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食品与发酵工业  2019, Vol. 45 Issue (5): 1-7    DOI: 10.13995/j.cnki.11-1802/ts.018768
  研究报告 本期目录 | 过刊浏览 | 高级检索 |
采用饱和突变提高谷氨酰胺酶的热稳定性
陈笑1,2,李江华1,2*,刘松1,2,堵国成1,2
1(江南大学 生物工程学院,江苏 无锡,214122)
2(工业生物技术教育部重点实验室(江南大学),江苏 无锡,214122)
Improved thermal stability of glutaminase through saturated mutations
CHEN Xiao 1,2, LI Jianghua 1,2*, LIU Song 1,2, DU Guocheng 1,2
1(School of Biotechnology, Jiangnan University, Wuxi 214122, China)
2(Key Laboratory of Industrial Biotechnology, Ministry of Education(Jiangnan University), Wuxi 214122, China)
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摘要 为提高谷氨酰胺酶(glutaminase,EC 3. 5. 1. 2)的热稳定性,利用Discovery studio 2017对Bacillus subtilis谷氨酰胺酶(YbgJ)中具有不利相互作用的49个氨基酸进行虚拟突变,确定了影响酶热稳定性的关键氨基酸位点E3、E55、D213。对上述3个位点分别进行饱和突变,筛选得到55 ℃半衰期(t1/2)较野生酶分别提高58%、69%和41%的突变体E3C、E55F和D213T。构建复合突变体E3C/E55F/D213T、E3C/E55F、E3C/D213T和E55F/D213T,其t1/2分别较野生酶提高1.73、1.44、1.22和0.97倍。其中,E3C/E55F比酶活较野生酶提高23%,达到693 U/mg。作用力分析发现,突变体E3C/E55F/D213T、E3C/E55F、E3C/D213T和E55F / D213T 分别较野生酶增加30、23、11和8个氢键及减少5、4、4和3个不利相互作用。上述结果表明,酶分子内部关键氨基酸的替换对YbgJ热稳定性有较大的影响,且分子内部不利相互作用的减少和氢键的增加可能是YbgJ热稳定性提高的重要原因。
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陈笑
李江华
刘松
堵国成
关键词:  谷氨酰胺酶  热稳定性  饱和突变  不利相互作用  氢键    
Abstract: Glutaminase is an important food enzyme. In order to improve the thermal stability of glutaminase, Discovery studio 2017 was used to virtually mutate forty-nine amino acids in Bacillus subtilis glutaminase (YbgJ) that have adverse interactions. Key amino acid loci E3, E55, and D213 that affect the thermal stability of YbgJ were identified and saturated mutated. Compared with wild type enzyme, half-lives (t1/2, 55 ℃) of the mutants E3C, E55F, and D213T increased by 58%, 69%, and 41%, respectively. The t1/2 of the compounded mutants E3C/E55F/D213T, E3C/E55F, E3C/D213T, and E55F/D213 were 1.73, 1.44, 1.22, and 0.97 times higher than that of the wild type enzyme, respectively. Among them, the specific activity of E3C/E55F raised by 23%, reaching 693 U/mg. Structural analysis indicated that E3C/E55F/D213T, E3C/E55F, E3C/D213T, and E55F/D213T had thirty, twenty-three, eleven, and eight more hydrogen bonds, respectively, than that in wild type enzyme. Meanwhile, their adverse interactions decreased by 5, 4, 4, and 3, respectively. The results revealed that substituting key amino acids in the enzyme molecule had great influences on the thermal stability of YbgJ. Decreases in intramolecular adverse interactions and increases in hydrogen bonds may account for enhanced thermal stability of YbgJ.
Key words:  glutaminase    thermal stability    saturated mutation    adverse interaction    hydrogen bonds
               出版日期:  2019-03-15      发布日期:  2019-03-25      期的出版日期:  2019-03-15
基金资助: 国家自然基金面上项目(31771913);江苏省重点研发计划社会发展项目(BE2016629)
作者简介:  硕士研究生(李江华教授为通讯作者,E-mail:lijianghua@jiangnan.edu.cn)。
引用本文:    
陈笑,李江华,刘松,等. 采用饱和突变提高谷氨酰胺酶的热稳定性[J]. 食品与发酵工业, 2019, 45(5): 1-7.
CHEN Xiao,LI Jianghua,LIU Song,et al. Improved thermal stability of glutaminase through saturated mutations[J]. Food and Fermentation Industries, 2019, 45(5): 1-7.
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