Molecular Cloning And Characterization Of Thermophilic Cellulase

With the shortage of human resources growing, the invention of renewable resources isimminent. As the sum of organic substances formed by photosynthesis of green plants,Biomass has lots of characteristic, such as sustainability, low pollution and extensivedistribution and others. Therefor, Taking full advantage of biomass resources, that canprovide an inexhaustible energy treasury. The cellulose can be degraded into fermentablesugar by cellulase, and then converted into alcohol as biofuel. It has extremely importantresearch significance, since it is the most abundant and bargain renewable resources. Thecellulases from thermophiles are of high hydrolytic activity at high temperature and highstability against high temperature, extreme pH and organic solvents. The cellulases fromthermophiles are low energy consumption and cost than traditional cellulases, so it hasimportant research value.Acidothermus cellulolyticus11B is one of the thermophiles in extreme environment,and its genome sequencing has been completed. In the present study, three predictivecellulase genes were chosen as the target. The analysis of amino acid sequence indicate thatthey are members of the glycosyl hydrolase family1,12,48. Based on the above results, weamplified three genes by PCR, ligated it with plasmid pET-20b, and then transformed theminto the host E. coli BL21-（DE3）. According to the general nomenclature rules, nativecellulase was named as AcCel12A.For recombinase AcCel12A, the protein purified route was established. Therecombinant E. coli BL21was cultured in2YT medium, and the induction was carried outby adding IPTG until OD600reached1.0. SDS-PAGE showed that a little of the cellulasewas expressed in the form of soluble protein. The crude enzyme solution was obtainedthrough cell disintegration, thermopreciptated and centrifugation. Then AcCel12A waspurified by Ni-NTA affinity chromatography. The properties were systematicallycharacterized using AcCel12A as the target.The optimum temperature and pH of AcCel12A were70℃and3.5, respectively. Theactivity of AcCel12A was totally inhibited by Zn²⁺（relative remain activity11.27%）, andpartially inhibited by Ni²⁺, Co²⁺and K+（relative remain activity68.91%,76.96%and83.81%）. The relative activity decreased to ca.73%in the presence of EDTA. TritonX-100had no significant effect on activity. The anionic surfactant SDS （0.25%w/v）, could inhibit AcCel12A completely.The properties characterization was of great significance to the research and applicationof the enzyme. In all, the enzyme AcCel12A was highly thermal stable, and had the vitalapplication potential in future.