Expression And Enzymatic Properties Of Novel Bifuntional Genes In Uncmitured Microorganisms

bgl1D gene (Genbank accession number FJ686869) is an encodingβ-glucosidase activity of the new genetic resources which come from a metagenomic library. It was been constructed that recombinant strains pGXAG801 which can showβ-glucosidase activity.Bioinformatics analysis indicate that:In the amino acid level, Bgl1D show 31% similarities according to lipase from Clostridium botulinum, which indicated that the Bgl1D may have lipase activity. There may be encoded protein is a moonlighting proteins.In the nucleotide level, there is no similarity whit the lipase gene according to GenBank database.After Purified,We make a preliminary study such PNPB as substrate for the lipase enzymatic properties, and the conclusions are following:The optimum pH and temperature of enzymes were 9.0 and 25℃; Km may 0.54 mmol/L; Vmax may be 3.17μmol/min; Enzymatic activity is 8.2U/mg; The lipase activity was strongly stimulated by Al3+ Li+, Cu2+,while was inhibited by Fe2+, Fe3+,It is indicated that the gene is a bifunctional genes.The mutant library was constructed by error-prone PCR.We were screened from 5000 to 10000 clones and obtained after screening mutants pGXAG801-1 and pGXAG801-2, verification of the purified mutant enzyme activity, found that dual activity were decreased,1,β-glucosidase activity in 30% of the original strains, lipase was 20%. Sequencing showed that the amino acid sequence mutation 3 sites,2 strains ofβ-glucosidase activity in 20% of the original strain, the enzyme activity of fat loss. Sequencing showed that the amino acid sequences of 6 amino acid mutation sites, which analysis may be speculated that mutations at the conserved region of the enzyme.Protein by coding with bgl1D genes has a moonlighting proteins. It is also bifunctional enzymes.while its encoded protein may be a new family of lipases. The conservative amino acid sequence play an importance part in the enzymes of activity through mutant library was constructed.It is a great explanation to clarify the catalytic mechanism of enzymes.