Expression Of Human ALDH2 Gene In Saccharomyces Cerevisiae And Study On Its Enzymatic Properties

Researches have fully demonstrated that acetaldehyde is highly toxic,carcinogenic,and mutagenic.Therefore,the International Agency for Research on Cancer?IARC?has classified acetaldehyde as a class I carcinogen.In human body,acetaldehyde dehydrogenase?ALDH,EC 1.2.1.10?exists as a tetramer,and can oxidize the harmful exogenous or endogenous acetaldyde to non toxious acetic acid,using nicotinamide adenine dinucleotide as a coenzyme factor.Almost 60%of the acetaldehyde in human body is metabolized by the aldehyde deoxygenase 2?ALDH2?in hepatocyte mitochondria.The For ALDH2 production,Escherichia coli and Pichia pastoris are the most commonly used expression systems.However,E.coli is a kind of pathogenic bacteria,and P.pastoris needs methanol for exogenous gene induction,leading certain safety problems.In this study,the food safe strain Saccharomyces cerevisiae W303-1A was used for integrated expression of ALDH2,and properties of the recombinant enzyme were characterized.The human ALDH2 gene was codon-optimized according to the codon bias of S.cerevisiae and synthesized.The recombinant plasmid pYX212-ALDH2 containing the target ALDH2 gene and the expression module TRP1L-URA3-TPIp-ALDH2-TPIt-TRP1R were constructed and transformed into the host S.cerevisiae W303-1A for free and integrated expression,respectively.The engineered strains were named E-ALDH2 and F-ALDH2.For free expressed ALDH2 in E-ALDH2,the crude enzyme liquid enzyme activity of the engineered strain was 82.41 U·mg^-1,61.07 U·mg^-11 higher than that in the parental strain.However,the free plasmid pYX212-ALDH2 is not stable in yeast cells and lost finally.For integrated expressed ALDH2 in F-ALDH2,the specific enzyme activity was 25.89 U·mg^-1,which was 4.55 U·mg^-1higher than the original strain.The fermentation conditions for maximum enzyme production were optimized using orthogonal experiments:pH 6.5,temperature 30?,and inoculation amount 6%.Under the optimum fermentation condition,the enzyme activity increased by 19.2%.The recombinant ALDH2 was then purified with a 1 mL HisTrap^TMM excel affinity column using AKTA avant 25 protein purification system for property characterization.The enzyme showed optimum reaction pH and temperature of 5.0,and 35?respectively.The enzyme will retain 20%of its activity after incubation under acidic conditions for 2 h.In addition,the enzyme exhibited good thermal stability below 40?,and the enzyme activity decreases rapidly above 45?.Furthermore,the activity of ALDH2 was slightly inhibited by Na⁺but activated by K⁺,Ca²⁺,Mg²⁺,and Mn²⁺etc,among which K⁺is the best promoter.The michaelis constant K_m of the coenzyme NAD⁺is 0.282 mmol·L^-1,and the maximum reaction rate V_maxax is 58.82 U·mg^-1.The Michaelis constant K_m of ALDH2 substrate acetaldehyde was0.908 mmol·L^-1,and the maximum reaction rate V_maxax was 114.94 U·mg^-1.