米黑根毛霉甘露聚糖酶的定向进化、高效表达与应用

doi:10.3969/j.issn.1673-1689.2022.07.011

首页 > 过刊浏览>2022年第41卷第7期 >120-129. DOI:10.3969/j.issn.1673-1689.2022.07.011

米黑根毛霉甘露聚糖酶的定向进化、高效表达与应用
DOI:
                        10.3969/j.issn.1673-1689.2022.07.011
                    
作者:
                        
                        
                    
作者单位:
作者简介:
通讯作者:
中图分类号:Q814.4
基金项目:

Directed Evolution, High Level Expression and Application of Mannanase from Rhizomucor miehei

Author:

Affiliation:

Fund Project:

摘要

图/表

访问统计

参考文献

相似文献

引证文献

资源附件

文章评论

摘要:

为提高米黑根毛霉甘露聚糖酶（RmMan5A）对魔芋粉的水解效率，利用定向进化技术对该酶进行分子改造，经两轮筛选获得了一个对甘露聚糖酶催化效率显著提升的突变体（RmMan5AM2）。该突变体的最适pH为7.0，最适温度为65 ℃，较野生型甘露聚糖酶提高了10 ℃。该突变体继承了野生型甘露聚糖酶的高比酶活力，对槐豆胶的比酶活力达10 371.4 U/mg。在最适条件下，该突变体对槐豆胶、魔芋粉和瓜尔胶的催化效率分别提高了37.4%、28.9%和34.4%。进一步将该突变体在毕赤酵母中进行高效表达，经过156 h高密度发酵，发酵液胞外酶活力达176 000 U/mL，是目前产甘露聚糖酶的最高水平。利用该突变体水解魔芋粉成功制备魔芋甘露寡糖，产物主要为聚合度2~6的甘露寡糖（相对峰面积>85%），总得率为88.5%。该突变体具有优良的酶学性质，对魔芋粉的水解效率显著提升，在魔芋甘露寡糖的酶法制备中具有很大的应用潜力。

Abstract:

To improve the hydrolysis efficiency toward konjac powder, the mannanase from Rhizomucor miehei was successfully modified through directed evolution. After two rounds of screening, a mutant (RmMan5AM2) with improved catalytic efficiency was obtained. The optimal pH of the mutant was pH 7.0, and its optimal temperature was 65 ℃, which was increased by 10 ℃ compared with RmMan5A. RmMan5AM2 inherited the high specific activity of RmMan5A. Its specific activity toward locust bean gum was 10 371.4 U/mg. At the optimal condition, the catalytic efficiencies of the mutant toward locust bean gum, konjac powder, and guar gum were improved by 37.4%, 28.9% and 34.4%, respectively. RmMan5AM2 was further expressed in Pichia pastoris for high cell density fermentation. After 156 h, the expression level of the mutant was up to 176 000 U/mL, which is the highest expression level of mannanases as reported so far. The mutant was then used to hydrolyze konjac powder for manno-oligosaccharides production. The products were mainly manno-oligosaccharides with a degree of polymerization of 2~6(relative peak area>85%), and the total yield was 88.5%. RmMan5AM2 showed excellent enzymatic properties, and its hydrolysis efficiency toward konjac powder was significant improved. Thus, the mannanase has great potential in manno- oligosaccharide production.

参考文献

相似文献

引证文献

引用本文

李延啸,马俊文,闫巧娟,武建,张伟,江正强.米黑根毛霉甘露聚糖酶的定向进化、高效表达与应用[J].食品与生物技术学报,2022,41(7):120-129.

LI Yanxiao, MA Junwen, YAN Qiaojuan, WU Jian, ZHANG Wei, JIANG Zhengqiang. Directed Evolution, High Level Expression and Application of Mannanase from Rhizomucor miehei[J]. Journal of Food Science and Biotechnology,2022,41(7):120-129.

复制

文章指标

点击次数:
下载次数:
HTML阅读次数:
引用次数:

历史

收稿日期:
最后修改日期:
录用日期:
在线发布日期: 2022-08-05
出版日期: 2022-07-25

地址：江苏省无锡市蠡湖大道1800号邮政编码：214122

电话：0510-85913526 电子邮件：xbbjb@jiangnan.edu.cn

技术支持：北京勤云科技发展有限公司

引用本文

分享

文章指标

历史

微信公众号二维码

手机版网站二维码