黑曲霉天冬氨酰氨肽酶的分子克隆与酶学性质解析  

Molecular Cloning and Biochemical Characterization of An Aspartyl Aminopeptidase from Aspergillus niger

作　　者：乔雅丽 董自星[1,2] 宋鹏 刘晓光 路福平[1] 

机构地区：[1]天津科技大学生物工程学院,天津300457 [2]天津科技大学化工与材料学院,天津300457

出　　处：《食品研究与开发》2017年第24期181-187,共7页Food Research and Development

摘　　要：以黑曲霉CICIM F0510的互补DNA(complementary DNA,c DNA)为模板,通过PCR扩增出一个新的天冬氨酰氨肽酶基因(vacuolar aspartyl aminopeptidase,vaap),并成功将其在毕赤酵母GS115中进行表达。摇瓶水平上,重组菌GS115(p PIC9K-vaap)的氨肽酶酶活达到39.7 U/mL。该酶的最适反应温度和pH值分别为55℃和9.5;在90℃或pH 8.0~10.0孵育1 h后,该酶仍能保持20%或80%以上的酶活;Sn^(2+)和Co^(2+)对其酶活有明显促进作用,Fe^(3+)、Ca^(2+)、EDTA和SDS对其有抑制作用;该酶对L-亮氨酰对硝基苯胺(Leu-p NA)的Km和Vmax分别是12.96 mmol/L和2.14μg/(mL·min)。在所测的8种底物中,Leu-p NA是Vaap最适底物,且它对丙氨酸对硝基苯胺(Ala-p NA·HCl)和赖氨酸对硝基苯胺(Lys-p NA·2HCl)也有一定的水解能力。In the present study, using complementary DNA （cDNA） of Aspergillus niger CICIM F0510 as the template, a novel gene （vaap） encoding aspartyl aminopeptidase was amplified by PCR and then successfully expressed in Pichia pastoris GS115. At the shake-flask level, the aminopeptidase activity of recombinant strain GS115 （pPIC9K-vaap） was 39.7 U/mL. The optimum temperature and pH of recombinant enzyme Vaap were shown to be 55℃and 9.5, respectively. After incubation at 90℃or pH 8.0-10.0 for 1 h, this enzyme retained 20%or 80%of its initial activity. The activity of Vaap was significantly enhanced by Sn2＋and Co2＋, but inhibit-ed by Fe3＋, Ca2＋, EDTA and SDS. Its Km and Vmax values towards L-leucine-pnitroanilide （Leu-pNA） were de-termined to be 12.96 mmol/L and 2.14μg/（mL·min）, respectively. Among the eight substrates tested, Leu-pNA was the preferred substrate for Vaap, and it also hydrolyzed alanine-pnitroanilide （Ala-pNA·HCl） and Lysine-pnitroanilide（Lys-pNA·2HCl）.

关 键 词：天冬氨酰氨肽酶 黑曲霉 分子克隆 酶学性质 

分 类 号：Q936[生物学—微生物学]