于木聚糖酶的协同酶解。结果表明,氨水结合双氧水预处理能有效脱除大米草中的木质素(63.81%),球磨处理能对纤维素结晶结构有所破坏,两者均可有效提高木聚糖酶的水解率。酶水解物主要成分为木糖(12.54%)、木二糖(40.38%)及少量阿拉伯糖(5.50%)。碱预处理结合球磨预处理,对木聚糖酶协同水解大米草产木寡糖具有明显的促进作用。     

Molecular dynamics studies on the thermostability of family 11 xylanases

Twelve members of the family 11 xylanases, including both mesophilic and thermophilic proteins, were studied using molecular dynamics (MD). Simulations of xylanases were carried out in an explicit water environment at four different temperatures, 300, 400, 500 and 600 K. A difference in thermotolerance between mesophilic and thermophilic xylanases became clear: thermophilic xylanases endured heat in higher simulation temperatures better than mesophilic ones. The unfolding pathways seemed to be similar for all simulations regardless of the protein. The unfolding initiates at the N-terminal region or alternatively from the alpha-helix region and proceeds to the 'finger region'. Unfolding of these regions led to denaturated structures within the 4.5 ns simulation at 600 K. The results are in agreement with experimental mutant studies. The results show clearly that the stability of the protein is not evenly distributed over the whole structure. The MD analysis suggests regions in the protein structure which are more unstable and thus potential targets for mutation experiments to improve thermostability.     

Crystallographic and mutational analyses of an extremely acidophilic and acid-stable xylanase: biased distribution of acidic residues and importance of Asp37 for catalysis at low pH

Xylanase C from Aspergillus kawachii has an optimum pH of 2.0 and is stable at pH 1.0. The crystal structure of xylanase C was determined at 2.0 A resolution (R-factor = 19.4%). The overall structure was similar to those of other family 11 xylanases. Asp37 and an acid-base catalyst, Glu170, are located at a hydrogen-bonding distance (2.8 A), as in other xylanases with low pH optima. Asp37 of xylanase C was replaced with asparagine and other residues by site-directed mutagenesis. Analyses of the wild-type and mutant enzymes showed that Asp37 is important for high enzyme activity at low pH. In the case of the asparagine mutant, the optimum pH shifted to 5.0 and the maximum specific activity decreased to about 15% of that of the wild-type enzyme. On structural comparison with xylanases with higher pH optima, another striking feature of the xylanase C structure was found; the enzyme has numerous acidic residues concentrated on the surface (so-called 'Ser/Thr surface' in most family 11 xylanases). The relationship of the stability against extreme pH conditions and high salt concentrations with the spacially biased distribution of charged residues on the proteins is discussed.      

低(无)纤维素酶活的木聚糖酶制备途径与潜在应用

低 (无 )纤维素酶活的木聚糖酶在制浆造纸工业中有着巨大的应用潜力 ,特别是在纸浆漂白中的应用。但大多数微生物在自然条件下会同时产生木聚糖酶和纤维素酶。文章对低 (无 )纤维素酶活的木聚糖酶在制浆造纸工业中的应用潜力及其制备途径进行了综述     

绿色木霉木聚糖酶在燕麦木聚糖上的亲和吸附和解析

研究了液相环境对绿色木霉木聚糖酶在水不溶性燕麦木聚糖上的亲和吸附及木聚糖酶―木聚糖复合体的解吸的影响。结果表明,燕麦木聚糖对木聚糖酶的亲和吸附作用主要依赖于它们之间的静电作用和氢键作用,疏水作用对吸附没有贡献。以pH3.0的柠檬酸―柠檬酸钠缓冲溶液为吸附体系的液相时,每克燕麦木聚糖的最大吸附容量为77IU的木聚糖酶。用含0.025mol/LNaCl、pH为7.0的柠檬酸―磷酸氢二钠缓冲溶液作为洗脱液,能将96%的被吸附的木聚糖酶洗脱下来,此时木聚糖酶活回收率为74.3%。     

