(1.华南理工大学 食品科学与工程学院, 广东 广州 510641;2.华南理工大学 生物科学与工程学院, 广东 广州 510006)
(1.School of Food Science and Engineering, South China University of Technology, Guangzhou 510641, China;2.School of Biology and Biological Engineering, South China University of Technology, Guangzhou 510006, China)
从烟曲霉Aspergillus fumigatus GIM3.20中克隆获得了一种脂肪酶B基因(AFLB)。通过序列比对,发现AFLB与南极假丝酵母脂肪酶B(CALB)的氨基酸序列有31%的一致性,两者同属CALB家族脂肪酶；AFLB 含有与家族其他成员类似的保守五肽序列(SWSQG),预测AFLB的催化三联体分别为S233、D318和H361,其中Ser233位于保守五肽序列中；此外AFLB含有一段N-端延长序列(由第1~128位氨基酸组成),是曲霉属脂肪B的特有序列。在毕赤酵母中成功地异源表达了脂肪酶AFLB,并通过亲和层析进行了纯化。SDS电泳结果显示,该酶存在部分高度N-糖基化和部分无糖基化的两种形态,对应分子量分别为45~66kDa和42kDa。酶学性质表征结果显示,重组脂肪酶AFLB最适反应pH 值为7.0,最适反应温度为40℃,并在50℃以下具有较好稳定性,其最适底物为肉豆蔻酸对硝基苯酯。脂肪酶AFLB与CALB有高度亲缘性,具有潜在的工业应用前景。
A lipase gene (AFLB) was cloned from Aspergillus fumigatus GIM3.20. Sequence alignment analysis showed that the AFLB shared 31% amino acid sequences identities with that of Candida antarctica lipase B (CALB) , and both of them were belonged to the CALB-like family. AFLB contained a similar conserved pentapeptide (SWSQG) that the member of CALB-like family shared. Its catalytic triad was supposed to be made up by residues S233,D318, and H361, and the catalytic serine locates in the pentapeptide. What’s more, AFLB hosted a special N-terminal extended sequence, which was a feature of Aspergillus spp. lipase B. AFLB was successfully expressed in Pichia pastoris and purified by affinity chromatography. The result of SDS-PAGE showed that AFLB had partial N-glycosylation, displaying molecular weights of 45-66kDa and 42kDa. Characterization of AFLB lipase showed that the optimum reaction pH was 7.0 and the optimum reaction temperature was 40℃. The optimum substrate for AFLB was p-nitrophenol myristate. AFLB was the homologue of lipase CALB, and might have good industrial application prospect.