Expression And Characterization Of An Endo-Glucanase From Symbiotic Bacteria Of Reticulitermes Labralis

Cellulose,one of the most abundant biomass resources in China,is difficult to degrade because of its insoluble structure.Low-temperature endo-?-glucanase(EglC)which has great potential applications in long cold period regions of china can efficiently degrade the cellulose to glucose and oligosaccharide in a cold environment.The symbiotic bacteria of wood-feeding termites are important for degradation cellulose.However,the secretion of low-temperature EglC from termite bacteria was at low level and it was hard to purify.Currently,protein expression technology is an available way to increase expression level and purify the target protein.In the present study,the Citrobacter farmeri A1 that secreted low-temperature EglC was isolated from the wood-feeding termite(Reticulitermes labralis).The low-temperature EglC gene was cloned and expressed in the Escherichia coli and Pichia pastoris.Furthermore,the EglC gene was designed by codon optimization to increase the expression level.The recombinant endo-?-glucanases were fully detected.1 Isolation,purification and characterization of the low-temperature endo-p-glucanase(EglC)from symbiotic bacteria of Reticulitermes labralisThe glucanase strain A1 which could efficiently degradated cellulose was isolated from the gut of the wood inhabiting termite Reticulitermes labralis,and the activity of this EglC was low(0.8 U/ml).Then 16S rRNA of strain A1(1,402 bp)was isolated to identify the species by PCR.Blast analysis revealed that this nucleotide sequence had the highest identity(99%)with the Citrobacter farmeri W17-1 and Citrobacter farmeri 17.7KSS.Moreover,the phylogenetic tree showed that the glucanase strain A1 was genetically similar to Citrobacter farmeri than other bacterium strains.Thus,the strain A1 was deemed to be a strain of Citrobacter farmeri,and the obtained 16S rDNA sequence was deposited in the GenBank database(GenBank accession no.KT313001).The chromatography technology was used to purify the EglC of Citrobacter farmeri Al.SDS-PAGE reveled that a single protein with the MW(38 kDa)was present in the gel.The EglC was active at pH 6.5-8.0 with an optimum pH of 7.0.It was stable at pH 3.5-pH 6.5 and maintained>60%of maximal activity in these buffers at 30 min.EglC had an optimium temperature of 30-40 ?and showed>50%relative activity even at 5 ?.These properties suggest that this enzyme is a low-temperature endoglucanase.2 Amplification,cloning and sequencing of the low-temperature endo-?-glucanase(EglC)gene of Citrobacter farmeri A1About 600 bp core region of EglC was amplified from Citrobacter farmeri A1 by touchdown PCR.Thermal asymmetric interlaced PCR(TAIL-PCR)was used to amplify the flanking sequences of EglC according its core region.Results showed that EglC gene was 1107 bp(G+C%= 58.08%)which encoded 368 amino acids.This nucleotide sequence was deposited to the GenBank database(accession number:KT313000).The deduced EglC had 86.4%,43.4%,44.7%,39.7%,and 44.3%amino acid sequence identity with endo-?-1,4-glucanases from E.coli CFT073,Burkholderia sp.CCGE1002,Cupriavidus taiwanensis,Pseudomonas fluorescens SBW25,and Xanthomonas campestris pv.vesicatoria strain 85-10,respectively.The EglC belongs to glycoside hydrolase family 8 with a signal peptide of 1-21 amino acids at its N-terminal.3.Expression and characterization of the low-temperature endo-p-glucanase(EglC)gene from Citrobacter farmeri A1 in E.coliIn the present study,the EglC gene was cloned and expressed in the E.coli to increase production level.The EglC gene was cloned to pET22b vector and transformed to E.coli BL21.The activity of E.coli BL21-pET22b-EglC crude extract was 9.5 U/ml.The crude EglC22b was purified using Ni2+-NTA affinity chromatography,as a protein with the expected MW(42 kDa)was present in the SDS-PAGE gel.The specific activity of the purified enzyme was 8.7 