The Cloning And High Level Expression Of Laccase Isoenzyme Genes From Trametes Versicolor And Application In Degradation

The laccase as the crucial enzyme shows great potential in degradation of environmentalpollutants. In the present paper, genetic engineering technology was used to clone three laccasegenes from Trametes versicolor for increasing laccase production and improving its enzymaticproperties. After expressed in Pichia pastoris, single factors and multiple factors for lacAproduction were optimized. Directed mutations were made for lacB. Research results are asfollows:Laccase genes lacA, lacB and lacC encoding for isoenzymes were cloned by RT-PCR fromtotal RNA of Trametes versicolor. The sequence analysis indicated that lacA, lacB and lacCgenes consisited of1560bp,1563bp and1584bp, and their open reading frames encoded519,520and527amino acids, respectively. The lacB and lacC had72.7%degree of identity innucleotides and86.7%in amino acids.The three genes were inserted into the Pichia pastoris expression vector and transformedinto Pichia pastoris GS115, KM71H and X33competent cells, respectively. The maximumactivity was obtained with the factor signal peptide in Pichia pastoris GS115with the highestactivity of2635U/L （lacA）,2291U/L （lacB） and2375U/L （lacC） when ABTS was used as asubstrate. The optimal pH and temperature for the LacA and LacB were3.0and55oC withABTS as a substrate, while LacC exhibited optimal pH and temperature at3.5and60oC. All ofthem were stable at30oC,40oC and50oC and at a pH range from3.0to8.0.The laccase gene lacB as a modified template was studied by this paper，the result showedthat both α-factor signal peptide and laccase signal peptide could successfully lead to laccasesecretion, with the former showed better effect. SV40enhancer was added to pPICZαC-lacB,which succeeded to produce laccase, however, failed to achieve the desired enhancement effect.Single factors and multiple factors of fermentation conditions for pPICZαB-lacA productionwere optimized. The results showed optimal culture conditions were as follows: medium initialpH7.1, Cu²⁺concentration0.5mmol/L, methanol additive amount0.6%with51mL liquidvolume and shaker rotate speed180r/min. Furthermore, induction at low temperature andaddition of appropriate amount peptone （4%） in culture medium were more suitable for lacAsecretion which provided the highest enzyme activity of11972U/L, doubled that of conditionsbefore multiple factors optimization. Based on the control of dissolved oxygen between20%-40%, enzyme activity of18123U/L was obtained by high-density fermentation usingmedium FM21at28oC, pH6.0.The recombinant laccase was used to degrade hydroquinone in several laccases andshowed the excellent degradation of hydroquinone with the degradation rate up to100%. Therecombinant laccases were also able to decolorize azo and anthraquinones dyes such asReactive Blue KN-R, Acid Violet43, and Acid Red35, et al. All of the three laccase isoenzymes had good degradation ability with more than80%of the decolorization rate. Thus,this article provides useful novel laccases from Trametes versicolor for application in thedecolorization of dyes.xynlanase gene xynB and laccase gene lacA were cloned by gene splicing by overlapextension PCR. The sequence analysis indicated that xynB-lacA and xynB-linker-lacA genesconsisited of2181bp and2196bp, and their open reading frames encoded726and731aminoacids, respectively. The two genes xynB-lacA and xynB-linker-lacA were inserted into thePichia pastoris expression vector and transformed into Pichia pastoris GS115sucessfully.