【作者】 周龙珍； 翁凌； 蔡秋凤； 刘光明； 曹敏杰；

【Author】 Longzhen Zhou Ling Weng Qiufeng Cai Guangming Liu Minjie Cao (Department of bio-engeneering,Jimei University,Xiamen,361021,Fujian Province,China)

【机构】 集美大学生物工程学院；

【摘要】 我国是南美白对虾的主产国。对虾加工过程中会产生大量的虾头下脚料,这些下脚料中含有各种营养成分和高活性的酶,因此,对下脚料的高效利用尤为重要。本研究从南美白对虾虾头下脚料中分离纯化了一种高活性的丝氨酸蛋白酶。纯化方法包括硫酸铵盐析、DEAE-Sepharose阴离子交换层析、Sephacryl S-200 HR凝胶过滤层析和DEAE Sepharose-Fast Flow阴离子交换层析等。SDS-PAGE结果显示该酶分子量约为28 kDa。酶的最适温度为40℃,最适pH为9.0,在45℃以下、pH6.5-11.0之间该酶较稳定。底物特异性实验结果表明,该酶对胰蛋白酶底物Boc-Phe-Ser-Arg-MCA等有较高的分解作用,对P1位为Arg-的底物的分解能力比Lys-的底物更强。丝氨酸蛋白酶抑制剂Pefabloc SC、MBTI、Benzamidine、STI等都能有效抑制该酶的活性。该酶的N末端序列为IVGGTDAKPGELPYQLSFQDI,肽指纹图谱结果显示该酶与旗鱼胰蛋白酶（gi|136429）有58个氨基酸同源性达到100%。动力学实验显示,该酶对Boc-Phe-Ser-Arg-MCA荧光底物的亲和力最强,K_m为0.96μmol/L,k_cat为261.4 S^-1,k_cat/K_m为2.73×10⁸（mol/L）^-1S^-1。本研究为高值化利用水产加工下脚料提供了理论依据。更多还原

【Abstract】 In aquatic products processing,a large number of scraps which contains a great quantity of nutrients and high activity enzymes were left.A serine proteinase was purified from the hepatopancreas of Pacific white shrimp by a series of procedures,including ammonium sulfate precipitation,DEAE-Sepharose, Sephacryl S-200 HR,and DEAE Sepharose-Fast Flow.As estimated by SDS-PAGE,the molecular masse of the proteinase was 28 kDa.The optimum pH and temperature of the proteinases were 40℃and 9.0,respectively. The enzyme was stable below 40 ?C and at the pH range from 6.5 to 11.0.Substrate specificity result suggested that this enzyme was a trypsin-like serine proteinase,and it can hydrolyze higher Arg- than Lys-at P1 site.Serine proteinase inhibitors,such as Pefabloc SC,MBTI,Benzamidine,STI,can effectively inhibit the enzyme activity.The N-terminal sequence of the enzyme was IVGGTDAKPGELPYQLSFQDI,the peptide mass ?ngerprint result showed that 58 amino acids were matched with high 100%identity with the trypsin of flags（gi|136429）.Using Boc-Phe-Ser-Arg-MCA as substrate,the K_m of the proteinase was 0.96μmol/L,the k_cat was 261.4 S^-1 and k_cat /K_m was 2.73×10⁸（mol/L）^-1S^-1.This research provide theoretical basis for recycle of aquatic scraps.更多还原