米曲霉AAP新基因的异源表达及其酶学性质研究  

Heterologous expression of a novel AAP gene from Aspergillus oryzae and characterization of the enzyme

作　　者：宋婷婷 吴珍珍 宗红 陆信曜[1,2,3] 诸葛斌 SONG Tingting;WU Zhenzhen;ZONG Hong;LU Xinyao;ZHU GEBin(Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education (Jiangnan University), Wuxi 214122, China;Key Laboratory of Industrial Biotechnology, Ministry of Education (Jiangnan University), Wuxi 214122, China;Research Centre of Industrial Microbiology, School of Biotechnology, Jiangnan University, Wuxi 214122, China)

机构地区：[1]糖化学与生物技术教育部重点实验室(江南大学),江苏无锡214122 [2]工业生物技术教育部重点实验室(江南大学),江苏无锡214122 [3]江南大学生物工程学院工业微生物研究中心,江苏无锡214122

出　　处：《食品与发酵工业》2019年第5期38-44,共7页Food and Fermentation Industries

基　　金：国家科技支撑计划(2015BAD16B00;2015BAD16B 01)

摘　　要：通过生物信息学分析发现了米曲霉(Aspergillus oryzae)中属于氨肽酶M18家族的新成员天冬酰胺氨肽酶(AAP)基因,经反转录克隆获得该基因,实现了其在毕赤酵母GS115中的表达,同时研究了重组AAP的酶学性质及其在大豆蛋白水解中的应用。结果表明,重组AAP的分子质量约54.0 kDa,在较广的pH范围内有活性(pH 6.0～9.5),最适反应温度和pH分别为50℃和7.0,Zn^(2+)和Ca^(2+)对该酶起促进作用,EDTA能够显著抑制该酶的酶活。以Asp-pNA为底物时,K_m为0.42 mmol/L,V_(max)为21.57μmol/(mL·min),该酶具有水解N末端为天冬氨酸和谷氨酸的底物特异性。重组AAP可显著降低大豆多肽的苦味,提高水解度,具有在食品中的应用潜力。A novel gene AAP that encodes aspartyl aminopeptidase from the peptidase family M18 in Aspergillus oryzae was discovered by bioinformatics analysis. The gene AAP was cloned and overexpressed in Pichia pastoris GS115. Enzymatic properties of recombinant AAP and its application in the hydrolysis of soybean protein were investigated. The results showed that the recombinant AAP had a molecular weight of about 54 kDa. It was active over a broad pH range (6.0-9.5). Its optimal reaction temperature and pH were 50 ℃ and 7.0, respectively. Moreover, Zn 2+ and Ca 2+ promoted the activity of recombinant AAP, while EDTA significantly inhibited its activity. Using Asp- p NA as a substrate, the K m and V max of the enzyme were 0.42 mmol/L and 21.57 μmol/(mL·min), respectively. Recombinant AAP exhibited a substrate specificity of hydrolyzing N -terminal aspartyl and glutamyl residues. In addition, recombinant AAP significantly decreased the bitterness of soybean pepides and improved its hydrolysis degree, which indicated its potential applications in food industries.

关 键 词：米曲霉 天冬酰胺氨肽酶 酶学性质 

分 类 号：TS201.3[轻工技术与工程—食品科学]