High-level Expression And Fermentation Optimization Of Laccase

Laccases are multi-copper polyphenol oxidase which can catalyze lots of phenolic compounds efficiently,they are widely used in environmental bioremediation,biofuel production,biological pulping,textile industry as catalysts for compound synthesis and structure modification.In this study,the laccase gene from wild strain Cerrena sp.WR1 in Pichia pastoris was expressed,then molecular chaperones were co-expressed and four different self-assembling amphipathic peptides which were attached into the N-terminus of the laccase were expressed to further increase its production and improve enzymatic properties.The principal study results are as follows:(1)Construction of recombinant P.pastoris for laccase productionThe laccase gene sequence Lcc3 from Cerrena sp.WR1 was optimized according to the condon bias of P.pastoris,then the gene Lcc3' which excluded itself signal peptides were obtained by PCR.Then the genes Lcc3 and Lcc3' were cloned into the expression vector pPIC9 K,and transferred them into P.pastoris GS115,thus the strains GS115-Lcc3 and GS115-Lcc3' were obtained.Through G418 resistance,the 48-well screening and shake flasks fermentation validation,two recombinant strains whose enzyme activities were relatively high were selected.It turned out that the laccase activity of GS115-Lcc3' was two times of GS115-Lcc3.The yield of GS115-Lcc3'(PP-L)reached 555 U·L-1 by the optimization of fermentation conditions in shake flasks.(2)Improving the secretion of laccase by co-expressing molecular chaperonesOn the base of the strain PP-L,six chaperones which included protein folding(BIP and ERO1),transportation(SEC53 and SEC1)and stress response(HAC1 and GCN4)were conducted and co-expressed.The results indicated that the co-expressions of chaperones had no effect on the growth of laccase secretion strain PP-L.The extracellular enzyme activities increased by 359% with BIP,decreased by 22% with ERO1.The other four chaperones had no significant effects on the extracellular activities(increased by 18%-53%).The extracellular enzyme activity of strain P1 with both BIP and HAC1 increased by 602% compared with strain PP-L,reached 3896 U·L-1.The results showed that the protein folding abilities and the unfold protein response were the key factors that limited the efficient expression of laccase in P.pastoris.(3)The effects of attaching SAPs into the N-terminus of laccase on the recombinant strainsAttaching four different structural characteristics of peptides SAP-1,SAP-2,SAP-3,SAP-4 into the N-terminus of Lcc3' by PT linker,then cloned into the vector pPIC9 K and then transferred them into P.pastoris GS115,obtained recombinant strains S1,S2,S3,S4,the yields were 1.42,1.47,0,0.64 times compared with PP-L.Based on the effects of coexpression molecular chaperones on the production of laccase,pGAPZB-HAC1 and pGAPZB-BIP were further trasferred into the strains S1,S2,S4,and obtained the recombinant strains S1',S2',S4',and the laccase activities were increased by 484%,497%,515%,respectively.The laccases produced by the recombinant strains P1,S1',S2',S4' were purified,then determined their enzymatic properties.The specific activity of laccase produced by S2' improved 36.9% compared to P1.The optimal temperature were 55,60,60,65?,respectively.The optimal pH were 3.4,3.0,3.4,3.0,respectively.The t1/2 of the laccase of S2' at 50? were 253 min.Incubated the laccases at pH 3-4 for 100 h,the activities decreased significantly;Incubated the laccases at pH 5-8 for 100 h,the activities decreased slowly.The kcat of the laccases were 948.3 s-1,1021.3 s-1,1034.5 s-1,827.6 s-1,respectively.The affinities of S1',S2' for ABTS were improved.They were all stable in methyl alcohol and ethyl alcohol.