一株海洋来源铜绿假单胞菌Gxun-7角蛋白酶基因的克隆、表达及重组酶酶学性质

Cloning and expression of keratinase gene from a marine-derived Pseudomonas aeruginosa Gxun-7 and enzymatic properties of the recombinant enzyme

【背景】角蛋白酶是一类特异性降解角蛋白的水解酶,在动物饲料、生物肥料、医学、洗涤、制革及环境治理等方面具有重要的应用潜力。【目的】对前期从海洋环境筛选出的一株铜绿假单胞菌Gxun-7的角蛋白酶基因进行克隆、表达,并探究重组酶酶学性质,为角蛋白酶在工业生产中的应用奠定基础。【方法】以铜绿假单胞菌Gxun-7基因组推定的角蛋白酶基因为基础,设计引物克隆获得角蛋白酶基因kp2,构建重组表达质粒pET22b-kp2,并转化到E.coli RosettagamiB(DE3)中进行诱导表达,同时对重组表达菌株的表达条件进行优化。利用镍柱分离纯化重组角蛋白酶并研究其酶学性质。【结果】重组角蛋白酶的分子量约为33 kDa,最适温度和pH值分别为40℃和8.0,在温度30−60℃和pH 6.5−8.0具有较好的稳定性。金属离子Co^(2+)、Cu^(2+)和化学试剂十二烷基磺酸钠(sodium dodecyl sulfonate,SDS)、乙二胺四乙酸(ethylenediaminetetraacetic acid,EDTA)、苯甲基磺酰氟(phenylmethylsulfonyl fluoride,PMSF)对酶活力有抑制作用,而Mg^(2+)、K^(+)、巯基乙醇和二硫苏糖醇(dithiothreitol,DTT)对酶活力有促进作用。重组角蛋白酶具有良好的耐盐性,在12.5%的NaCl作用下相对酶活为87.55%。以酪蛋白为底物时,酶的K_(m)值为60.92 mg/mL、V_(max)值为9.70 U/mL。【结论】海洋来源铜绿假单胞菌Gxun-7的重组角蛋白酶具有良好的温度、碱、盐稳定性,可应用于工业生产中。

[Background]Keratinase is a kind of hydrolase that specifically degrades keratin,and has important application potential in animal feed,biological fertilizer,medicine,washing,tanning,and environmental treatment.[Objective]The keratinase gene of Pseudomonas aeruginosa Gxun-7 from marine environment was cloned and expressed,and the enzymatic properties of recombinant enzyme were investigated,which laid a foundation for the application of keratinase in industrial production.[Methods]Based on the putative keratinase gene of P.aeruginosa Gxun-7 genome,primers were designed to obtain keratinase gene kp2.The recombinant expression plasmid pET22b-kp2 was constructed,and transformed into E.coli RosettagamiB(DE3)for induced expression.Meanwhile,the expression conditions of the recombinant expression strain were optimized.The recombinant keratinase was isolated and purified by nickel column and its enzymatic properties were studied.[Results]The molecular weight of recombinant keratinase was about 33 kDa,and the optimal temperature was 40℃ at pH 8.0.The recombinant keratinase had good stability at temperature of 30−60℃and pH 6.5−8.0.Metal ions including Co^(2+)and Cu^(2+),and chemical reagents including sodium dodecyl sulfonate(SDS),ethylenediaminetetraacetic acid(EDTA),and phenylmethylsulfonyl fluoride(PMSF)inhibited the enzyme activity,whereas Mg^(2+),K^(+),mercaptoethanol,and dithiothreitol(DTT)promoted the enzyme activity.The recombinant keratinase showed good salt tolerance,and the relative enzyme activity was 87.55%under the action of 12.5%NaCl.When casein was used as substrate,the K_(m) and V_(max) of the enzyme were 60.92 mg/mL and 9.70 U/mL.[Conclusion]The recombinant keratinase from the marine-derived P.aeruginosa Gxun-7 has good stability in temperature,alkali and salt,which can be applied to industrial production in the future.