黑曲霉羧肽酶CapA的克隆表达与酶学性质解析  

Cloning,Expression and Biochemical Characterization of a Novel Carboxypeptidase CapA from Aspergillus niger

作　　者：冯玮[1] 宋鹏 FENG Wei;SONG Peng(School of Life Sciences,Liaocheng University,Liaocheng 252000,Shandong,China)

机构地区：[1]聊城大学生命科学学院,山东聊城252000

出　　处：《食品研究与开发》2021年第17期189-195,共7页Food Research and Development

摘　　要：通过对A.niger CBS 513.88基因组信息进行阅读和比对分析,发现一个新的丝氨酸羧肽酶基因CapA,并在毕赤酵母中进行克隆表达。在摇瓶发酵水平,重组菌GS115(pPIC9K-CapA)的羧肽酶酶活达到109.7 U/mL。酶学性质研究表明该酶最适温度和pH值分别为45℃和6.0;在30℃~50℃或pH 4.0~8.0孵育1 h,仍可保持80%或60%以上的活力;Mg^(2+)可以显著提高酶活,Cu^(2+)、Fe^(2+)、Co^(2+)和苯甲基磺酰氟则对酶活有明显的抑制作用。在6种被检测的底物中,CapA可以水解其中的5种,具有较广泛的底物特异性,并且偏好水解羧基端疏水性氨基酸亮氨酸和赖氨酸,苄氧羰基-苯丙氨酰-亮氨酸是其最适底物。CapA较好的耐热性和pH值稳定性以及水解底物特征表明其在食品和生物技术领域有较好的应用基础。A novel serine carboxypeptidase gene CapA was identified in Aspergillus niger CBS 513.88 by screening based on genomic information and sequence alignment.The gene was cloned and expressed in Pichia pastoris.In shake flask cultures,the enzyme activity of recombinant strain GS115(pPIC9K-CapA)reached 109.7 U/mL.The optimum temperature and pH of the enzyme was 45℃and 6.0,respectively.After incubation at 30℃to 50℃,or pH 4.0 to 8.0 for 1 h,more than 80%and 60%,respectively,of the initial enzyme activity was retained.Mg^(2+)enhanced the activity of CapA.Cu^(2+),Fe^(2+),Co^(2+),and phenylmethylsulfonyl fluoride inhibited the activity.Among the six substrates tested,five were hydrolyzed by CapA.The broad substrate specificity of CapA included a preference for c-terminal hydrophobic amino acids leucine and lysine.The preferred substrate of CapA was N-carbobenzoxy-L-phenylalanyl-L-leucine.The demonstrated thermostability,pH stability,and hydrolysis characteristics of CapA could be valuable for food and biotechnology applications.

关 键 词：丝氨酸羧肽酶 黑曲霉 分子克隆 异源表达 酶学特征 

分 类 号：Q78[生物学—分子生物学] Q936