Heterogenous Expression Of A Laccase Gene In Trichoderma Reesei And Basic Study On Its Application

The laccase produced by Pycnoporus sanguineus with good thermostability has great potential in industrial applications,however,low production capacity limits its large-scale production.In this study,gene recombination technology was used to achieve high expression of heterologous laccase gene in Trichoderma reesei.Furthermore,the recombinant T.reesei was studied with respect to its enzyme production performance and applications in degradation of straw and organic pollutants.The major results are as follows:The laccase gene from P.sanguineus was codon-optimized according to the codon usage bias of T.reesei,and then linked to T.reesei strong promoter Pcbhl,its terminator Tcbh1 and hygromycin B resistance marker to obtain recombinant plasmid pCH-lac,which was subsequently transformed into the conidia of T.reesei via Agrobacterium-mediated transformation.The study of the main factors affecting transformation efficiency showed that the highest transformation efficiency was observed when co-cultivation temperature was 24 °C,the pH of the co-cultivation medium was 5.2,T.reesei spore concentration was 107/mL,and the cultivation time was 60 h.Nine transformants were obtained through hygromycin resistance screening and secondary screening.After that,the chromosomal DNA of transformants was confirmed by polymerase chain reaction(PCR)detection,showing that the foreign laccase gene had been integrated into the chromosomal DNA of T.reesei and could be inherited steadily.SDS-PAGE analysis of fermentation broth of recombinant T.reesei showed an obvious protein band(approximately 66 kDa)corresponding to the expressing product of target laccase gene from P.sanguineus,which proved the successful extracellular expression of the laccase gene.Laccase production by recombinantT.reesei was performed in shaking flasks.Via optimization of medium and fermentation conditions,the laccase activity peaked at 8.8 IU/mL after 96 h.Fed-batch fermentation was further employed,and the resulting laccase activity was increased to 20.3 IU/mL.These results lay the foundation for large-scale production of laccase.The study of enzymatic properties of the recombinant laccase showed that it exhibited good catalytic activity during 50-75 °C with the optimum reaction temperature of 65 °C.At 70 °C,its thermostability was largely improved compared with that produced by the original P.sanguineus.It is possibly due to the mature glycosylation system of T.reesei,which enables the protein to maintain stable.The optimum pH of the recombinant laccase was 2.2 and it had obvious catalytic activity between pH 2.0-2.6.After being kept at pH 2.2-4.2 for 4 h,its residual activity was still over 80%.In the meantime,the recombinant laccase presentedgreattolerance towards certain types of metal ions and organic solvents,and good affinity for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS).During the solid state fermentation(SSF)using plant fiber materials like bagasse and rice straw as substrate,the recombinant T.reesei could produce not only cellulase and xylanase but also high-activity laccase,which enabled it to degrade lignocellulose raw materials effectively.When using bagasse as the major content of SSF medium,the laccase activity,the cellulase activity(Filter paper activity,FPA)and the xylanase activity per gram dry basis produced by recombinant T.reesei could reach 16.4 IU,89.2 IU and 3830 IU,respectively;on the 12th day of fermentation,the degradation rates of cellulose,hemicellulose and lignin were 80.2%,58.7%and 31.7%,respectively,and the total loss of bagasse was 57.1%,which was 1.5-fold of that achieved by the original T.reesei.When using rice straw as the major content of SSF medium,the laccase activity,the cellulase activity(FPA)and the xylanase activity per gram dry basis produced by recombinant T.reesei could reach 8.1 IU,132.2 IU and 5504 IU,respectively;on the 12th day of fermentation,degradation rates of cellulose,hemicellulose and lignin were 75.6%,57.2%and 28.6%,respectively,and the total loss of rice straw was 55.4%,which was 1.6-fold of that achieved by the original T.reesei.The laccase-mediated dye decolorizationexperiment was performed.The recombinant laccase could efficiently decolorizeanthraquinone dyes such as weak acid violet N-FBL and reactive brilliant blue KN-R,whose decolorization rates were both over 91%without the addition of mediator.As for Amaranth,an azo-dye,the decolorization rate was up to 92.2%with the addition of 0.1 mM syringaldehyde,a natural mediator.Furthermore,the laccase-ABTS system was used to degrade endocrine disruptors(bisphenol A and triclosan),and the degradation rates of bisphenol A and triclosan reached 95%and 81%,respectively.