Isolation Of Cellulase Bacteria From Apriona Germari And Expression Of Endoglucanase Gene In Lactobacillus

In recent years,the construction of beneficial and safe cellulase-secreting Lactobacillus recombinant bacteria was expected,and it was expected to be used as a feed microbial additive,which can not only maintain animal intestinal health but also improve ruminant roughage utilization.However,the gene source of efficient cellulase secretion had always been one of the research difficulties and hot spots.The purpose of this test was to obtain cellulose-degrading bacteria from the Apriona germari,clone the cellulase gene,express it in E.coli,which laid a foundation for the later construction of engineering bacteria that efficiently decompose cellulose.The recombinant lactobacillus containing cellulase gene was constructed to study its degradation effect on straw plant feed.The main findings are as follows:1. Three strains of cellulose producing bacteria（C1,C2,C3）were isolated fromApriona germari using cellulase selection medium,and were identified as Bacillus thuringiensis and Bacillus subtilis.The results showed that from the isolated strains,the cellulase activity of Bacillus subtilis was the highest,and the optimum fermentation temperature and p H of the strain was 42℃ and 5.5.The optimum temperature and p H of enzymatic reaction was 60℃ and 5.0,under this condition,the cellulase activity reached2.205 U/m L.2. The endo-β-1,4-glucanase gene Nqm andβ-glucosidase gene Bq T were isolated and exogenously expressed in Escherichia coli to construct cellulase producing recombinant strains.Gene cloning results indicated that Bacillus subtilis contained two cellulase genes Bq T and Nqm.Recombinant expression of pro Bq T showed no cellulase activity,whilst in pro Nqm cultured at 70℃,p H 5.5,cellulase activity reached 2.366 U/m L,and at 50℃ and p H 5.5β-glucosidase activity reached 1.428 U/m L.The Bq T and Nqm gene products were 27k Da and 54 k Da,respectively and a fusion protein of～35 k Da was identified.At 60℃ and p H 6.5,the cellulase activity of the fusion product reached 2.517 U/m L.3. This study amplified a endo-β-1,4-glucanase gene Nqm from the Bacillus subtilis which was isolated from the Apriona germari in vivo.Through the restriction enzyme digestion and connection method,the promoter element(P₃₂),signal peptide(SP_lp0373)and cellulase gene are connected with Lactobacillus expression vector p LEM-415.The optimum enzyme producing time and temperature of recombinant lactobacillus were 24 h and 37℃.Under the optimum enzyme producing conditions the activity of CMCase was 2.06 U/m L.The optimum temperature and p H of enzymatic reaction is 60℃,6.0 respectively.Under the inhibitory effects.4. The fermentation test showed that engineering bacteria has certain effect on cornstraw,alfalfa hay and wheat straw,and the content of neutral detergent fiber（acid detergent fiber）decreased by 10.42%（21.12%）,4.99%（7.85%）,4.57%（9.53%）,respectively.Taken together,this study indicates that Bq T interacts with the Nqm gene to produce a protein with high cellulase activity and a smaller molecular weight.The artificial recombinant Lactobacillus based on the Nqm gene has certain degradation ability to common straw feed.