以黑曲霉CICIM F0510的互补DNA(complementary DNA,cDNA)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(pPIC9K-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;Sn2+和Co2+对其酶活有明显促进作用,Fe3+、Ca2+、EDTA和SDS对其有抑制作用;该酶对L-亮氨酰对硝基苯胺(Leu-pNA)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-pNA是Vaap最适底物,且它对丙氨酸对硝基苯胺(Ala-pNA·HCl)和赖氨酸对硝基苯胺(Lys-pNA·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).
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