Cloning And Expression Of Lignocellulase Genes From Termites And Their Endosymbionts

As the energy crisis and environmental problems becoming more and more serious,the use of renewable lignocellulosic biomass resources to produce biofuels is attracting more and more attention.Termites are well recognized for their ability to digest lignocellulose,for their importance as models for bioenergy production and as potential sources for novel lignocellulases.The fungus-growing termite Macrotermes barneyi was used in this study.In previous transcriptome sequencing of M.barneyi,we found some lignocellulase genes,which include:laccase,P-glucosidase and endoglucanase unigenes.The ?-glucosidase(MbBG)and endoglucanase(MbEG)with high expression were successfully heterolougous expressed and studied before.While cDNA of laccase is still not expressed.In addition,two bacteria strains with laccase and xylanase activity were isolated from M.barneyi,while the role of xylanase and laccase from the symbiont microbes were not clear.In order to study the role of these proteins in the degradation of lignocellulose,we carried out the experiments as follows:1.Cloning and expression of laccase gene from M.barneyi.In previous transcriptome sequencing of M.barneyi,a laccase gene MbLac was predicted.Through sequence alignment and phylogenetic tree analysis,it is speculated that the gene is from M.barneyi.The MbLac gene was amplified by PCR from the cDNA of the salivary-foregut gland of M.barneyi.Then the cDNA of MbLac was expressed in E.coli JM109,E.coli BL21(DE3)and Pichia pastoris GS115 respectively.The MbLac could be recombinantly expressed in E.coli JM109 and E.coli BL21(DE3),but the recombinant protein was inactive.Probably because E.coli does not have the ability to modify the secondary structure of the protein.MbLac could not be expressed in Pichia pastoris GS115,with no signal detected by western blotting.The laccse from termite couldn't expressed in Pichia pastoris,which probably caused by the codon bias.2.Cloning and expression of laccase gene from M.barneyi symbionts.A Bacillus sp.with laccase activity was isolated from hindgut of M.barneyi.The laccase gene CotA was cloned from Bacillus sp.genome DNA.The full-length of the CotA fragment was 1533 bp,encoding 510 amino acids and one stop codon.This gene was heterologously expressed and purified in E.coli JM109.The optimum temperature and pH of recombinant CotA were 70 ? and 5.0,respectivly.The enzyme in the alkaline environment is relatively stable.The Km of the recombinant CotA was 0.278 mM for ABTS and the value of Vmax was 555.55 U/mg.Recombinant CotA has strong decolorization ability to indigo,crystal violet and malachite green,and the decolorization rate is over 80%after 3 h.3.Cloning and expression of xylanase gene from M.barneyi symbionts.The XylMbl gene from Paenibacillus sp.Mbl was overexpressed in E.coli JM109.The recombinant protein was purified by Ni-NTA column.The specific activity of the recombinant XylMb1 towards birch xylan wood was 3203.12 U/mg.4.Synergism of lignocellulase on filter paper and PASC.The ?-glucosidase mutant BGDS-5 and the endoglucanase mutant EG71 were co-expressed in E.coli BL21.The co-expressed BGDS-5 and EG71 can synergistically degrade filter paper and PASC.In addition,we studied the synergistic degradation of CotA,XylMbl,BGDS-5 and EG71 on filter paper and PASC.The synergistic factors of these four enzymes toward filter paper and PASC were 1.63 and 1.4375,respectively.In conclusion,the laccase gene MbLac from M.barneyi,the xylanase gene Xy.lMbl and the laccase gene CotA from the intestinal microbes were cloned and expressed in this paper.Then,synergism of lignocellulase from termites and symbiotic microbes on cellulosic materials were studied.This study deepens our understanding of the mechanism of termite lignocellulosic degradation and provides a theoretical basis for the application of lignocellulosic degradation enzymes from termite and its microbes.