低聚木糖生产用里氏木霉木聚糖酶选择性合成的研究现状

对低聚木糖生产用里氏木霉木聚糖酶选择性合成的研究现状进行了较全面的总结和评述。系统地介绍了里氏木霉木聚糖酶的的多样性以及碳源、pH和碳氮比等培养条件对合成内切木聚糖酶和木糖苷酶的影响,提出了调控这些培养条件选择性合成低木糖苷酶活的木聚糖酶的方法。     

New insights into the role of the thumb-like loop in GH-11 xylanases

GH-11 xylanases are highly specific and possess a thumb-shaped loop, a unique structure among enzymes with a jelly-roll scaffold. To investigate this structure, in vitro mutagenesis was performed on a GH-11 xylanase (Tx-Xyl) from Thermobacillus xylanilyticus. Targets were the conserved amino acids Pro(114)-Ser(115)-Ile(116) that are located at the thumb's tip and Thr(121) and Tyr(111), linker residues that connect the thumb to the main enzyme scaffold. Site-saturation mutagenesis provided an active variant that possesses a new triplet (Pro(114)-Gly(115)-Cys(116)), not found in naturally occurring GH-11 xylanases. The k(cat) value for xylan hydrolysis catalysed by this mutant was increased by 20%. Re-positioning of the thumb through the deletion of the linker residues produced different effects. As predicted by in silico analyses, deletion of Thr(121) had drastic consequences on activity, whereas deletion of Tyr(111) only affected (4-fold decrease) k(cat). Finally, deletion mutagenesis was used to create a thumbless variant that was almost catalytically inactive. Fluorescence titration with xylotetraose and xylopentaose revealed that this thumb-deleted xylanase retained the ability to bind substrates. This binding was comparable to that of the wild-type enzyme. Additionally, unlike wild-type Tx-Xyl, the thumb-deleted xylanase efficiently bound cellotetraose, although no cellulose hydrolysing activity was detected. Overall, these data show that the thumb is a key determinant for substrate selection and support previous data that suggest that it plays a role in the catalytic process.     

Enzymatic saccharification of canola meal

For the enzymatic saccharification of canola meal by enzyme preparations from Trichoderma reesei as well as by commercially available hemicellulase and multienzyme preparations, a pretreatment consisting of autoclaving is necessary. These enzyme preparations hydrolysed over 20% (w/w) of pretreated canola meal, which constitutes over 70% saccharification of the total polysaccharides present in canola meal. The results show that saccharification of canola meal is mainly brought about by hemicellulases capable of degrading arabinogalactan, arabinoglucan, galactan and galactomannan, while cellulases and xylanases play a minor role. These hemicellulases were found to be more stable at 50°C than cellulases or xylanases. This pretreatment also released water-soluble polysaccharides consisting mainly of arabinose and glucose. Trichoderma reesei was unable to produce enzymes capable of hydrolysing this polysaccharide when cultivated on canola meal as substrate.     

Module shuffling of a family F/10 xylanase: replacement of modules M4 and M5 of the FXYN of Streptomyces olivaceoviridis E-86 with those of the Cex of Cellulomonas fimi

To facilitate an understanding of structure–function relationships,chimeric xylanases were constructed by module shuffling betweenthe catalytic domains of the FXYN from Streptomyces olivaceoviridisE-86 and the Cex from Cellulomonas fimi. In the family F/10xylanases, the modules M4 and M5 relate to substrate bindingso that modules M4 and M5 of the FXYN were replaced with thoseof the Cex and the chimeric enzymes denoted FCF-C4, FCF-C5 andFCF-C4,5 were constructed. The k_cat value of FCF-C5 for p-nitrophenyl-ß-D-cellobiosidewas similar to that of the FXYN (2.2 s^–1); however, thek_cat value of FCF-C4 for p-nitrophenyl-ß-D-cellobiosidewas significantly higher (7.0 s^–1). The loss of the hydrogenbond between E46 and S22 or the presence of the I49W mutationwould be expected to change the position of Q88, which playsa pivotal role in discriminating between glucose and xylose,resulting in the increased k_cat value observed for FCF-C4 actingon p-nitrophenyl-ß-D-cellobioside since module M4directly interacts with Q88. To investigate the synergisticeffects of the different modules, module M10 of the FCF-C4 chimerawas replaced with that of the Cex. The effects of replacementof module M4 and M10 were almost additive with regard to theK_m and k_cat values.