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.
[1] LACHENMEIER D W, KANTERES F, REHM J.Carcinogenicity of acetaldehyde in alcoholic beverages:Risk assessment outside ethanol metabolism[J].Addiction (Abingdon, England), 2009, 104(4):533-550.
[2] 曹婳. 改造益生大肠杆菌降解乙醇和乙醛以减轻过度饮酒小鼠机体损伤[D].兰州:兰州大学, 2021.
CAO H.Genetically encoded probiotic Nissle 1917 to alleviate alcohol-induced acute mice body injury[D].Lanzhou:Lanzhou University, 2021.
[3] MIZUMOTO A, OHASHI S, HIROHASHI K, et al.Molecular mechanisms of acetaldehyde-mediated carcinogenesis in squamous epithelium[J].International Journal of Molecular Sciences, 2017, 18(9):1943.
[4] LYU Y B, ZHONG L, LIU Y N, et al.Protective effects of Lactococcus lactis expressing alcohol dehydrogenase and acetaldehyde dehydrogenase on acute alcoholic liver injury in mice[J].Journal of Chemical Technology & Biotechnology, 2018, 93(5):1 502-1 510.
[5] 张娟, 陈坚, 汪冉, 等.一株乙醛脱氢酶高产菌株的分离与鉴定:CN113046252B[P].2022-07-05.
ZHANG J, CHEN J, WANG R, et al.Isolation and characterization of a high-producing strain of acetaldehyde dehydrogenase:CN113046252A[P].2022-07-05.
[6] MANOW R, WANG C, GARZA E, et al.Expression of acetaldehyde dehydrogenase (aldB) improved ethanol production from xylose by the ethanologenic Escherichia coli RM10[J].World Journal of Microbiology & Biotechnology, 2020, 36(4):59.
[7] O’GRADY I, ANDERSON A, O’SULLIVAN J.The interplay of the oral microbiome and alcohol consumption in oral squamous cell carcinomas[J].Oral Oncology, 2020, 110:105011.
[8] WANG W J, WANG C G, XU H X, et al.Aldehyde dehydrogenase, liver disease and cancer[J].International Journal of Biological Sciences, 2020, 16(6):921-934.
[9] SEITZ H K, STICKEL F.Acetaldehyde as an underestimated risk factor for cancer development:Role of genetics in ethanol metabolism[J].Genes & Nutrition, 2010, 5(2):121-128.
[10] 吴桂英. 酿酒酵母产乙醇脱氢酶和乙醛脱氢酶的研究[D].武汉:武汉工程大学, 2007.
WU G Y.Study on alcohol dehydrogenase and aldehydedehydrogenase from Saccharomyces cerevisiae[D].Wuhan:Wuhan Institute of Technology, 2007.
[11] LU J, LYU Y B, LI M T, et al.Alleviating acute alcoholic liver injury in mice with Bacillus subtilis co-expressing alcohol dehydrogenase and acetaldehyde dehydrogenase[J].Journal of Functional Foods, 2018, 49:342-350.
[12] 杨伟伟, 高秀峰, 包海生, 等.法尔凯干酵母乙醛脱氢酶的提取和纯化研究[J].酿酒科技, 2010(6):32-34.
YANG W W, GAO X F, BAO H S, et al.Extraction and purification of aldehyde dehydrogenase from faerkai dry active yeast[J].Liquor-Making Science ＆ Technology, 2010(6):32-34.
[13] 陈贵佺. 雪峰干酵母中乙醛脱氢酶提取及其酶学性质研究[D].广州:华南理工大学, 2012.
CHEN G Q.Studies on the extraction of ALDH from Xuefeng dry yeast and enzymatic properties of the ALDH[D].Guangzhou:South China University of Technology, 2012.
[14] 周百灵, 高秀峰, 吴晓婷, 等.酿酒酵母胞浆中乙醛脱氢酶的提取和研究[J].酿酒科技, 2009(6):28-30.
ZHOU B L, GAO X F, WU X T, et al.Extraction of and research on cytosolic aldehyde dehydrogenase from Saccharomyces cerevisiae[J].Liquor-Making Science ＆ Technology, 2009(6):28-30.
[15] YAO Z Y, ZHANG C, LU F X, et al.Gene cloning, expression, and characterization of a novel acetaldehyde dehydrogenase from Issatchenkia terricola strain XJ-2[J].Applied Microbiology and Biotechnology, 2012, 93(5):1 999-2 009.
[16] XU X, NIU C T, LIU C F, et al.Unraveling the mechanisms for low-level acetaldehyde production during alcoholic fermentation in Saccharomyces pastorianus lager yeast[J].Journal of Agricultural and Food Chemistry, 2019, 67(7):2 020-2 027.
[17] 许鑫. 基于增变因子选育低乙醛啤酒酵母及机制解析[D].无锡:江南大学, 2020.
XU X.Application of perturbation factor for breeding lager yeast with lower acetaldehyde production and related mechanisms analysis[D].Wuxi:Jiangnan University, 2020.
[18] 刘春凤. 啤酒酿造酵母M14低产乙醛的研究[D].无锡:江南大学, 2019.
LIU C F.Research of brewing yeast M14 with low acetaldehyde production[D].Wuxi:Jiangnan University, 2019.
[19] 沈楠. 低产乙醛啤酒酵母的选育[D].无锡:江南大学, 2013.
SHEN N.Screening of brewer’s yeast of low acetaldehyde production[D].Wuxi:Jiangnan University, 2013.
[20] 豆欣喜, 张明, 林材, 等.乙醛脱氢酶的克隆表达及其酶学性质[J].食品与发酵工业, 2018, 44(4):22-28.
DOU X X, ZHANG M, LIN C, et al.Expression and enzymatic properties of acetaldehyde dehydrogenase[J].Food and Fermentation Industries, 2018, 44(4):22-28.
[21] 豆欣喜. 人源ALDH2基因在酿酒酵母中的表达及其酶学性质研究[D].无锡:江南大学, 2018.
DOU X X.Expression of human ALDH2 gene in Saccharomyces cerevisiae and study on its enzymatic properties[D].Wuxi:Jiangnan University, 2018.
[22] GUZ S.The yeasts:A Taxonomic Study, 5th edition [J].Library Journal, 2011, 136(18):108-108.
[23] 周冠宇, 李江华, 彭政, 等.定点突变提高枯草芽孢杆菌角蛋白酶的低温催化活性[J].微生物学通报, 2022, 49(1):1-13.
ZHOU G Y, LI J H, PENG Z, et al.Improving the low-temperature activity of Bacillus subtilis keratinase by site-directed mutagenesis[J].Microbiology China, 2022, 49(1):1-13.
[24] 姚正颖. Issatchenkia terricola XJ-2乙醛脱氢酶的纯化和性质及其基因的克隆与表达[D].南京:南京农业大学, 2011.
YAO Z Y.Purification, characterization, gene cloning and expression of acetaldehyde dehydrogenase from Issatchenkia terricola strain XJ-2[D].Nanjing:Nanjing Agricultural University, 2011.
[25] LIU Y C, TANG Y Q, KENJI K.Application of OMICS technology in construction of Saccharomyces cerevisiae strains for ethanol production[J].China Biotechnology, 2014, 34:118-128.
