Degradation Of Cellulose By Bacillus Subtilis And Construction Of Bacillus Subtilis Overexpressing Vector Of Cellulase Gene

Cellulose is a natural polymer polysaccharide,only a small portion of cellulose-containing substances in the natural environment have been decomposed and utilized.Cellulase contained in microorganisms can degrade cellulose at room temperature and atmospheric pressure.Therefore,microbial fermentation degradation is a hotspot in cellulose utilization research at present.However,different microorganisms have different degradation characteristics,and their comprehensive degradation ability is often low.There are many unclear aspects in the study of degradation mechanism and process,therefore,at present,it is difficult to enhance the utilization value of microorganisms in cellulose degradation.Met,Lys and Leu,the contents of these three amino acids are generally not high in animal and plant proteins,though they play an important role in husbandary.In the application of animal feed,the demand of these three amino acids is very large,but the industrial synthesis of these three amino acids is not only expensive,furthermore the products also have chiral molecular structure problems,so they are not suitable for direct addition.Probiotics are a group of microorganisms that can effectively improve the composition of microbial flora in animal organisms and produce various substances beneficial to animal organisms.Bacillus subtilis is a kind of probiotics.Its probiotic effect has been reported in many literatures at home and abroad.It is not only included in the list of microbial additives for safe feed by the Ministry of Agriculture of China,but also recognized as one of the microorganisms safe for food and medicine by the FDA of the United States.Adding Bacillus subtilis to livestock feed can significantly enhance the immunity of livestock and poultry,improve the balance of flora and digestibility of organism,improve the absorption rate of feed,and then improve the quality of livestock and poultry.Bacillus subtilis is also an excellent enzyme-producing microorganism,which can secrete various degrading enzymes including cellulase.In this study,first,a strain of Bacillus subtilis was isolated and purified from probiotic products,and identified by PCR.The degradation of cellulose-containing substances by Bacillus subtilis was studied and analyzed,including the detection of its ability to degrade cellulose,hemicellulose and lignin,the detection of corresponding enzyme activity,the change of total protein and the contents of Met,Lys and Leu.Then the gene of endo-beta-1,4-glucanase(CE)of Bacillus subtilis was obtained by genetic engineering.The CE gene was linked to the expression vector p HT43 of Bacillus subtilis,and the recombinant expression vector p HT43-CE was constructed.Then,the natural competent method was used to prepare the competent Bacillus subtilis,and the recombinant vector was transformed into Bacillus subtilis,and the expression of the recombinant vector in the target bacteria was verified.Finally,the enhancement of cellulose degradation ability and cellulase activity of Bacillus subtilis recombinant bacteria were detected.The results showed that Bacillus subtilis had a certain ability to degrade cellulose and hemicellulose,in which cellulose was degraded significantly(21.3%),did not degrade hemicellulose and lignin.The total protein content of fermentation products was higher than that of control group without adding cellulose-rich experimental sample substrates.The contents of Met,Lys and Leu increased by 31.4%,42.2% and 4.9% respectively.The analysis showed that Bacillus subtilis synthesized more proteins and restrictive essential amino acids from cellulose substrates.The transformation of Bacillus subtilis into p HT43-CE was validated.The transformation of p HT43-CE into Bacillus subtilis was successfully carried out and the genetically engineered Bacillus Subtilis expressing transferred cellulase gene was successfully constructed.After induction by IPTG,the expression levels of m RNA and protein of target gene were both significantly higher than that of non-transformed Bacillus subtilis and non-transformed Bacillus subtilis.The ability of cellulose degradation of recombinant bacteria expressing transferred p HT43/CE was tested.The ability of cellulose degradation of recombinant bacteria induced by IPTG was significantly improved compared with that of non-transformed Bacillus subtilis and transformed bacteria not induced by IPTG.Our experiment data provide theoretical and technical basis for the further application of the recombinant bacteria as feed additives.