Cloning Laccase Gene, Consturcting Of Engineering Strains And Producing Recombinant Enzyme

Laccase, an extracellular polyphenol oxidase, has been widely using in the fields of paper pulp, paper making and cellulose ethanol production and so on. Discovering new laccase gene and studying production recombinant laccase are the hot topics now since the natural Laccase which coming from the whit lot fungus with long production cycle and lower yield couldn’t meet the requirement in the Laccase market.Here a DNA segment in 1542bp was obtained by PCR amplification from the bacillus subtilis. The result of BLAST proved the DNA sequence is maximal similar to the laccase of AL009126.3 (GenelD), a gene from bacillus subtilis subsp. subtilis str.168, but the deoxyribose nucleotide homology between the bslac2 and AL009126.3 is 86%, and also their amino acid homology is only 93.57%. The protein coded by this new sequence shows laccase activity. Those result indicated that the bslac2 is a new laccase gene sequence,which was named as bslac2.For studying expression recombinant bslac2 protein in Pichia pastoris, the gene of bslac2 was cloned to the MCS of pPIC9K and pGAP9K and the pPIC9K-bslac2 and pGAP9K-bslac2 were transformed into the genome of Pichia pastoris GS115 by electroporation. The recombinants containing his gene were proved by MD plate and the high expressed strains were selected by adding the fermentation broths on the plate to analyze the Laccase activity. High-density cell culture of GS115(pPIC9K-bslac2) was progressed in a 50 L bioreactor with a 20 L working volume for 73 h, the biomass is A600=266.5 and Laccase activity is 1097.5U/L. While using glycerol as the only carbon source to ferment 30 h in the bioreator, the GS115(pGAP9K-bslac2) grown to A600=140.4 and secreted 136.7U/L of Laccase in the fermentation broth. The recombinant Laccase was purified to 93.65% by SP-Sepharose FF ion-exchange chromatography.The results from testing of the bslac2 activity indicated its best laccase activity is at pH 4.0 and 25℃. Cu2+ induces the bslac2 gene expression and O.1mmol/L is the proper concentration, but regardless at any concentration, ABTS、Tannic acid、Benzoic acid and Hydroxybenzoic acid no display any role on inducement the laccase gene expression.