• 主题
高级搜索  >
历史搜索 清空历史
首页> 外文学位 >Purification, molecular cloning and expression of endoglucanase and beta-glucosidase from the edible straw mushroom, Volvariella volvacea.
【24h】

Purification, molecular cloning and expression of endoglucanase and beta-glucosidase from the edible straw mushroom, Volvariella volvacea.

机译：食用草菇沃尔沃菌丝菌的内切葡聚糖酶和β-葡萄糖苷酶的纯化，分子克隆和表达。

获取原文
获取原文并翻译 | 示例
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

Five forms of endoglucanase were isolated from culture fluid of V. volvacea grown on crystalline cellulose following ion-exchange, gel filtration, hydrophobic interaction (Phenyl-Sepharose) chromatography, and preparative polyacrylamide gel electrophoresis. All had the same molecular mass and identical N-terminal amino acid sequences suggesting that they consist of the same core protein. One of these endoglucanases, designated EG1, had a molecular mass of 42 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and an isoelectric point of 7.65.; The cDNA of the eg1 gene was cloned and shown to contain an ORF of 1167 by encoding for 389 amino acids. Alignment of the deduced amino acid sequence of EG1 with deduced amino sequences of other fungal endoglucanases showed highest overall homology with endoglucanases from Macrophomina phaseolina (53%), Aspergillus niger (52%), Emericella nidulans (52%) and Humicola insolens (51%).; Endoglucanase expression in V. volvacea is regulated at the transcriptional level by both induction and catabolite repression. Transcripts of eg1 were detected by Northern blot in total RNA from mycelium grown on cellulose but not from mycelium grown on glucose. Cellobiose also induced eg1 expression but the signal intensity was less than that obtained with cellulose. Sophorose and gentiobiose showed little induction of eg1 expression. Catabolite repression was observed 24 hours after addition of 1% (w/v) glucose, xylose, mannose, sorbose or fructose to medium containing 1% (w/v) crystalline cellulose. α-Lactose and β-lactose were both inducers and repressors of eg1 expression in V. volvacea.; A cell-associated β-glucosidase (BGL-A) was also purified from the extracts of V. volvacea using DEAE-Sepharose, gel filtration, native PAGE and Mono-P chromatography. Cellobiose and crystalline cellulose strongly induced the expression of the bgl-A gene, and induction of bgl-A expression was also with α-lactose, β-lactose, cellobiose, xylose, sorbose, genitobiose and sophorose.; EG1 was expressed functionally in the methylotrophic yeast Pichia pastoris. Secretion was obtained by fusing eg1 to the alpha-factor secretion signal sequence of Saccharomyces cerevisiae under the control of the alcohol oxidase gene (AOX1) promoter. Attempts to express bgl-A in Pichia pastoris were unsuccessful although high levels of mRNA corresponding to bgl-A were observed. (Abstract shortened by UMI.)
机译：通过离子交换，凝胶过滤，疏水相互作用（苯基-琼脂糖）色谱法和制备型聚丙烯酰胺凝胶电泳，从生长在结晶纤维素上的 V.volvacea 培养液中分离出五种形式的内切葡聚糖酶。它们都具有相同的分子量和相同的N末端氨基酸序列，表明它们由相同的核心蛋白组成。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE），这些内切葡聚糖酶之一称为EG1，分子量为42kDa，等电点为7.65。克隆了 eg1 基因的cDNA，并通过编码389个氨基酸显示其ORF为1167。 EG1的推导氨基酸序列与其他真菌内切葡聚糖酶的推导氨基酸序列比对显示与 Macrophomina phaseolina （53％），黑曲霉（italic）>（52％）的内切葡聚糖酶具有最高的整体同源性）， Emericella nidulans （52％）和 Humicola insolens （51％）。内切葡聚糖酶在中的表达。在诱导转录和分解代谢物阻抑下，菌丝体均在转录水平上受到调节。通过Northern印迹在来自纤维素生长的菌丝体的总RNA中检测到 eg1 的转录本，而不是葡萄糖生长的菌丝体。纤维二糖还诱导了 eg1 表达，但信号强度低于纤维素。槐糖和龙胆二糖几乎没有诱导 eg1 表达。向含有1％（w / v）结晶纤维素的培养基中添加1％（w / v）葡萄糖，木糖，甘露糖，山梨糖或果糖后24小时观察到分解代谢物阻抑。 α-乳糖和β-乳糖都是 eg1 在 V中表达的诱导物和抑制物。菌纲。还从<斜体> V的提取物中纯化了细胞相关的β-葡萄糖苷酶（BGL-A）。 DEAE-Sepharose，凝胶过滤，天然PAGE和Mono-P色谱法测定菌丝体。纤维二糖和结晶纤维素强烈诱导 bgl-A 基因的表达，而 bgl-A 表达的诱导也来自α-乳糖，β-乳糖，纤维二糖，木糖，山梨糖，genitobiose和槐糖。 EG1在甲基营养酵母 Pichia pastoris 中表达。在酒精氧化酶基因（AOX1）启动子的控制下，通过将 eg1 与 Saccharomyces cerevisiae 的alpha因子分泌信号序列融合来获得分泌。尽管观察到高水平的 bgl-A mRNA表达，但尝试在巴斯德毕赤酵母中表达 bgl-A 的尝试并未成功。 （摘要由UMI缩短。）

著录项

  • 作者

    Ding, Shaojun.;

  • 作者单位

    Chinese University of Hong Kong (People's Republic of China).;

  • 授予单位 Chinese University of Hong Kong (People's Republic of China).;
  • 学科 Biology Microbiology.; Biology Molecular.
  • 学位 Ph.D.
  • 年度 2002
  • 页码 200 p.
  • 总页数 200
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 微生物学;分子遗传学;
  • 关键词

相似文献

  • 外文文献
  • 中文文献
  • 专利
获取原文

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号