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文章摘要
定点突变提高食品新型L-天冬酰胺酶的活力及热稳定性
Improving the Activity and Thermal Stability of Pyrococcus yayanosii CH1L-asparaginase by Ste-directed mutagenesis
投稿时间:2018-09-02  修订日期:2018-10-04
DOI:
中文关键词: L-天冬酰胺酶  定点突变  比酶活  热稳定性
英文关键词: L-asparaginase  site-directed mutagenesis  specific activity  thermal stability
基金项目:中国博士后科学基金资助项目(2015M570407,2016T90421);国家自然科学基金(31500065);江苏省青年自然科学基金(BK20150142);国家轻工技术与工程一流学科自主课题资助(LITE2018-06);宁夏回族自治区科技创新团队项目(KJT2017001);江苏高校品牌专业建设工程资助项目;江苏高校优势学科建设工程资助项目
作者单位E-mail
徐书琴 江南大学 18861830310@163.com 
胡静怡 江南大学  
张恒维 江南大学  
赵浩东 江南大学  
刘鹏 江南大学  
席毓淋 江南大学  
李谞 江南大学  
张显 江南大学 zx@shengwu@126.com 
饶志明 江南大学  
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中文摘要:
      摘要:L-天冬酰胺酶(L-Asparaginase II ,EC 3.5.1.1)可将L-天冬酰胺转化为L-天冬氨酸,减少高温加工食品中丙烯酰胺的形成,因而受到人们的广泛关注。该酶在食品加工及预处理阶段的使用已受到人们广泛的关注,但是由于食品加工及预处理过程中环境的复杂性,对L-天冬酰胺酶的性质、热稳定性和酶活等方面有较高的要求。在本实验的研究中,我们通过序列对比和同源模拟对嗜热菌Pyrococcus yayanosii CH1来源的编码L-天冬酰胺酶的基因PyAsnase进行了3个位点的突变,并在B. subtilis 168 中进行表达,提高了该酶的热稳定性及比酶活。其中突变株E22K较原始菌株相比所得突变体比酶活提高了约37.3%,突变株R111L较原始菌株相比所得突变体的比酶活提高了约31.1%,突变株M92A较原始突变菌株相比所得突变体在85 ℃时的半衰期延长了约35 min。本研究为探索L-天冬酰胺酶结构和功能的相互关系提供了借鉴,提高了其在食品工业中的应用前景。
英文摘要:
      Abstract: L-asparaginase II (EC 3.5.1.1) which can convert L-asparagine, the precursor of carcinogenic acrylamide, to L-aspartic acid, has now attracting more attentions in food industry. The use of L-asparaginase II can reduce the formation of acrylamide during the pretreatment process of foods. However, the application of this enzyme is limited due to variant requirements of commercial use, especially for its activity and thermostability. In this study, by sequence aligning and homology modeling, three mutants of PyAsnase gene from the thermophilus Pyrococcus yayanosii CH1was constructed and over-expressed in Bacillus subtilis. According to the results, the specific activities of the mutant E22K and R111L were increased by 37.3% and 31.1%, respectively, and the half-life of the mutant obtained by the mutant M92A was extended by 35 min at 85℃. This work provides references for studying the interrelationship between function and structure of L-asparaginase, and also develops the application of L-asparaginase in food industry.
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