Structure-Based Engineering of Methionine Residues in the Catalytic Cores of Alkaline Amylase from Alkalimonas amylolytica for Improved Oxidative Stability

被引:34
作者
Yang, Haiquan [1 ,2 ]
Liu, Long [1 ,2 ]
Wang, Mingxing [1 ,2 ]
Li, Jianghua [1 ,2 ]
Wang, Nam Sun [3 ]
Du, Guocheng [1 ,2 ]
Chen, Jian [4 ]
机构
[1] Jiangnan Univ, Minist Educ, Key Lab Ind Biotechnol, Wuxi, Peoples R China
[2] Jiangnan Univ, Sch Biotechnol, Wuxi, Peoples R China
[3] Univ Maryland, Dept Chem & Biomol Engn, College Pk, MD 20742 USA
[4] Jiangnan Univ, Natl Engn Lab Cereal Fermentat Technol, Wuxi, Peoples R China
关键词
ALKALIPHILIC BACILLUS ISOLATE; ALPHA-AMYLASE; ENZYMATIC-PROPERTIES; PURIFICATION; RESISTANT; THERMOTOLERANT; ENZYMES; GENE;
D O I
10.1128/AEM.01307-12
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
This work aims to improve the oxidative stability of alkaline amylase from Alkalimonas amylolytica through structure-based site-directed mutagenesis. Based on an analysis of the tertiary structure, five methionines (Met 145, Met 214, Met 229, Met 247, and Met 317) were selected as the mutation sites and individually replaced with leucine. In the presence of 500 mM H2O2 at 35 degrees C for 5 h, the wild-type enzyme and the M145L, M214L, M229L, M247L, and M317L mutants retained 10%, 28%, 46%, 28%, 72%, and 43% of the original activity, respectively. Concomitantly, the alkaline stability, thermal stability, and catalytic efficiency of the M247L mutant were also improved. The pH stability of the mutants (M145L, M214L, M229L, and M317L) remained unchanged compared to that of the wild-type enzyme, while the stable pH range of the M247L mutant was extended from pH 7.0 to 11.0 for the wild type to pH 6.0 to 12.0 for the mutant. The wild-type enzyme lost its activity after incubation at 50 C for 2 h, and the M145L, M214L, M229L, and M317L mutants retained less than 14% of the activity, whereas the M247L mutant retained 34% of the activity under the same conditions. Compared to the wild-type enzyme, the k(cat) values of the M145L, M214L, M229L, and M317L mutants decreased, while that of the M247L mutant increased slightly from 5.0 x 10(4) to 5.6 x 10(4) min(-1). The mechanism responsible for the increased oxidative stability, alkaline stability, thermal stability, and catalytic efficiency of the M247L mutant was further analyzed with a structure model. The combinational mutants were also constructed, and their biochemical properties were characterized. The resistance of the wild-type enzyme and the mutants to surfactants and detergents was also investigated. Our results indicate that the M247L mutant has great potential in the detergent and textile industries.
引用
收藏
页码:7519 / 7526
页数:8
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