Abstract: Nattokinase (NK, EC220.127.116.11) is a bacterial serine protease derived from the traditional Japanese food natto with strong fibinolytic activity. However, the thermal stability of NK is too low to ensure high enzyme activity in the production process, and thus limits its production and application. Proteins deamidation process converts asparagine (Asn) and glutamine (Gln) residues into negatively charged aspartate (Asp) and glutamic acid (Glu),which may change the local structure of protein and affect the enzyme activity, pH optimum, and stability. Therefore, simulating this process can efficiently modify the target enzyme. In order to improve the activity and stability of NK, Asn and Gln located on the surface were mutated to Asp and Glu, respectively. The mutant Q59E with increased activity (about 1.54 times of the wild type) and mutant N218D with increased thermal stability were obtained. The thermal stability of the double mutant Q59E-N218D was further improved, and its half-life (t1/2, 33 min) was 2.75 times of that of the wild type NK (t1/2, 12 min). It provides a method for enzyme engineering and a new enzyme material for the industrial application of NK.
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