Tyr78-loop对鱼腥藻来源苯丙氨酸脱氨酶活性的影响  

Influence of Tyr78-loop on phenylalanine ammonia-lyase from Anabaena variabilis

作　　者：郭军玲[1,2] 张帆[1,2] 黄楠 周丽[1,2] 周哲敏[1,2] GUO Jun-Ling;ZHANG Fan;HUANG Nan;ZHOU Li;ZHOU Zhe-Min(School of Biotechnology, Jiangnan University, Wuxi, Jiangsu 214122, China;Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, Jiangsu 214122, China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122 [2]江南大学工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《微生物学通报》2018年第6期1171-1179,共9页Microbiology China

基　　金：国家重点研发计划项目(2016YFE0127400);中央高校基本科研业务费专项资金(JUSRP51713B)~~

摘　　要：【背景】MIO(Methylidene-imidazol-5-one)依赖型酶中,催化因子Tyr所在Loop(Tyr78-loop)的灵活性显著影响酶学性质。【目的】探讨Tyr78-loop对鱼腥藻来源苯丙氨酸脱氨酶酶活的影响,以提高其反应活性。【方法】将该酶的Tyr78-loop进行分子改造,筛选出酶活提高的突变体,并对突变体的酶学性质进行研究。【结果】突变体S73N、E95V、E95K和S73N/E95K在37°C、pH 8.5下比活分别比原酶提高了34%、30%、18%和35%。蛋白三维结构模拟推测在突变体S73N、E95V和E95K中,位于α螺旋与Tyr78-loop交界处的Asn73、Val95和Lys95与附近氨基酸的氢键作用力数目减少,一定程度上增加了Tyr78-loop的柔性。【结论】Ser73位和Glu95位氨基酸的突变增加了Tyr78-loop的灵活性,提高了苯丙氨酸脱氨酶的酶活。[Background] Enzymatic property of the MIO-dependent（methylidene-imidazol-5-one） enzyme is significantly affected by the flexibility of the special loop that includes the catalysis Tyr（Tyr78-loop）. [Objective] The effect of Tyr78-loop on activity of phenylalanine ammonia-lyase（PAL） from Anabaena variabilis was investigated to improve PAL catalytic activity. [Methods] Tyr78-loop was genetically modified. Positive mutants with enhanced activity were selected, followed by characterization. [Results] The specific activities of S73 N, E95 V, E95 K and S73 N/E95 K mutants were improved by 34%, 30%, 18% and 35%, respectively, compared with that of the wild type at 37 °C and p H 8.5. According to protein structure simulation, the amino acid sites Asn73, Val95 and Lys95 in the mutants S73 N, E95 V and E95 K, which located at t he junction of α-helix and Tyr78-loop, had fewer hydrogen bonds with the nearby amino acids. This would increase the flexibility of Tyr78-loop and result in enhancement of the enzyme activity. [Conclusion] Catalytic activity of AvPAL could be improved by increasing the flexibility of Tyr78-loop through mutation on the Ser73 and Glu95 sites.

关 键 词：苯丙氨酸脱氨酶 酶活 定点突变 稳定性 

分 类 号：Q55[生物学—生物化学]