U/mg.The optimum pH of the recombinant enzyme was 7.0,and it had high pH stable(pH 3.5?6.5).EglC had an optimium temperature of 30-40 ? and showed>50%relative activity even at 5 ?.Its activity was enhanced by Co2+,but inhibited by Zn2+,Li+,Triton X-100,DMSO,Tween 80,SDS and EDTA.4 Expression and characterization of the low-temperature endo-p-glucanase(EglC)from Citrobacter farmeri A1 by co-expression of Myxococcus xanthus protein SThe EglC gene of Citrobacter farmeri was expressed in E.coli as an N-terminal fusion to protein S(ProS)from Myxococcus xanthus to improve its expression level.A novel expression vector,pET(ProS-EglC),was successfully constructed for the expression of Citrobacter farmeri EglC gene in E.coli.SDS-PAGE showed that the recombinant protein(ProS-EglC)was approximately 60 kDa.The activity of crude extract was 12.4 U/mL,which was higher than that of the nature EglC(0.8 U/mL)and EglC22b(9.5 U/mL).ProS-EglC was active at pH 6.5-pH 8.0,with optimum activity at pH 7.0.The recombinant protein was stable at pH 3.5-pH 6.5.The optimal temperature for activity of ProS-EglC was 30?-40?.It showed greater than 50%of maximum activity even at 5?,indicating that the ProS-EglC is a cold-active enzyme.Its activity was increased by Co2+,but decreased by Zn2+,Li+,Triton X-100,DMSO,Tween 80,SDS and EDTA.5 Expression and characterization of the low-temperature endo-?-glucanase(EglC)gene from Citrobacter farmeri A1 in P.pastorisThe primers(PEglC-F and PEglC-R)which were used in the P.pastoris expression system were designed according to properties of expression plasmid pPICZaA.The P-EglC gene was cloned to pPICZaA vector and transformed to P.pastoris X-33 to improve expression level.P.pastoris pPICZaA-PEglC was induced by 0.8%methanol.The maximal activity of crude enzyme is 21.6 U/mL after 96 h induction.SDS-PAGE revealed that the molecular mass of recombinant enzyme(P-EglC)was about 38 kDa.The optimum pH of P-EglC was 7.0,and kept>85%relative activity at pH 6.0-8.0.The optimum tempature of P-EglC was 30 ?-40 ?,and kept>50%relative activity at 5?-20?.It was highly temperature stability at 40 ?-50 ?.Its activity was enhanced by Co2+,but inhibited by Zn2+,Li+,Triton X-100,DMSO,Tween 80,SDS and EDTA.6.Codon optimization increased the expression level of the endo-?-glucanase(EglC)gene in P.pastorisFor higher expression level,the mature EglC gene was optimized based on the codon bias of Pichia genome and synthesized by whole gene assembly.The optimized gene SEglC was designed by choosing most-preferred codons of P.pastoris.In addition,the second-preferred codons of P.pastoris were alternatively selected to eliminate the stable secondary structures of mRNA and balance A+T%and G+C%.The optimized gene SEglC showed 70.89%identity with the native EglC gene.The G+C%of SEglC was 41.1%.After optimization,the minimal free energy of the SEglC gene was raised from-296.03 kJ/mol to-184.08 kJ/mol.The S-EglC gene was cloned to pPICZaA vector and transformed to P.pastoris X-33.P.pastoris pPICZaA-SEglC was induced by 0.8%methanol.After 96 h induction,the maximal activity of crude enzyme is 36.2 U/mL,which was 1.66 fold higher than P-EglC.The expression level was increased to 633 ?g/mL in the shake flask fermentations.The transformants of recombinant strains presented quite good genetic stability.In summary,the low-tempature endo-p-glucanase which secreted by Citrobacter farmeri A1 was isolated and purified from the wood-inhabiting termite Reticulitermes labralis.To improve the production level,SEglC gene was successfully expressed in the Pichia pastoris.Pichia pastoris pPICZaA-SEglC secreted more low-tempature endo-?-glucanase than the native EglC gene expressed in Escherichia coli and Pichia pastoris.The present study enriched the gene resource of endo-?-glucanase in termite gut microorganism,provided available approaches for further study on expression of endo-?-glucanase,and laid the foundations for its applications.