Application Of Vanadium Chloride Peroxidase Catalyzed Amino Acid Synthesis Of Nitrile Compounds

Object:(1)Through literature review and database investigation,a broad spectrum enzyme that can catalyze amino acid synthesis of nitrile compounds was discovered,and its heterologous expression in industrial microorganism Escherichia coli was realized.(2)Optimize the culture conditions of the engineered strain to improve the expression and catalytic activity of vanadium chloride peroxidase(VCPO),and explore the enzymatic properties of VCPO to find out its optimal catalytic conditions;(3)By studying the product spectrum corresponding to the action of VCPO on different substrates,the broad spectrum of the enzyme was determined,and the best substrate was identified and the product of the substrate was purified.Methods:(1)Plasmid p ET28a-VCPO was constructed using molecular biology technology,and VCPO expression strains were further screened.The activity of the enzyme was preliminatively determined according to the color change of phenol red aqueous solution caused by the crude enzyme of VCPO.(2)The culture conditions of VCPO expression bacteria were optimized by adjusting the induction temperature,induction time and concentration of the inducer.Meanwhile,the enzyme expression was detected by SDS-PAGE,and the protein concentration was detected by BCA protein concentration assay kit.The optimal reaction conditions of VCPO were determined by analyzing the concentration of sodium bromide,the time,pH and temperature of adding hydrogen peroxide.(3)The catalytic activity of VCPO on L-homoserine,L-alanine,L-tyrosine,L-isoleucine,L-tryptophan,L-glutamic acid,L-aspartic acid and L-2-aminobutyric acid was preliminatively tested.The optimal substrate of VCPO was determined by high performance liquid chromatography(HPLC),and the reaction product was isolated and purified.Results:(1)The optimal expression strain of VCPO was determined by screening.According to the results of reaction of VCPO protein solution with phenol red aqueous solution,the activity of the enzyme was preliminatively proved.(2)The maximum protein expression was 12.72 mg/mL and the maximum total enzyme activity was 0.42 U when the inducer IPTG supplemental level was 1m M,induction temperature was 20 ℃ and induction time was 20 h.The enzymatic activity analysis showed that the catalytic activity of the enzyme was the best when the concentration of sodium bromide was 10 m M,hydrogen peroxide was added 5 m M every 5 min,pH was 5.6 and 25 ℃,and the optimum catalytic conditions were determined.(3)By expanding the substrate spectrum of VCPO and detecting their products by HPLC,it was found that the conversion rate of 1.272 mg/mL VCPO to L-homoserine with substrate concentration of 50 m M was the highest 58.32 %.1.11 g/L intermediate 3-hydroxypropanitrile with purity of 68.37 % was prepared by using L-homoserine as substrate in laboratory.Conclusions: The expression vector and host bacteria of VCPO from Marine organisms were identified by screening,and the heterologous expression of VCPO in industrial microorganism Escherichia coli was successfully realized.The optimal induction conditions were obtained as follows: IPTG concentration of 1m M,induction temperature of 20 ℃,induction time of 20 h,sodium bromide concentration of 10 m M,pH=5.6,adding 5 m M hydrogen peroxide every 5 min,temperature of 25 ℃.In this reaction condition,the concentration of 3-hydroxypropionitrile was 1.11 g/L and the purity was 68.37 %.It provides reference for industrial application of amino acid catalyzed synthesis of nitrile compounds.