食品科学

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北京棒杆菌天冬氨酸激酶G377定点突变及酶学性质表征

朱运明,王晓飞,闵伟红*,詹冬玲,王隆洋   

  1. 小麦和玉米深加工国家工程实验室,吉林农业大学食品科学与工程学院,吉林 长春 130118
  • 出版日期:2014-05-15 发布日期:2014-05-19

Site-Directed Mutagenesis and Characterization of Aspartate Kinase G377 from Corynebacterium pekinense

ZHU Yun-ming, WANG Xiao-fei, MIN Wei-hong*, ZHAN Dong-ling, WANG Long-yang   

  1. National Engineering Laboratory on Wheat and Corn Further Processing, College of Food Science and Engineering, Jilin Agricultural University, Changchun 130118, China
  • Online:2014-05-15 Published:2014-05-19

摘要:

采用饱和定点突变和高通量筛选技术,对北京棒杆菌天冬氨酸激酶(aspartate kinase,AK)AK Gly377位点进行突变,并筛选获得高活力突变体G377F,分离纯化后对G377F AK酶学性质进行表征。结果表明:突变体AK最适pH值为9.0,较突变前野生型(wide type,WT)最适pH值为8.5有所提高;最适反应温度与WT一致,均为25 ℃;半衰期由WT的2.6 h提高到5.3 h;pH耐受性在5 h内保持其活力50%以上,与WT 3 h内酶活力保持能力相同。G377F对金属离子和有机溶剂均表现出良好的抗性。其中,Lys的抑制作用在一定程度上被解除,当Lys和Met同时存在时对AK具有明显的激活作用。动力学研究显示:G377F AK Vmax较WT提高9.3 倍;n值为1.0明显低于WT的2.7,说明AK正协同性降低,趋向于米氏酶。

关键词: 北京棒杆菌, 天冬氨酸激酶, 定点突变, 酶学性质

Abstract:

 Aspartate kinase (AK) is the first critical enzyme in the biosynthesis pathway of vital amino acids, in which
AK is strictly regulated by metabolites. In the present study, AK from Corynebacterium pekinense was mutated using sitedirected
mutagenesis combined with high-throughput screening and G377F with high activity was successfully constructed.
Then, the purified AK from G377F was characterized. The results showed that the optimum reaction pH of G377F was 9.0,
which was slightly higher than that of the wild-type strain (WT). The optimum reaction temperature of G377F was 25 ℃,
which was consistent with that of WT. The half-life period of G377F increased from 2.6 h (WT) to 5.3 h. The pH tolerance
of G377F maintained more than 50% of its original vitality in 5 h, which was identical to that of WT (3 h). In addition, AK
from G377F had a good tolerance to metal ions and organic solvents. To some extent, the inhibition by Lys could be released and
the co-presence of Lys and Met had an active effect on the G377F. Kinetic studies showed that the Vmax of AK from G377F was
10.3 times higher than that of WT. The n value was 1.0, which was significantly lower than that of WT, indicating that the positive
cooperativity of AK from G377F decreased, which showed that AK from G377F tended to be a Michaelis enzyme.

Key words: Corynebacterium pekinense E31, aspartate kinase, site-directed mutagenesis, enzymatic activity