Study On The Rare Earth Directed Breeding And Mechanism Of High-yield Xylanase Bacillus Subtilis

Xylan is the main component of plant cell wall and the second largest natural polysaccharide in the world,with its content second only to cellulose.As the main enzyme that catalyzes the hydrolysis of xylan,xylanase can transform xylan-rich biomass resources and has great potential for development.Xylanase is mainly derived from the metabolic process of microorganisms,so the breeding of high-yielding xylanase strains is of great significance.In this paper,rare earth was used as an additive to study the effects of different concentrations and types of rare earth on the growth and xylanase production of Bacillus subtilis 1.108,and then B.subtilis with high xylanase production was selected.The results showed that when the final concentration of Er³⁺in the medium was 1×10^-4mol/L,the highest xylanase activity was 133.79 U/m L,which was 10.48%higher than that of the original strain.Adding rare earth ions when the strain was cultured for 0-6 h could significantly increase the activity of xylanase,and the increase of xylanase activity has no obvious correlation with the growth of the strain.B.subtilis after rare earth treatment was subcultured eight times,the results showed that the high yield strain had genetic stability.The mechanism of high xylanase production of rare earth breeding B.subtilis was studied.Scanning electron microscopy results showed that the cell morphology of the strain did not change after rare earth treatment,but the cell surface was slightly changed,and the holes in the cell wall were generally larger.It was speculated that it led to the increase of xylanase secretion,thereby improving the xylanase activity.Two xylanase genes xyn A and xyn D were amplified by PCR and sequenced.The results showed that the two xylanase gene sequences of the strain after rare earth treatment were changed.Among them,13 bases of xylanase gene xyn A changed,including 6 transitions,4 transversions and3 insertion mutations.However,only one base of xylanase gene xyn D changed,which was an insertion mutation,that is,base T was inserted once.The analysis of the changes in the structure of xylanase protein showed that there was no significant difference in the relative molecular mass and theoretical isoelectric point of xylanase protein before and after rare earth treatment.However,the addition of rare earth ions changed the active,catalytic site,secondary structure of protein and the connection and arrangement of amino acids of xylanase,which may be one of the important reasons for the improvement of xylanase activity.The modified xylanase gene was recombined with p AX01 plasmid,and then transferred into the original strain B.subtilis 1.108.The results showed that the xylanase activities of the recombinant strains B.subtilis 1.108-A and B.subtilis 1.108-D were127.65 U/m L and 118.79 U/m L,respectively.Compared with the original strain,the xylanase activity of B.subtilis 1.108-D was not increased,and the xylanase activity of B.subtilis 1.108-A was increased by 5.5%.Therefore,the change of gene sequence of xylanase gene xyn A is the reason for the increase of xylanase activity.