纳豆激酶(Nattokinase,NK,EC3.4.21.62)是日本传统食品纳豆在发酵过程中产生的具有很强溶栓活性的枯草杆菌蛋白酶,热稳定性较差,不利于在工艺生产中高温环节保证酶活,限制了其生产应用。在蛋白质中,脱酰胺过程将天冬酰胺和谷氨酰胺转化为带负电的天冬氨酸和谷氨酸,可能改变蛋白质的结构进而影响酶的活性、最适pH值和稳定性等。因此,模拟此过程可以高效改造目的酶。为提高纳豆激酶的酶活及稳定性,将位于纳豆激酶表面的天冬酰胺和谷氨酰胺分别突变为天冬氨酸和谷氨酸。通过筛选得到酶活提高突变体Q59E(约为野生型酶的1.54倍)以及热稳定性提高的突变体N218D。双突变体Q59E-N218D的热稳定性进一步提高,半衰期(t 1/2, 33 min)提高为野生型酶(t 1/2, 12 min)的2.75倍,并且酶活达到与原始酶相似水平。
Nattokinase (NK, EC3.4.21.62) 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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