Improvement Of Enzyme System Of Bacillus Subtilis And Application In Degumming Of Ramie

Ramie,a member of hemp fiber family,is one of the excellent textile materials.It must be degummed before application to remove the pectin in the bast fiber and separate the fibers,whichgive the fiber excellent textile properties and quality.Compared with traditional chemical degumming,the enzymatic degumming method,using microbial enzymes instead of chemical degradation of colloidal composites,has become an inevitable trend in the development of hemp degumming technology due to its environmental friendliness and elimination of fiber damage.The alkaline pectinase produced by Bacillus subtilis 7-3-3 in our laboratory has a good degumming effect on ramie.Strategies including the optimization of medium composition and fermentation conditions,regulation of fermentation process and overexpression of pectin lyase PelA,have significantly improved the ability of this strain to produce polygalacturon lyase(PGL).Due to the complexity of both composition and pectin structure in hemp fiber,its degradation requires a complex enzyme system with variousinvolved enzymes.In order to further improve the enzyme system and improve the enzymatic degumming efficiency,this paper studied the degumming of ramie with different degumming related enzymes PelA,xylanase(Xyn)and mannanase(Man)based on the preliminary research of the laboratory.For the first time,the homologous overexpression of the above different enzymes in B.subtilis 7-3-3 was obtained,the activity of three enzymes in the crude enzyme solution was improved.The composition change significantly improved the degumming effect of ramie.In addition,the paper also expressed the rhamnogalacturonanase from B.subtilis and discussed its role in ramie degumming.Firstly,pectin lyase A(PelA),xylanase(Xyn)and mannanase(Man)were homologously overexpressed in B.subtilis 7-3-3 using the shuttle plasmid pNW33N.The enzyme activities reached 870 U/mL,400 U/mL and 43 U/mL,which were 10,20 and 2 folds higher than the original strain,respectively.On this basis,replacing the promoter of mannanasefurther improvedthe activity of mannanase by about 40%to reach 68 U/mL.What's more,the gene fragment of two pectin lyase A was expressed in tandem to make the PGL enzyme activity reached 1324 U/mL,which was increased by about 30%compared to the PelAsingle overexpression strain.The degumming efficiency of the crude enzyme solution of different engineering strains on ramie fiber was studied.After examining the degumming rate,tensile strength and reducing sugar content in waste liquid,it was found that thecrude enzyme of strain OE PelA(overexpressing PelA)had the best degumming effectwith highfiber dispersion.The crude enzyme solutions of OE Xyn and OE Man have a little effect on degumming compared with the starting strain B.subtilis 7-3-3,indicating that PelA is more criticalin the ramie degumming process.Secondly,Xyn and PelA were both overexpressed in B.subtilis 7-3-3,and the enzyme activities of xylanase and PGL were improved in the fermentation broth of OE PelA+Xyn.The degumming effect of the liquid is obviously better than that of the crude enzyme solution of OE PelA,which proves that the combined action of PelA and xylanase is beneficial to the degradation and removal of pectin in ramie.Further,the three enzyme components of Xyn,Man and PelA were successfully overexpressed in B.subtilis 7-3-3 simultaneously,generating the engineering strain OE PXM.The enzyme activities of pectin lyase,xylanase mannanase in the fermentation broth reached 1028 U/mL,502 U/mL and 25 U/mL,respectively.Compared with the starting strain B.subtilis 7-3-3,the production levelof these three enzymes was increased by 10,20 folds and 67%,respectively.The proportion of degumming-related enzymes in the whole secreted protein was increased,and the degumming enzyme composition of B.subtilis was optimized.Finally,the enzymatic properties of the crude enzyme solutionof co-overexpressing strain OE PXM were preliminarily studied.The optimum temperature of pectin lyase,xylanase and mannanase were found to be 60 0? 50? and 50?,respectively.Theoptimum pH values are 9.5,8.0,and 8.5,respectively.The degumming of ramie was carried out by using the crude enzyme solution of the co-expressing strain OE PXM.The results showed that the degumming effect for the crude enzyme solution of OE PXM was better than that of other single-overexpressing strains,and other engineering strains such as OE PeIA+Xyn.The fiber has a higher strength and the fiber also becomes softer.Scanning electron microscopy showed that the surface of ramie fiber after degumming with OE PXM crude enzyme solution became smoother and the fiber dispersion was higher.Infrared analysis showed that some of the chemical bonds of hemicellulose in ramie were broken after degumming.In addition,degradationof the extracted colloidal complexes(including hemicellulose and pectin components)from ramie found that the crude enzyme solution of strain OE PXM is better than single enzyme overexpressing strains OE PelA,OE Xyn,OE Man and starting strain,which demonstrated the combined degradation ability of hemicellulase and pectin lyase in degumming.Rhamnogalacturonan is a polysaccharide formed by the alternate connection of rhamnose and galacturonic acid,which counts for about 35%of pectin.It is also speculated that thedegradation of the rhamnogalacturonanase in pectin may play an important role in degumming.Previously,a gene cluster of rhamnogalacturonanase was foundin the genome of B.subtilis 7-3-3,which was about 26 kbp in size.The gene cluster contained a set of genes related to the degradationof rhamnogalacturonan.Four gene fragments were selected,in which the gene fragments 3851,3856 and 3864 without signal peptide were expressed in E.coli,and the 3859 fragment with signal peptide was expressed in B.subtilis,and the target proteins were obtained.Furthermore,based on different degumming enzyme systems,the above different proteins were added for degumming experiments.The preliminary results showed that the addition of proteins 3851,3856 and 3864 did not significantly promote the degumming effect on ramie,while the over-expression of protein 3895 in B.subtilis did promote the degumming effect of ramie.This result provides a novel reference for the optimization of the degumming enzyme system in the future.