Effect Of Molecular Chaperone CpkA On D-aminoacylase Activity

Molecular chaperones are a class of highly conserved proteins that have high levels of expression in cells.They play a decisive role in the folding,assembly,transportation,and degradation of proteins,and can effectively protect the protein folding structure.D-aminoacylase(D-aminoacylase)is a class of hydrolytic enzymes that can specifically hydrolyze N-acyl-D-amino acids to obtain high purity.In this study,the gene(DAA fragment)encoded by D-aminoacylase DAA(1488 bp)produced by M.natoriense TNJL143-2 and the enzyme(JM109-pUC19-DAA)expressed in E.coli JM109 were used Research object.To study the effect of molecular chaperone CpkA on D-amino acid enzyme activity.The expression identification results showed that each mutant enzyme was a soluble protein,and the activity identification results showed that:Compared with M.natoriense143-2 activity,the activity of the mutant enzyme was reduced by 0.15?0.33times;compared with JM109-pUC19-DAA activity,the activity of the V125 L mutant enzyme was increased by 1.66 times.After each mutant enzyme was co-expressed with its molecular chaperone DE3-pET21a-CpkA,compared with the activity of M.natoriense TNJL143-2,the activity of JM109-pUC19-DAA and each mutant enzyme increased by1.95-3.37 times;compared with JM109-pUC19-DAA,the activity of each mutant enzyme increased by 1.00?1.72 times.In general,after co-expression with the molecular chaperone DE3-pET21a-CpkA,the activity of each mutant enzyme increased by 3.92?20.30 times.The target gene DAA was recombined with DE3-pACYCDuet1-CpkA to obtain recombinant strain DE3-pACYCDuet1-CpkA-DAA,which made the expression product active.The results of this experimental study show that:The mutation site selected in the experiment contains amino acids that affect the amount of enzyme activity.According to the enzyme activity of each mutant.Presumably:In the D-aminoacylase derived from M.natoriense TNJL143-2,Valine(V)at position 125 replaces Leucine(L),can increase the activity of JM109-pUC19-DAA.The Arginine(R)at position 103 in its amino acid sequence replaces Alanine(A)and Lysine(K).Valine(V)at position 125 replaces Leucine(L)and Lysine(K),Alanine(A)at position 196 replaces Lysine(K),Valine(V)at position296 replaces Lysine(K)and Leucine(L),That is,all the mutant enzymes and molecular chaperones in the study can help improve their enzyme activity.The mutant enzyme expressed by JM109-pUC19-DAA is at a relatively low level for the activity of M.natoriense TNJL143-2,and the enzyme activity is greatly improved after binding to the molecular chaperone.It is speculated that the binding of molecular chaperones to DAA can help its enzyme protein form the correct high-level structure to ensure its increased activity,and also enhance the value of DAA heterologous expression activity research.