乙醛脱氢酶高产菌株的筛选及其酶学性质  

Screening and enzymatic properties of high-producing acetaldehyde dehydrogenase strains

作　　者：汪冉 彭政 张娟[2,3] WANG Ran;PENG Zheng;ZHANG Juan(School of Biotechnology,Jiangnan University,Wuxi 214122,China;Key Laboratory of Industrial Biotechnology,Ministry of Education/Science Center for Future Foods,Jiangnan University,Wuxi 214122,China;Engineering Research Center of Ministry of Education on Food Synthetic Biotechnology/Jiangsu Province Engineering Research Center of Food Synthetic Biotechnology,Wuxi 214122,China)

机构地区：[1]江南大学生物工程学院,江苏无锡214122 [2]工业生物技术教育部重点实验室(江南大学)/未来食品科学中心,江苏无锡214122 [3]食品合成生物技术教育部工程研究中心(江南大学)/江苏省食品合成生物技术工程研究中心,江苏无锡214122

出　　处：《食品与发酵工业》2023年第3期38-45,共8页Food and Fermentation Industries

基　　金：国家重点研发计划项目(2019YFC1605800)。

摘　　要：乙醛广泛存在于食品和环境中,具有高毒性、诱变性和致癌性,对人类健康有很大的危害。乙醛脱氢酶(aldehyde dehydrogenase, ALDH)能催化多种脂肪族和芳香族的醛类转化为相应的酸,以此降低醛类物质特别是乙醛对人体的危害。以葡萄为材料,通过双硫仑抗性筛选结合酶活性测定获得1株高产ALDH的菌株,并研究了该菌株产的ALDH乙醛降解能力及酶学性质。利用形态学特征及ITS rRNA基因序列的测定与分析对其种属进行鉴定。根据NADH在340 nm的吸光值变化量计算出ALDH活性,并通过顶空气相色谱测定其乙醛降解能力。鉴定菌株为热带假丝酵母,命名为Candida tropicalis LBBE-W1。采用超声波破碎法提取菌株LBBE-W1的粗酶液,随后分离纯化得到乙醛脱氢酶(Candida tropicalis acetaldehyde dehydrogenase, CTALDH),比酶活力为181.17 U/mg。CTALDH的最适反应温度为60℃,最适反应pH为8.0~9.0,在30~60℃和pH 5.0~9.0下处理2 h显示较好的稳定性。Na+、K+、Ba2+金属离子对CTALDH的活性具有明显促进作用;Ni2+、Cu2+、Mn2+对CTALDH具有较强的抑制作用。CTALDH的底物特异性研究表明,乙醛是其最适底物。CTALDH在2 h内对乙醛的降解率达到95.12%。通过研究获得了1株C.tropicalis LBBE-W1菌株,其产生的ALDH具有较高活性、稳定性和乙醛降解能力,为食品和环境中的乙醛降解提供了可能。Acetaldehyde with high toxicity, mutagenicity and carcinogenicity exists widely in food and environment, which threatened human health. Aldehyde dehydrogenase can catalyze the conversion of various aliphatic and aromatic aldehydes into corresponding acids, thereby reducing the hazard of aldehydes, especially acetaldehyde. To obtain a high-producing acetaldehyde dehydrogenase strain from grapes, disulfiram resistance screening combined with enzyme activity assay were used, and the acetaldehyde degradation ability and enzymatic properties of acetaldehyde dehydrogenase were studied. Combining morphological features and ITS rRNA gene sequence, its species was identified. The acetaldehyde dehydrogenase activity was calculated according to the change in the absorbance of NADH at 340 nm, and its acetaldehyde degradation ability was determined by headspace-gas chromatography. The strain was identified as Candida tropicalis and named it as LBBE-W1. The crude enzyme solution of strain LBBE-W1 was extracted by ultrasonic and purified to obtain Candida tropicalis acetaldehyde dehydrogenase(CTALDH) with a specific enzyme activity of 181.17 U/mg. The optimum temperature of CTALDH was 60 ℃ and the optimum pH was 8.0-9.0. It showed good stability after being endured for 2 h at 30-60 ℃ and pH 5.0-9.0. The metal ions Na+, K+and Ba2+can obviously promote the activity of CTALDH, while Ni2+, Cu2+and Mn2+showed strong inhibitory effect on its activity. Acetaldehyde is the most suitable substrate, and the degradation rate of acetaldehyde by CTALDH reaches 95.12% within 2 h. In this study, a strain of Candida tropicalis LBBE-W1 was obtained, which produced acetaldehyde dehydrogenase with high activity, stability and acetaldehyde degradation capacity, offering the possibility of acetaldehyde degradation in food and environment.

关 键 词：乙醛脱氢酶 热带假丝酵母 筛选 蛋白纯化 

分 类 号：TQ925[轻工技术与工程—发酵工程]