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[1]张亚波,刘连盟,徐荣燕,等.嗜热子囊菌光孢变种内切葡聚糖酶I基因在毕赤酵母中的表达及部分酶学性质[J].应用与环境生物学报,2009,15(03):419-422.[doi:10.3724/SP.J.1145.2009.00419]
 ZHANG Yabo,LIU Lianmeng,XU Rongyan,et al.Expression of Thermoascus aurantiacus var. levisporus Endo-β-glucanase I Gene in Pichia pastoris and Anylysis of Enzymic Properties[J].Chinese Journal of Applied & Environmental Biology,2009,15(03):419-422.[doi:10.3724/SP.J.1145.2009.00419]
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嗜热子囊菌光孢变种内切葡聚糖酶I基因在毕赤酵母中的表达及部分酶学性质()
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《应用与环境生物学报》[ISSN:1006-687X/CN:51-1482/Q]

卷:
15卷
期数:
2009年03期
页码:
419-422
栏目:
生物质能源专辑
出版日期:
2009-05-15

文章信息/Info

Title:
Expression of Thermoascus aurantiacus var. levisporus Endo-β-glucanase I Gene in Pichia pastoris and Anylysis of Enzymic Properties
作者:
张亚波刘连盟徐荣燕李多川
1山东农业大学环境生物系 泰安 271018
2中国水稻研究所 杭州 310006
Author(s):
ZHANG YaboLIU LianmengXU RongyanLI Duochuan
1Department of Environmental Biology, College of Plant Protection, Shandong Agricultural University, Tai’an 271018, Shandong, China
2China National Rice Research Institute, Hangzhou 310006, China
关键词:
纤维素酶内切葡聚糖酶嗜热子囊菌光孢变种嗜热真菌 毕赤酵母
Keywords:
cellulase endo-14-β-glucanase Thermoascus aurantiacus var. levisporus thermophilic fungus Pichia pastoris
分类号:
Q556.2 : Q949.325.06
DOI:
10.3724/SP.J.1145.2009.00419
文献标志码:
A
摘要:
摘 要 嗜热子囊菌是一种嗜热真菌,可以产生具有很高工业价值的内切葡聚糖酶. 本研究成功表达了嗜热子囊菌内切葡聚糖酶Ⅰ基因,并获得热稳定的重组内切葡聚糖酶. 提取嗜热子囊菌光孢变种(Thermoascus aurantiacus var. levisporus)总RNA,通过RT-PCR方法克隆出内切-β-葡聚糖酶eg1基因的成熟肽编码序列. 采用基因重组的方法构建该基因的巴斯德毕赤酵母Pichia pastoris分泌型表达载体pPIC9K-eg1,经线性化后采用电穿孔法将其导入毕赤酵母GS115中,大量筛选后获得高效表达内切葡聚糖酶Ⅰ的毕赤酵母工程菌株GpN24. 该菌株采用甲醇诱导120 h后,内切葡聚糖酶Ⅰ的活力可达570.7 U/mL,最适温度为55 ℃,在90 ℃的条件下保温30 min后仍具有60%的酶活力;最适pH为5.0,在pH 3.0~5.0的条件下酶活力保持稳定. 图6 表2 参16
Abstract:
Abstract The thermophilic fungus Thermoascus aurantiacus var. levisporus was studied and it was found able to produce thermostable endo-1,4-β-glucanase with high industrial value. The thermostable endo-1,4-β-glucanase was obtained through Pichia pastoris expression. The eg1 gene was isolated from T. aurantiacus var. levisporus by RT-PCR. A DNA fragment coding for eg1 without signal sequence was cloned into P. pastoris expression vector pPIC9K for extracellular expression in P. pastoris. The resulted recombinant plasmid pPIC9K-eg1 was then linearized and transformed into P. pastoris GS115 by electroporation. The eg1 gene was integrated into the genome of P. pastoris through homologous recombination and a multi-copy expression strain, named GpN24, was obtained through high throughput screening with G418. The endo-1,4-β-glucanase secreted by GpN24 reached a final yield of 570.7 U/mL after methanol induction for 120 h. The optimum pH and temperature of the recombinant enzyme activity were 5.0 and 55 ℃, respectively. The recombinant enzyme remained 90% of its original activity after 30 min at 90 ℃. Fig 6, Tab 2, Ref 16

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备注/Memo

备注/Memo:
*国家“863”计划项目(Nos. 2006AA10Z304, 2008AA05Z403)资助 Supported by the National High-Tech R & D Program of China (863 Program, Nos. 2006AA10Z304, No. 2008AA05Z403)
**通讯作者 Corresponding author (E-mail: lidc20@sdau.edu.cn)
更新日期/Last Update: 2009-07-03