吸水链霉菌谷氨酰胺转氨酶突变体的构建和酶学性质表征

Construction and characterization of transglutaminase mutants from Streptomyces hygroscopicus

根据茂源链霉菌谷氨酰胺转氨酶点突变的结果,用重叠延伸PCR法构建了3个吸水链霉菌谷氨酰胺转氨酶突变体Tyr81ValGly82Asn、Tyr133Phe、Tyr360Ala,在巴斯德毕赤酵母GS115中表达并对其酶学特性进行了表征。结果表明,野生型和3个突变体的最适温度为40℃,最适pH 7;除了甘油外,其他有机试剂都降低了TGase的酶活力;与野生型相比,突变体Tyr81ValGly82Asn、Tyr133Phe和Tyr360Ala的比活力分别提高了9%、85%和30%;3个突变体与野生型相比,K_(m)、K_(cat)和K_(cat)/K_(m)值有所升高,表明突变可能降低了TGase与底物的亲和力,却加速了其催化效率。上述结果为通过点突变提高吸水链霉菌谷氨酰胺转氨酶酶活力提供了试验依据和途径。

According to the results of the point mutations of Streptomyces mobaraensis,three mutants(Tyr81ValGly82Asn,Tyr133Phe and Tyr360Ala) of transglutaminase gene were constructed using overlapping extension PCR from Streptomyces hygroscopicus.The mutants were expressed in Pichia pastoris GS115 and their enzymatic properties were characterized.The results showed that the optimum temperature and pH of the wild type and three mutants were 40℃and pH7,respectively.All organic reagents except for glycerol reduced the activity of TGase.Compared with the wild-type,the specific activity of mutants Tyr81ValGly82Asn,Tyr133Phe,and Tyr360Ala increased by 9%,85%,and 30%,respectively.The K_(m),K_(cat),and K_(cat)/K_(mwas) also increased,indicating that the mutations may cause the reduction of the affinity of mutant TGase to the substrate and the increment of its catalytic efficiency.These results provide an experimental basis and approach for improving the TGase enzyme activity of S.hygroscopicus by point mutation.