Expression And Characterization Of Three Cellulases From Bacillus Licheniformis ATCC 14580

As the main component of plant cell wall,cellulose is the most widely distributed abundant renewable resources on the earth,plant produced up to one hundred million tons cellulose through photosynthesis every year.With the petrochemical energy increasingly scarce and agricultural waste cellulose production growing background,finding a way to use cellulose efficiently in the cell wall is very significant for our country's economic sustainable development.Rely on biological produce cellulose enzyme degradation of cellulose had became a trend of degradation technology.According to the similarity of structure of amino acid sequences,cellulose enzyme was divided into different glycoside hydrolase in the family.According to the different sources,microbes was divided into sources of fungi and bacteria.Due to fungal cellulase system complete and high output,people focused on fungi cellulase for a long time;But research which get the attention of people had shown that bacteria cellulose enzyme resource was rich,and tend to be suitable for industrial application characteristics in recent years.With the development of genome sequencing technology,more and more bacterial genome was determined and resolved these forecasts genetic information for the development of novel enzymes provide a broad resource.Bacillus licheniformis ATCC14580 means one strain genome had been sequenced and analyzed strain.It was found that its genome contained a large number of genes related to enzymatic degradation of cellulose,so in this thesis the cellulase genes of bacteria were studied using Molecular cloning and expression techniques and the following results were obtained.1.According to the prediction of genomic information,three cellulose enzyme genes derived from Bacillus licheniformis ATCC 14580 by PCR amplification method was belonging to glycoside hydrolases GH5,GH9 and GH12 family,named blcel5 A,blcel9A and blcel12 A.2.blcel5 A fragment consist of a conserved sequence GH5 family and a CBM_X2domain configuration,with the length of 1581 bp,encoding protein relative molecular mass of about 60 kDa.The gene cloning to pET-22 b carrier and Escherichia coli Rosetta which host to expression,inducted by IPTG and nickel column affinity chromatography,BlCel5 A recombinant protein was obtained.The optimum reaction pH of recombinant enzyme was pH5,pH stability was better.The optimum reaction temperature of recombinant enzyme was 40 ?,temperature stability was better.Enzyme activity was reduced to 65.54% and 79.57% with Mn²⁺and Cu²⁺of 1 mM concentration.1%TritonX-100 made BlCel5 A recombinant activity up to 114.03%.1 M urea made recombinant activity reduced to 77.6%.0.1% SDS made enzymes completely inactivated.The Km and Vmax of BlCel5 A on CMCare were 6.75 mg/mL and 125 ?mol/?min?g?,kcat value was 2403.85 /s.Enzyme activity of BlCel5 A on CMC was 102.84 U/mg and on xylan was 3.07 U/mg.3.blcel9 A fragmentsis consisit of a conservative sequence and a CBM₃ GH9 family structure domain,with the length of 1854 bp,encoding protein relative molecular mass of about 70 kDa.The gene cloning to pET-22 b carrier and Escherichia coli Rosetta which host to expression,inducted by IPTG and nickel column affinity chromatography,BlCel9 A recombinant protein was obtained.The optimum reaction pH of recombinant enzyme was pH6,pH stability was better.The optimum reaction temperature of recombinant enzyme was 60 ?,temperature stability was better.Activity of recombinant enzyme rose to113.33% with 1 mM Ca²⁺,dropped to 63.31% and 75.73% with 1 mM Mn²⁺ and 1 m M Fe3+.Enzyme activity increased to more than 110% with 1% of Tween80,TritonX-100,isopropyl alcohol and n-hexane,decreased to 77.48% with 1% of glycerol.0.1% SDS made enzymes completely inactivated.The Km and Vmax of BlCel9 A on CMC were 6.75mg/m L and 125 ?mol/?min?mg?,kcat value was 2403.85 /s.Activity on CMC was 111.36U/mg,and activity on xylan,seaweed polysaccharides and straw were 5.10 U/mg,1.72U/mg and 6.9 U/mg,respectively.4.blcel12 A segmentonly contained a GH12 family conservative sequence,without CBM domain structure.The length of gene was 639 bp,and the mass of protein was about25 kDa.The gene cloning to pET-22 b carrier and Escherichia coli Rosetta which host to expression,inducted by IPTG and nickel column affinity chromatography,BlCel12 A recombinant protein was obtained.The optimum reaction pH of recombinant enzyme was pH4,pH stability was better.The optimum reaction temperature of recombinant enzyme was 60 ?,temperature stability was better.Activity of recombinant enzyme rose to117.07% and 117.07% with 1 mM Fe²⁺ and Ni+.Activity dropped to 57.77%,45.53% and57.77% with Fe3+,Mn²⁺and Cu²⁺respectively.1% of Tween80 could improve the enzyme activity of recombinant enzymes to 169.36%.Activity dropped to 23.72%,23.72% and34.25% with Ethanol,acetone and isopropanol.Activity dropped to 29.78% with 1 M urea.0.1% SDS made enzymes completely inactivated.The Km and Vmax of BlCel12 A on CMC were 4.464 mg/mL and 31.65 ?mol/?min?mg?,and kcat value was 597.17 /s.Enzyme activity of BlCel9 A on CMC was 25.15 U/mg,and 1.93 U/mg and 10.19 U/mg on xylan and seaweed polysaccharide.In this paper,three bacterial origin endoglucanase were directly cloned by Genome sequencing information of Bacillus licheniformis ATCC 14580 strain,providing a newsource for theoretical research and practical application of bacterial cellulase enzymes.At the same time,the construction of mature expression system and the characterization of the enzyme,laid the foundation for the follow-up study of the structure and function of cellulase experimental basis.