Improving the activity of the subtilisin nattokinase by site-directed mutagenesis and molecular dynamics simulation.
Nattokinase (NK), a bacterial serine protease from Bacillus subtilis var. natto, is a potential cardiovascular drug exhibiting strong fibrinolytic activity. To broaden its commercial and medical applications, we constructed a single-mutant (I31L) and two double-mutants (M222A/I31L and T220S/I31L) by site-directed mutagenesis. Active enzymes were expressed in Escherichia coli with periplasmic secretion and were purified to homogeneity. The kinetic parameters of enzymes were examined by spectroscopy assay and isothermal titration calorimetry (ITC), and their fibrinolytic activities were determined by fibrin plate method. The substitution of Leu(31) for Ile(31) resulted in about 2-fold enhancement of catalytic efficiency (Kcat/KM) compared with wild-type NK. The specific activities of both double-mutants (M222A/I31L and T220S/I31L) were significantly increased when compared with the single-mutants (M222A and T220S) and the oxidative stability of M222A/I31L mutant was enhanced with respect to wild-type NK. This study demonstrates the feasibility of improving activity of NK by site-directed mutagenesis and shows successful protein engineering cases to improve the activity of NK as a potent therapeutic agent.
Jiangxi University of Traditional Chinese Medicine, Nanchang 330004, Jiangxi, PR China; National Key Laboratory of Virology, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China.
Meizhi
M
Weng
Hongdu Chinese Medical Hospital, Nanchang 330008, Jiangxi, PR China.
Xiongwei
X
Deng
National Key Laboratory of Virology, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China.
Wei
W
Bao
National Key Laboratory of Virology, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China.
Li
L
Zhu
National Key Laboratory of Virology, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China.
Jieyuan
J
Wu
National Key Laboratory of Virology, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China.
Yongjun
Y
Cai
National Key Laboratory of Virology, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China.
Yan
Y
Jia
National Key Laboratory of Virology, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China.
Zhongliang
Z
Zheng
National Key Laboratory of Virology, Department of Biochemistry and Molecular Biology, College of Life Sciences, Wuhan University, Wuhan 430072, PR China. Electronic address: zouguolin@whu.edu.cn.
Guolin
G
Zou
D004789:Enzyme Activation; D004926:Escherichia coli / Q000502:physiology; D008956:Models, Chemical; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D015202:Protein Engineering / Q000379:methods*; D013329:Structure-Activity Relationship; D013381:Subtilisins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D013381:Subtilisins; C053771:nattokinase
Enzyme kinetics; Isothermal titration calorimetry; Oxidative stability; Site-directed mutagenesis; Subtilisin NAT