Discovery And High-efficiency Expression Of The Family GH45 Cellulases

The family 45 glycoside hydrolase(GH)was an important endo-cellulase applied in the textile for its high defibrillation activity on the fabric,which was widely used in the biofinishing and biostoning.However,the characterized GH45 endo-cellulases were much less than the other family cellulases,and most of these characterized enzymes were directly purified from the wild-type microorganisms,and the productivity was very low,which limited its application and functional research.In addition,the effective GH45 endo-cellulases have been patented.Our research targets in this study were exploring the effective enzyme and the construction of high productive strains.Using the degenerate primers and the Thermal Asymmetric Interlaced PCR(TAIL-PCR),a GH45 gene nmGH45 was directly cloned from the metagenomic DNA of the alkaline lake in Inner Mongolia.And the other GH45 gene ncGH45 was cloned from the type-strain Neurospora crassa.After successfully expressed these two genes in P.pastoris,the recombinant proteins both displayed single band on the SDS-PAGE.The yield of rNMgh45 and rNcGH45 were 660 mg/ml and 80 mg/ml in the flask fermentation,respectively.Besides,the characteristics of the four enzymes had little change.Both of these two enzyme showed excellent pH and temperature stability,and the optimal pH of rNMgh45 and rNcGH45 was fit for industrial application.The dominant products were mainly cellopentose and trace cellobiose for CMC-Na,barley(3-glucan.Most of the metal ion had trace impact on the enzymatic activities,while SDS had great inhibit ability on the two enzymes.The reduction reagents DTT and the(3-Mercaptoethanol had some positive effect on the two enzyme.Furthermore,the rNMgh45 had the ability to resistant the high concentration salt and the ionic liquid.The result of molecular dynamic simulation showed that the conformation of the molecule and the shift of the amino acids were very subtle,which may indicate the reason of the stability of rNMgh45 in salt and ionic liquid.Furthermore,the defibrillation activities on dyed cotton of rNMgh45 and rNcGH45 were investigated,when 10 U enzyme were added,the weight-loss ratio reached 1.81 and 1.55%,respectively.In addation,a series of researches were conducted to improve the activity and productivity of the rNMgh45.Firstly,the cysteine residuals formed the disulfide bonds were mutated into alanine,which caused the complete activity loss,this may be attributed to the conformation changed.Secondly,the chaperons were co-expressed to investigate their impacts on the protein production.The results showed that BiP increased the yield of rNMgh45 by 20%,while PDI and HAC1 had no enhancement effect on the yield.Finally,the fermentation condition of methanol concentration,original pH,initial inoculum on the protein production were compared,and its shown that 1%methanol can improve the production by 90%than the 0.5%methanol.And pH and initial inoculum have on impact on the yield.Besides,four reported GH45 cellulases Egl3,STCE1,MCE1,RCE1 with excellent application efficiency in textile were chosen for production optimization,through codon optimization and hetero-overexpression in P.pastoris.The four protein displayed about 20 U/ml enzymatic activity,which was much better than that of in the literature.In this study,two novel GH45 cellulase with potential application in the textile was found,and at the same time,the impact factors on the protein expression level were conducted,all these works offer us an excellent theoretical and technical support for the application of family GH45 cellulase in the textile field.