双碱性氨基酸内肽酶末端剪切修饰显著提高酶活及其高效水解大豆蛋白  

Terminal truncation improves the activity and soybean protein hydrolysis efficiency of dibasic endopeptidase

作　　者：彭爱凤 张荣珍[1] 徐岩 PENG Aifeng;ZHANG Rongzhen;XU Yan(Key Laboratory of Industrial Biotechnology of Ministry of Education,School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu,China)

机构地区：[1]江南大学生物工程学院工业生物技术教育部重点实验室,江苏无锡214122

出　　处：《微生物学报》2023年第9期3602-3615,共14页Acta Microbiologica Sinica

基　　金：国家重点研发计划(2018YFA0900302);国家自然科学基金(31970045);江苏省研究生科研与实践创新计划(1012050205205974);国家轻工技术与工程一流学科(LITE2018-12);高等学校学科创新引智计划(111-2-06)。

摘　　要：【目的】将酿酒酵母(Saccharomyces cerevisiae)和毕赤亚库德里亚夫泽维(Pichia kudriavzevii)来源的双碱性氨基酸内肽酶基因sckex2和pkkex2克隆到大肠杆菌(Escherichia coli)BL21中,实现双碱性氨基酸内肽酶的异源表达,研究重组酶的酶学性质及其与碱性蛋白酶的协同高效水解大豆蛋白释放出小分子活性肽的作用。【方法】按照大肠杆菌的密码子偏好性,对S.cerevisiae和P.kudriavzevii的kex2基因进行优化,分析KEX2蛋白的非功能区域,对其C-末端、N-末端氨基酸进行剪切修饰获得4种突变酶基因sckex2Δ3、sckex2Δ4、pkkex2Δ3和pkkex2Δ4,构建在载体pGEX-6P-1上,转入E.coli BL21感受态细胞中,经DNA测序验证,获得重组菌株E.coli BL21/pGEX-ScKEX2Δ3、E.coli BL21/pGEX-ScKEX2Δ4、E.coli BL21/pGEX-PkKEX2Δ3和E.coli BL21/pGEX-PkKEX2Δ4。利用GST亲和层析柱和PreScission蛋白酶对重组酶进行分离纯化,研究纯酶pH和温度稳定性等酶学性质。以碱性蛋白酶单独水解大豆分离蛋白为对照,重组双碱性氨基酸内肽酶与碱性蛋白酶协同水解大豆蛋白,测定水解产物中小分子活性肽组分。【结果】重组双碱性氨基酸内肽酶野生型和突变酶在E.coli BL21中可溶性表达,SDS-PAGE分析表明纯化的重组酶显示单一条带,在最适条件下,野生型酶几乎没有酶活,突变体最高比酶活达到47.32 U/g,K_(m)值为23.61μmol/L,kcat值为50.18 s^(−1),k_(cat)/K_(m)值为2125.06 L/(mmol·s)。当重组酶在pH 5.0孵育2 d后,相对酶活力保留最高为40%以上。在35°C下孵育1 h后酶活力仍能保留60%以上。重组双碱性氨基酸内肽酶与碱性蛋白酶协同水解大豆分离蛋白,水解产物中超过39%为分子量小于500 Da的活性肽,且分子量小于100 Da的活性肽的含量比碱性蛋白酶单独水解时高50%。【结论】通过末端剪切修饰,获得在大肠杆菌中可溶性表达的双碱性氨基酸内肽酶截短突变体,其重组酶催化效率高,能够高效水解�[Objective]To realize the heterologous expression of dibasic endopeptidase genes sckex2 and pkkex2 from Saccharomyces cerevisiae and Pichia kudriavzevii in Escherichia coli BL21 and then study the properties of the recombinant KEX2 enzymes and their efficiency of hydrolyzing soybean protein in collaboration with alkaline protease.[Methods]The kex2 from S.cerevisiae and P.kudriavzevii was optimized according to the codon bias of E.coli.After analysis of non-functional region,we redesigned their C-terminal and N-terminal residues by truncation.Four truncated genes,sckex2Δ3,sckex2Δ4,pkkex2Δ3,and pkkex2Δ4,were respectively cloned into the vector pGEX-6P-1,and the recombinant plasmids were transformed into E.coli BL21 competent cells.After confirmed by DNA sequencing,the four recombinant strains,E.coli BL21/pGEX-ScKEX2Δ3,E.coli BL21/pGEX-ScKEX2Δ4,E.coli BL21/pGEX-PkKEX2Δ3,and E.coli BL21/pGEX-PkKEX2Δ4,were constructed.The recombinant enzymes were purified by GST affinity chromatography and PreScission protease and then their pH stability and thermal stability were determined.With the soybean protein hydrolysis by alkaline protease alone as the control,the yield of small-molecule peptides from the hydrolysis with the recombinant KEX2 in collaboration with alkaline protease was determined.[Results]The recombinant KEX2 and the wild type showed soluble expression in E.coli BL21.SDS-PAGE showed that the purified recombinant enzymes produced single bands.Under the optimal conditions,the wild type showed no enzyme activity,while the mutants showed the maximum specific activity of 47.32 U/g,K_(m)of 23.61μmol/L,kcat of 50.18 s^(−1),and k_(cat)/K_(m)of 2125.06 L/(mmol·s).After incubation at pH 5.0 for 2 d,the maximum relative activity of the recombinant enzymes was more than 40%.After incubation at 35°C for 1 h,the relative activity was still over 60%.When the recombinant KEX2 and alkaline protease were used together to hydrolyze soybean protein,more than 39%of the hydrolysates were active peptides with molecular w

关 键 词：双碱性氨基酸内肽酶 大肠杆菌 酶学性质 大豆蛋白 活性肽 

分 类 号：Q78[生物学—分子生物学] Q93