Mutation Design Of Oral Stable Acetaldehyde Dehydrogenase 2

It is known that,in human body,ethanol is transformed into aldehyde by ethanol dehydrogenase firstly,and then into acetic acid by aldehyde dehydrogenase.Aldehyde dehydrogenases 2(ALDH2)is a promising antialcoholic protease,which can effectively hydrolyze aldehyde produced by ethanol metabolism,thus protecting human liver and other organs and reducing the harm of aldehyde to human body.However,recombinant ALDH2 prepared by Escherichia coli fermentation usually exists as an inclusion body and has no activity.Meanwhile,wild-type aldehyde dehydrogenase 2 is easily hydrolyzed by proteases in the digestive tract.In this study,in order to improve the resistance of ALDH2 on protease's digestions,the wild type ALDH2 was subjected to mutation design,namely,the cleavage sites of pepsin and trypsin on ALDH2 were transformed into other amino acids,especially the hydrophilic amino acids.Meanwhile,hydrophobic amino acids were introduced into the inside of ALDH2 so as to promote the correct folding of ALDH2 and improve the soluble expression of recombinant ALDH2.Specific studies were as follows:1.Mutation design:the mutation design of acetaldehyde dehydrogenase 2 was carried out using Rosetta software.The cleavage sites of pepsin and trypsin on ALDH2 were mutated to other amino acids,especially the hydrophilic amino acids.Meanwhile,random mutations were introduced into the binding site of substrate and coenzyme NAD~+to enhance the interactions among substrate,coenzyme NAD~+and the surrounding amino acid residues,and to improve the stability and catalytic activity of mutant acetaldehyde dehydrogenase 2.At the same time,a few mutations were introduced to correct the main chain structure of the enzyme to ensure the correct conformation of the catalytic center.Finally,according to the Rosetta's score,ten mutants were selected.Their sequences were subjected to gene synthesis,and then cloned into plasmid pET44b and transformed into E.coli BL21(DE3)strain.2.Expression of recombinant proteins:the induction conditions were optimized by screening the IPTG concentration,the induction time and the induction temperature.The results showed that the optimal IPTG concentration was 0.5 mM,the optimal induction temperature was 37?,and the expression level reached the maximum level after 5 h of induction.Wild-type ALDH2 existed as inclusion body,while part of the mutant proteins were soluble.The soluble expression levels of mutant proteins DE-852,DE-64,DE-49 and DE-1437 were higher than that of DE-143,DE-1179 and DE-1013,while the remaining mutant proteins were still expressed ALDH2 as inclusion bodies.The recombinant proteins were identified as ALDH2 by Western Blot assay.3.Purification of recombinant proteins:the recombinant proteins were purified by Ni2~+-NTA affinity chromatography,and the purification conditions were optimized by changing the concentration of imidazole in the equilibrium buffer.Studies showed that an equilibrium buffer containing 20 mM imidazole could improve the final purities of recombinant proteins.The recombinant protein concentrations were determined using ultrahigh UV visible photometer,and the protein recovery rates of the mutant strains DE-852,DE-1437,DE-64,DE-143,DE-1179,DE-49 and DE-1013were 11.9%,35.7%,36.5%,31.6%,29.4%,29.6%,32.8%and 30.2%,respectively.The protein purity of the wild type ALDH2 was 72.67%,while the purities of mutant proteins DE-852,DE-64,DE-49 and DE-1013 were all higher than that of wild type ALDH2,with purities of 94.08%,92.75%,90.17%and 87.64%,respectively.The purities of mutant proteins DE-143,DE-1179 and DE-1437 were 50.43%,59.45%and43.98%,respectively.4.Stability study:the stabilities of wild type ALDH2 and mutant proteins were studied using artificial gastric solution and artificial pancreatic solution.In 80?g/mL artificial gastric solution,the soluble expressed mutant protein was more stable than the wild-type protein.In 2?g/mL artificial pancreatic solution,the stability of the mutant proteins were improved significantly.The wild type ALDH2 was hydrolyzed by trypsin at 15 min,but the mutant proteins were stable for at least 60 min.Through the mutation deisgn,the stability of ALDH2 in digestive solutions could be significantly improved.The optimum reaction temperature and pH of the enzymes were optimized,and the results showed that the optimum pH of the mutant proteins was 8.5,and the enzyme activity reached the highest at 30?.Under optimal reaction conditions,the enzyme activity of wild type ALDH2 was 1.21 U/mL,and that of mutant proteins DE-852,DE-64,DE-143,DE-1179,DE-49,DE-1013 and DE-1437were 0.70 U/mL,0.57 U/mL,0.51 U/mL,0.37 U/mL,0.61 U/mL,0.47 U/mL and0.39 U/mL.Studies on thermal stability showed that the recombinant enzymes were stable at temperatures not higher than 40?.In this study,the amino acid sequence of the wild-type ALDH2 was changed through mutation design.The expression and purification conditions of recombinant proteins were optimized,and most of the mutant ALDH2s were expressed in soluble form and stable in pepsin and trypsin solutions.This research laid a foundation for the development of ALDH2-based protein drugs or antialcoholic products.