摘要
为改善米曲霉(Aspergillus oryzae)11家族木聚糖酶AEx11A的催化特性,作者采用全质粒PCR方法对AEx11A基因(AEx11A)中编码Thr98、Asn100、Val124、Pro129和Ile132的密码子实施饱和突变,构建饱和突变文库。以木聚糖酶的酶活性为指标,采用DNS法从文库中筛选出酶活性提高30%以上的转化子。对其中酶活性提高最明显的两个位点Thr98和Val124实施迭代饱和突变,筛选出最优突变子AEx11AT98D-V124Q,其纯化后的比活性及催化效率分别为AEx11A的3.04和2.74倍。此外,突变酶AEx11AV124T的热稳定性较AEx11A有一定的提高,其余2个突变酶的温度特性与AEx11A差异不大。
In order to improve the catalytic properties of AEx11A,a glycoside hydrolase(GH)family 11 xylanase from Aspergillus oryzae,the Thr98,Asn100,Val124,Pro129and Ile132-encoding codons in AEx11A were selected for saturated mutagenesis by whole-plasmid PCR technique.Then,the mutagenesis libraries of AEx11A were constructed by transforming these variants into E.coli BL21(DE3),respectively.Using the enzyme activity of xylanase as criterion,transformants having an increase of more than 30%in activities were selected from the libraries by DNS method.Two mutation sites(Thr98 and Val124)with remarkably increased enzyme activities were subjected to iterative saturation mutation(ISM)and the optimal mutant,AEx11AT98D-V124Q,was selected.The specific activity and catalytic efficiency of purified AEx11AT98D-V124Qwere 3.04-and 2.74-fold those of AEx11A.An improvement in the thermostability of AEx11AV124Twas observed compared with that of AEx11A,while the temperature properties of the other two mutants almost had no changes.
作者
刘艳
张婷
阚婷婷
李剑芳
邬敏辰
LIU Yan;ZHANG Ting;KAN Tingting;LI Jianfang;WU Minchen(School of Food Science and Technology,Jiangnan University,Wuxi 214122,China;School of Pharmaceutical,Jiangnan University,Wuxi 214122,China;Wuxi Medical School,Jiangnan University,Wuxi 214122,China)
出处
《食品与生物技术学报》
CAS
CSCD
北大核心
2020年第5期31-37,共7页
Journal of Food Science and Biotechnology
基金
国家自然科学基金项目(21176117)
江苏省普通高校研究生科研创新计划项目(SJCX17_0467)
江苏省普通高校研究生科研创新计划项目(JSCX17_0503)。
关键词
饱和突变
米曲霉
木聚糖酶
催化特性
Saturated mutagenesis
Aspergillus oryzae
xylanase
catalytic properties
