Molecular Modification And Its Muttions Functionnal Characterizatjion Of A Novel Metagenome-Derived Peptidase Gene TRY1A

Proteases are everywhere, being found in a wide diversity of sources such as plants, animals, and microorganisms. Proteases not only play an important role in the cellular metabolic processes, but also have many applications in detergents,leather processing, silver recovery, medical purposes,food processing, feeds, the chemical industry, as well as waste treatment.In the previous studies, a compost metagennomic library had been constructed.And trylA,a novel gene of peptidase belong to trypsin had been screened and had a research of the property. This study will to take a molecular modifation through a high-through approach to screen positive mutants using error-prone polymerase chain reaction(epPCR).Then research the mutation enzymatic property for regonize the relationship about structure and function deeply.Fristly, optimize the epPCR reaction system of different combine concentration about Mg2+ and Mn2+ through the mutation sequencing result.The ensured epPCR reaction system contain 0.5mM Mg2+ and 4mM Mn2+.The epPCR products were digested with HindⅢ and EcoRI. insert into the pGEM-3Zf(+) colon vector. The ligestion products were introduced into E.coli DH5a.Pick up the positive clone with toothpich to LB plate with 2%(v/v) skim milk.Select the positive mutation exhibited larger zones than control strain. And inserted the mutated gene into pET-32a(+) expression vecoter introduced E.coli BL21(DE3)pLysS. To express and purificate the recombinant protein after induced, and measure the rude enzyme activity. Two mutation with inserted gene ep4 and ep30 were screened because of the improve of the enzyme activity. Through the aligent to TrylA, Ep4 and Ep30 had two amino acid site changed. Gln307Leu, Asp391Gly and Phe122Leu, Thr266Ile. respectively. The enzymatic property research showed that, Ep4 and Ep30 had the same optimal temperature and pH of 50℃ and 9.0 It was the same to TrylA.The thermostability of Ep4 in 50℃ for 1h increased 30% compare with TrylA. The metal ions effections showed that, the activity could be prompted by Mn2+ and exhibited by Cu2+ and Zn2+. Compared to TrylA,the specific of Ep4 was 14.9U/mg and the specific of Ep30was 11.1U/mg to the 9.2U/mg of Try1A.The higher value of kcat of Ep4 indicated it had a higher affinity for substrate.It make the catalytic property of trylA deeply through the enzymatic property of mutation by molecular modification. And it showed a good foundation to reveal the relationship about structure and function and the application in commercial.