Construction Of Genetic Engineering Strains About Alkaline Pectinase PelC Gene From Bacillus Subtilis And Application In The Dumming Of Ramie Fibers

Alkaline polygalacturonate lyase from Bacillus subtilis has wide application potential in degumming of ramie fibers.In order to construct a genentic engineering bacterium for industrial degumming of remie fibers,firstly,genetic modifications of Bacillus subtilis 7-3-3 were conducted in this thesis.pelC is one gene coding alkaline polygalacturonate lyase in Bacillus subtilis 7-3-3.The pelC gene was homologously overpressed in Bacillus subtilis 7-3-3 by increasing the gene copies,as the type of expression plasmid pNW33N-pelC,to enhance the PGL production.The resulting PGL activity was 169 U/mL after feimenting 96 h,which was 1,6 times to the PGL activity of Bacillus subtilis 7-3-3.We also analyzed other enzymes activities of the crude enzyme from strain B-pN-pelC,using the crude enzyme from strain Bacillus subtilis 7-3-3 as control.It was found tha that activities of xylanse and mannase were 1.555 U/mL and 0.525 U/mL respectively,increased by about 20%and 10%respectively compared to that of the crude enzyme from strain Bacillus subtilis 7-3-3.There was no obvious change,however,on the CMCase activity and Filter paper activity.It was known that alkaline polygalacturonate lyase PelA was one of the primary proteins,and its concentration was very abundant on the basis of SDS-PAGE of the crude enzyme from Bacillus subtilis 7-3-3,inferred the strength of the promoter and signal peptide were quite high.In order to further improve the expression of PelC,we successfully constructed a pelC gene homologously overexpressed strain B-pN-pA-pelC-pst by replacing the promoter and signal peptide sequence of pelC gene with that of pelA gene in Bacillus subtilis 7-3-3.The protein concentration in the crude enzyme was improved from 0.3682 mg/mL for strain B-pN-pelC to 0.5204 mg/mL for strain B-pN-pA-pelC-pst,increased approximately 40%,but the PGL activity of the crude enzyme from strain B-pN-pA-pelC-pst was 86.42%of that from strain B-pN-pelC.Besides,there were almost same activities of the xylanse,mannase,CMCase and FPase for the crude enzymes from strain B-pN-pA-pelC-pst and strain B-pN-pelC.According to our previous researches,there was synergism in the degumming of ramie fibers between the PelA and PelC.We constructed a recombinant bacteria B-pN-pelA-pelC by homologously overexpressing pelA gene and pelC gene in Bacillus subtilis 7-3-3 at the same time,which is not be reported in literatures.The PGL activity of the crude enzyme from strain B-pN-pelA-pelC was substantially increased to 1458 U/mL,which was 1.2 times the PGL activity of the crude enzyme from strain B-pN-pelA that be only homologously overexpressed the pelA gene.Enzymatic degumming of ramie fibers using crude enzymes from different recombinant bacteria and Bacillus subtilis 7-3-3 was studied and compared.The result showed that the over-expression of PelC enhanced enzymatic degumming of ramie fibers.By comparing with all of the recombinant bacteria,the strain B-pN-pelA-pelC gave the highest degumming rate.The brightness and dispersibility of the ramie fibers treated by the crude enzyme from strain B-pN-pelA-pelC were better than that by strain B-pN-peA.It was found from analysis of enzyme activities of the crude enzyme from B-pN-pelA-pelC that,besides PelC,the xylanase activity and mannase activity were also increased,which help to degradation of hemicellulose in the degumming process.