Expression Of Alkali-tolerant Cellulase And Mechanism Of Enzymatic Modification

Due to the shortcomings of poor drainage and low strength,the application of straw pulp is limited,so modifications of the drainage and strength are needed when the straw pulp is used for paper production.Using cllulase(mainly endo-glucanase,EG)to treat pulp is an environmentally friendly technique compared to the use of chemicals,and has been proved to be a good method to improve pulp drainage and paper strength.As the pulping and papermaking processes are often carried out in the neutral and alkaline conditions,which requires the enzymes used in the processes maintaining its activities in high pH conditions to ensure its catalytic effects.However,the existing cellulase in the market are mostly acidic enzymes.The enzyme activity is very low under alkaline conditions.Although the research on alkaline cellulase at home and abroad is intensively studied,there are still many problems.For example,the production of alkaline cellulase from bacterial strain is low,and the enzyme system is unreasonable.Therefore,it is of great value to raise the production and reduce the costs of alkaline cellulase for promoting the application of the enzymes in the paper industry.It is also important to study and optimize the enzyme system intensively for reducing the Enzyme modification costs.In this thesis,based on the previous research of our laboratory,we constructed several alkali-tolerant cellulase producing strains for increasing the cellulase production.At the meanwhile,we constructed an alkali-resistant cellulase and xylanase co-expression strain for optimizing the enzyme system and improving the enzymatic modification effects.We also studied the mechanism of enzymatic modification of pulp from different aspects.The main contents and results of this thesis are as follows:1.Expression of alkali-tolerant cellulase and its effect on pulp modificationIn order to obtain the alkali-tolerant cellulase which suitable for pulp modification,on the basis of previous research of our laboratory,three alkali-tolerant endo-cellulases H.EGI,H.EGII,H.EGV form Humicola insolens were cloned and successfully expressed in Pichia pastoris.Three endo-cellulases Y106-EG,z-16-EG and A30-EG derived from Bacillus subtilis were also successfully expressed in Escherichia coli.The properties of the recombinant alkali-tolerant cellulases were determined after purification.The results showed that the optimal pH values of the six EGs were about 6.5-7.5,the optimum temperatures were about 50??60?.The enzymes maintained more than 60%of the maximum activity when placed at pH 6.0-8.0 for 1 h.Notably,Y106-EG,z-16-EG and H.GEG can maintained more than 60%of enzyme activity when placed at pH 9.0 for 1 h.Applying the six alkali-tolerant enzymes to modify wheat straw pulp,The results showed that Y106-EG treatment at a low enzyme dosage of 0.2 IU/g could reduce 12.5%of the beating degree,and increase 14.6%14.3%and 10.7%of the tensile index,burst index and tear index respectively compared with the control.The effect of Y106-EG on pulp modification was significantly better than that of the other five kinds of alkali-tolerant endo-cellulases,showing good prospects for potential applications.2.Optimization of the enzyme treatment conditions with the recombinant Y106-EG for modification of different kinds of pulp and synergistic action of Y106-EG and other enzymes on pulp modificationIn order to improve the production of Y106-EG,homologous overexpression of Y106-eg was performed through genetic engineering method.The crude enzyme activity of CMCase was 8.45 IU/ml,which was 8.28 times of that of the original strain,by using the optimum fermentation medium.The poplar CMP(chemical mechanical pulp),pine CMP and wheat straw CP(chemical pulp)were modified by crude enzyme from the engineering strain Y106OE-EG.The results showed that the modification effects of wheat straw CP was better than that of poplar CMP,and the effect of poplar CMP was better than pine CMP.Enzymatic modification conditions such as treatment temperature,time and pH were optimized,and found that the tensile index,burst index and tear index of wheat straw CP were increased by 15.4%,16.9%and 11.8%respectively compared to the control under the treatment condition of Y106-EG enzyme dosage of 0.2 IU/g,pH 7.0 and temperature of 55?.The synergistic modification effects of crude enzyme of Y106OE-EG with other enzymes were studied.The results showed that crude enzyme of Y106OE-EG cooperation with SWO4(Cellulosic expansion agent)reduced the strength and cellulose crystallinity of the pulp.However,the tensile index,tear index and burst index were significantly improved when Y106OE-EG(0.2 IU/g)cooperated with?-glucosidase(1.2 IU/g)and xylanase(5 IU/g).At 55 0C and pH 7.0,using Y106OE-EG of 0.2 IU/g and P-glucosidase of 1.2 IU/g,the tensile index,tear index and burst index of the wheat straw pulp increased by 23.68%,34.10%and 20.82%respectively,compared with the control.When the dosage of xylanase was 5 IU/g,the tensile index,tear index and burst index of wheat straw pulp were increased by 32.65%,42.44%and 25.00%respectively compared with the control.In addition,the combined treatment of straw pulp with xylanase/SWO4 or xylanase/xylosidase also had a good modification effect.In summary,the synergistic treatment of endo-cellulase produced by Y106OE-EG with xylanase Xyn30 produced by Bacillus pumilusA30 had the best effect on the modification of pulp.This conclusion provides a theoretical basis for the further construction of Y106OE-EG and Xyn30 co-expressing strains.3.Construction of cellulase and xylanase co-expressing strains and optimization of culture conditions for enzyme productionIn order to achieve co-expression of alkali-tolerant cellulase(Y106-EG)and xylanase(Xyn30)in Y106-WT,we respectively used three methods such as cistronic co-expression,fusion expression and tandem co-expression,to improve the enzyme production of Bacillus subtilis Y106-WT.The results showed that tandem co-expression was the best method for co-expression of the two enzyme.The activity of the CMCase in the culture of co-expressing strain Y106OE-EG-Xyl was found to be the highest(8.2 IU/ml)at 56 hours,and the activity of xylanase reached the highest(60.4 IU/ml)at 64 hours.Enzyme analysis revealed that the filter paper activity(0.12 IU/ml),cellobiohydrolase activity(0.02 IU/ml),?