Construction And Fermentation Optimization Of High-yielding Strains Of Acetaldehyde Dehydrogenase

Acetaldehyde is widely present in food and the environment,and numerous cellular and animal studies have shown that acetaldehyde at high doses(40-1000μmol·L^-1)is highly toxic,carcinogenic and teratogenic to cells,and it is classified as a class I carcinogen by the International Agency for Research on Cancer（IARC）.Acetaldehyde dehydrogenase is a class of oxidoreductases widely found in biological cells in nature,which can catalyze the degradation of acetaldehyde to non-toxic acetic acid,thereby reducing the toxic effects of acetaldehyde on humans.In this thesis,five aspects of research were carried out,including screening of highly active acetaldehyde dehydrogenase producing strains,purification and enzymatic properties of acetaldehyde dehydrogenase,cloning and expression of ALDH gene,single point saturation mutation to improve the catalytic activity of substrate and fermentation optimization,to promote the research and practical application of acetaldehyde dehydrogenase.The following are the main findings.（1）Screening of acetaldehyde dehydrogenase-producing strains:A strain CT-20 with an enzyme activity of 20.06 U·m L^-1 was isolated from grape samples by disulfiram resistance screening combined with enzyme activity determination.acetaldehyde dehydrogenase produced by CT-20 was detected by headspace gas chromatography（HS-GC）to degrade acetaldehyde efficiently,and the catalytic activity of acetaldehyde dehydrogenase was verified.The phylogenetic tree and conventional morphological characteristics of yeast ITS r RNA sequences determined that CT-20 was a tropical pseudofilamentous yeast,and named it Candida tropicalis LBBE-W1.（2）Isolation and purification of acetaldehyde dehydrogenase and enzymatic properties:acetaldehyde dehydrogenase was purified by ammonium sulfate graded precipitation and DEAE-sephacel anion exchange chromatography,and the specific enzyme activity was181.17 U·mg^-1.Its optimum reaction temperature and p H were 60℃and 9.0,respectively,with good stability at 30-50℃and 5.0-9.0.The metal ions Na⁺,K⁺and Ba²⁺activated the enzyme activity,while Ni²⁺,Cu²⁺and Mn²⁺inhibited the enzyme activity,and its optimum substrate was acetaldehyde.The enzyme was able to degrade about 20.81%of 100 mmol·L^-1of acetaldehyde within 2 h.（3）Cloning and expression of ALDH gene:The total RNA of strain LBBE-W1 was extracted and double-stranded c DNA was obtained by RT-PCR,and the full-length 1500 bp ALDH gene fragment was amplified using this as the template.The expression vector ALDH-P43 was successfully constructed and transferred into B.subtilis WB600 to obtain the active expression with the enzyme activity of 47.26 U·m L^-1,specific enzyme activity of 44.83U·mg^-1 and molecular weight size of about 55 k Da.its optimum reaction temperature and p H were 70℃and 9.0,respectively,with good stability in the environment of 30-50℃and 5.0-9.0.good stability.The metal ions Ni²⁺and Cu²⁺have a strong inhibitory effect on acetaldehyde dehydrogenase,and acetaldehyde is the most suitable substrate for it.（4）Enhance the substrate catalytic activity of acetaldehyde dehydrogenase:seven amino acids,including K272,L427,S273,A428,G271,G426,and Y425,were selected for saturation mutation,and the mutant S273N was screened by NADH fluorescent probe with an enzyme activity of 119.82 U·m L^-1 and a specific enzyme activity of 65.46 U·mg^-1.The optimum reaction temperature and p H of S273N were 60°C and 9.0,respectively.p H was60°C and 9.0,respectively,with good stability in the environment of 30-50°C and 5.0-9.0.The metal ions Ni²⁺,Cu²⁺,Co²⁺,Zn²⁺and Mn²⁺had strong inhibitory effects on S273N,and acetaldehyde was the most suitable substrate.S273N was able to degrade about 78.31%of100 mmol·L^-1 acetaldehyde within 2 h.（5）Fermentation optimization:The inoculum level,medium p H and maltose content were determined to play a major role in the enzyme production of S273N fermentation through single-factor experiments.According to the orthogonal experiment,3%inoculum,p H9.0 and maltose content of 30 g·L^-1 were determined as the optimal fermentation conditions,and the enzyme activity was 263.52 U·m L^-1,which was 13.13 times higher than that of natural enzyme.When whole cells were utilized to react with acetaldehyde for 15 min,30 min,1 h and 2 h,the acetaldehyde degradation rate reached 36.05%,42.54%,63.08%and 87.34%,respectively.