Cloning,Expression,Synergy And Application Of Cellulases From Celluloytic Bacterium B.subtilis 1AJ3

In today’s society where the demand for new energy sources is growing,especially the study of alternative energy sources for petroleum energy is imminent.As a new type of green energy,bioethanol uses natural biomass raw materials such as agricultural and side crop wastes.Generates bioethanol through the process of saccharification and fermentation,which can be used as a broader energy source.The saccharification process is a key step in the bioenergy conversion process.How to convert cellulose efficiently,economically and environmentally into reducing sugars has always been a problem that needs to be solved.Biological method is based on microorganisms（fungi or bacteria）and enzymes.Bacteria have faster growth than fungi,as well as a widly sources,strong adaptability and easy gene manipulation,etc.which attracted researches attention in recent years.But degradation system of bacteria is not yet complete studied,and more bacteria and cellulase systems need further study.In this study,rotten wood from Qinling Mountains was used as an isolation material for bacterial strains,to explore the types of cellulose-degrading bacteria.After that,a potential bacterium was selected out to explore its degradation ability of biomass cellulose.Three different species cellulases were obtained from the strain by cloning and heterologous expression,to study its enzymatic properties,degradation mechanism and the synergy effect.The specific research content and main results are as follows.（1）Isolation of celluloytic bacteria from the rotten wood of Qinling Mountains.The 55strains of bacteria screened from the rotten wood of the Qinling Mountains via the Congo-red plate method are mainly Bacillus subtilis,as well as included the following species:Bacillus sp.,Pseudomonas aeruginosa,Bacillus licheniformis,Bacillus methylotrophicus,and Bacillus megaterium.Three strains were selected out based on the accumulated reducing sugar content of the cultural medium and the cellulase activity:B.subtilis 1AJ3,B.methylotrophicus 1EJ7,and B.subtilis 3BJ4,which had the degradation ability of unpretreated lignocellulose（wheat straw,corn stover and switchgrass）.（2）Bacterium B.subtilis 1AJ3 has the ability to switchgrass lignocellulose.The strain B.subtilis 1AJ3 was applied to switchgrass,and the degradation rates of cellulose,xylan and acid-insoluble lignin within 5 days at 37℃,150 r/min were 16.13%,14.24%and 13.91%,respectively.The content of aromatic compounds was determined by GC-MS to assist in demonstrating its ability to degrade lignin.Scanning electron microscopy observed structural changes on the surface of switchgrass,proving that the strain degraded switchgrass.Strain1AJ3 has the ability of acid and heat resistance.Moreover,acid-heat conditions are benefit to the degradation of biomass raw materials such as switchgrass,corn cob and coffee grounds,indicating that the strain has industrial application potential.（3）Recombinant endocellulase was obtain from the bacterium B.subtilis 1AJ3.Via cloning and heterologous expression,the recombinant cellulase Cel-A with a His-tagged was obtained.The enzyme contains a catalytic domain of the GH5 family and a carbohydrate binding domain of the CBM3 family,and showed different enzymatic properties when compared with single domain.Cel-A can obtain the highest enzyme activity under the conditions of p H 4.5 and 50℃.Metal ion Co²⁺can promote enzyme activity.Cel-A can efficiently degrade substrates of CMC-Na and barley glucan.It mainly acts onβ-1,3-1,4glycosidic bonds,but has no effect onα-glycosidic bonds.（4）Recombinant exocellulase Cbh-A was obtained from B.subtilis 1AJ3 through cloning and heterologous expression.The enzyme contains two domains:the catalytic domain Flg J and the carbohydrate-binding domain SH3＿8.The Flg J domain was first studied as an exocellulase,and the enzymatic properties were studied using Avicel as a substrate.The optimum temperature and p H of Cbh-A were 50℃and 6.4,respectively.Preheating for 4 hours at 50℃can maximize Cbh-A enzyme activity and maintain stability.Mn²⁺can enhance the activity of the recombinant enzyme Cbh-A,5 m M or 10 m M of Cu²⁺and the chemical reagent EDTA can inhibit the enzyme activity.Cbh-A can effectively hydrolyzeβ-1,3-1,4/β-1,3/β-1,4 glucan bonds andα-1,4 glucan bonds,but not forα-1,6 glucan bonds.（5）Recombinantβ-glucosidase Bgl-A was obtained from strain B.subtilis 1AJ3,which belongs to the GH16 family.Bgl-A has the maximam enzyme activity at 50℃and p H 8.6.The enzyme has a wide range of temperature and p H stability,and its enzyme activity can reach more than 80%in the temperature range of 0-50℃and p H 6.4-9.0.The metal ions and SDS had little effection on it,while EDTA inhibits the enzyme under high concentration conditions.It has no effect on the substrate of 4-nitrophenyl-β-D-glucopyranoside.（6）The three cellulases from bacteria B.subtilis 1AJ3 have a synergistic effect.Different types of cellulases（endocellulase Cel-A,exocellulase Cbh-A,andβ-glucosidase Bgl-A）from bacterium 1AJ3,have good synergistic effects in simple cellulose substrate（CMC-Na,Avicel,and filter paper）.Further more,it is speculated that exocellulase can act on the crystalline region of cellulose to loosen the structure of cellulose,providing more sites for endocellulase hydrolysis.β-glucosidase present similar synergy effect with exocellulase in different cellulose substrates,and the hydrolysis rate ofβ-glucosidase is limited by the activity of exocellulase.Switchgrass and corn cob are good raw materials for bioethanol because that both have high saccharification rate under cellulase hydrolysis.Three enzymes showed good synergistic effects in corn straw,wheat straw,ricehusk,sugarcane straw and coffee grounds.