-glucosidase(not detected)are low,indicating that the crude enzyme from the co-expressing strain was suitable for pulp modification.The Y106OE-EG-Xyl crude enzyme was used to treat wheat straw chemical pulp,and found that the optimum treatment pH was 7.0,the optimum treatment temperature was 55? and pulp concentration was 10%(w/v).Brightness and tensile index of wheat straw chemical pulp could be well improved by treatment with the crude enzyme from strain Y106OE-EG-Xyl when compared to the control(no adding enzyme)and the only addition of cellulase(0.2 IU/ml).Compared to the control,when the cellulase dosage was 0.2 IU/ml and xylanase dosage was 1.5 IU/ml,the pulp brightness was increased by 2.0%ISO,and the tensile index,brust index and tear index increased by 8.7%,16.6%and 9.3%respectively,and beating degree of pulp reduced by 15%.By optimizing the mixing proportion of carbon source and inducer in fermentation culture of engineering strain Y106OE-EG-Xyl,we found that when the dosage of wheat bran,corn cob and lactose was 3.33%,0.83%and 0.83%respectively,the enzyme production is the highest.The activity of CMCase reached 7.9 IU/ml,and Xylanase activity reached 74.5 IU/ml,which 14.1 IU/ml higher than original(before optimization).The ratio of xylanase to endoglucanase was increased from 7.5 to 9.4.4.Study on mechanism of pulp enzymatic modificationThe fiber characteristics,cellulose crystallinity(XRD)and infrared(FITR-ATR)spectra of pulps before and after treatment with crude enzyme of Y106OE-EG were studied.After treatment,the fiber qualities of poplar CMP and wheat straw CP were significantly improved,and the modification effect on polar CMP and wheat straw CP was better than that on pine CMP.The cellulose crystallinity of poplar CMP and wheat straw CP were significantly increased compared to the control after the enzyme treatment,but little effect on cellulose crystallinity of the pine CMP.The lignin components of wheat straw chemical pulp,pine CMP and poplar CMP were extracted out respectively,and the adsorption characteristics of lignin on the Y106OE-EG enzyme protein were studied.The result showed that the adsorption capacities of lignin from poplar CMP and pine CMP on the cellulase protein were significantly higher than that of wheat straw pulp.The irreversible adsorption of lignin on the enzyme protein affects the interaction of the enzyme with the fiber substrate.So Y106OE-EG crude enzyme is more suitable for the modification of wheat straw pulp.The crude enzymes from Y106OE-EG(EG dosage of 0.2 IU/g),Y106OE-Xyl(Xylanase dosage of 1.5 IU/g)and co-expressing strain Y106OE-EG-Xyl(EG 0.2 IU/g-Xylanase 1.5 IU/g)were respectively used to treat wheat straw chemical pulp,and found that,compared with the control and only addition of endo-cellulase or xylanase,treament with Y106OE-EG-Xyl crude enzyme resulted in the fiber length of pulp increase,but the fiber width almost unchanged,so the ratio of length to width increased.The fines content,curl index and kink index of wheat straw CP were also reduced after the enzymatic treatment.Relatively,the effect on pulp properties with crude enzyme from co-expression strain was better than that with single enzyme.FITR-ATR analysis of the wheat straw pulp before and after the treatment with Y106OE-EG-Xyl crude enzyme showed that hydrogen bond strength was significantly improved by the crude enzyme treatment compared with the control and single enzyme treatments.SEM observation of the fiber morphological found that the fines content was reduced and the fibers surface became flat compared to the control.The changes in fiber quality,cellulose crystallinity,hydrogen bond strength and surface morphology should be some internal causes that enzyme treatment improved strength and drainage of pulp.To understand why the enzyme Y106-EG treatment had better modification effect on wheat straw chemical pulp than other alkali-tolerant enzymes which respective from Humicola insolens(H.EGI,H.EGII and H.EGV)and other Bacillus(z-16-EG and A30-EG),the structure and characteristics of the enzymes,such as surface charge of protein,hydrophobicity of protein and adsorption of the protein onto the pulp fiber,were evaluated and compared.It was found that enzyme Y106-EG had a relatively strong affinity on the fiber substrate compared with the other five alkali-tolerant endo-cellulases.The amino acid of Y106-EG is liable to be positively charged at pH 7.0(pH used in pulp modification)becuase the pH is lower than its own isoelectric point.The Y106-EG protein had a low Zeta potential and low hydrophobic compared with the other alkaline endo-cellulases,which make the Y106-EG enzyme protein more easily to bind with the negatively charged small fibers.Besides,Y106-EG enzyme protein is a typical(p/a)8 barrel structure which is stable,and had wide open active center that make it accommodate to more types of polysaccharide branching,thus had advantages in degradation of complex substrates.The binding domain(CBM)of the Y106-EG belongs to type A and tends to bind to crystalline cellulose.These features make Y106-EG more suitable for application in pulp modification.