| Cellulose is the most widely distributed, and also the cheapest renewable resource in nature. Cellulase plays a key role in cellulosic biomass transformation. In recent years, research on cellulase becomes a focus all over the world.In this study, several cellulose degrading microorganism included bacteria, fungi, and actinobacteria were isolated from high temperature compost. Three of them, with high activities of cellulase were selected for further study. First, they were identified according to their morphological characteristics, colony characteristics, results of physiological and biochemical experiments, as well as their 16S rDNA sequences. Strain W-5 was identified as Thermobifida fusca, accordingly named as Thermobifida fusca W-5. Strain F-7 was identified as Thermobifida alba, accordingly named as Thermobifida alba F-7. Strain S-1 was identified as Saccharomonospora viridis, accordingly named as Saccharomonospora viridis S-1. Saccharomonospora viridis S-1 showed highest ability in the degradation of xylan, but lowest activity of cellulase among the three strains. Both Strain W-5 and F-7 showed clear holos on CMC plates, which indicated high cellulase activity. Both strains could also grow well in Hutchinson filter medium and the filter scrips could be completely smashed after the fermentation. So, both strains should have complex cellulose degrading enzymes, and would be very useful in the applications.The characteristics of the crude enzyme of Thermobifida fusca W-5 were studied. It showed optimal CMCase activity between pH 5.0 to pH 6.5, and retained more than 50 percent of its activity between pH 4 to pH 8.5. It showed optimal CMCase activity at 70℃, and retained more than 50 percent of its activity between 55℃to 85 "C. The CMCase activity of the crude enzyme did not change much after incubation at 70℃for 1 hour, which indicated the very good characteristic of the crude enzyme in heat stability. The components in the media that can influence the enzyme production were also investigated. The nature carbon source such as corncob residue and crop straw powder was its optimal carbon source, and yeast extract, urea and peptone was its optimal nitrogen source for enzyme production. Two cellulase genes, cel9b and cel5b, were cloned from Thermobifida fusca W-5 by PCR and sequenced. Then they were subcloned into vector pET22b(+) and the recombinant plasmids, pET22b-cel9b and pET22b-cel5b, were transformed to E. coli Rosetta(DE3)pLysS, respectively. The expressed Cel9B is about 100 kDa, with optimal reaction conditions at 74℃and pH 5.0. After incubation at 70℃for 1 hour, the CMCase activity of Cel9B still remained as same as the untreated. While the expressed Cel5B is about 65 kD, with optimal reaction conditions at 80℃and pH 5.0, and showed more than 70 percent of its activity between pH 4.5 to pH 9.0.The gene cel9b is composed of one catalytic domain and two cellulose binding modules, which locate at the N-terminal and C-terminal, respectively. The gene cel5b is composed of one catalytic domain and one cellulose binding module at its C-terminal. According to the structure characteristic of gene cel9b, it was truncated to study the function of each domains. Three recombinant plasmids, pET22b-cel9b-n-cbd-cd, pET22b-cel9b-cd and pET22b-cel9b-cd-c-cbm, were constructed and expressed in E. coli Rosetta(DE3)pLysS, respectively. The N-terminal CBM played an important role to its activity, and Cel9B-N-CBD-CD showed the highest cellulase activity among all mutant enzymes and wild type Cel9B. Both absence of CBM did not change its activity, because Cel9B-CD showed the same activity as that of wild type Cel9B. All mutant enzymes showed a little decreased optimal temperature and unchanged optimal pH.Site-directed mutagenesis protocol was carried out to study the mechanism of thermophily of Cel9B and alkali resistance of Cel5B, respectively. According to the alignment of amino acids sequences of related cellulases and homology modeling, some key amino acid residues were selected for further study. The mutant C715R and C464A of Cel9B-CD showed lower optimum reaction temperature than wild type Cel9B-CD, which indicated that the disulfur bonds played more important role than other interactions between residues in the stability of protein. The optimum pH of mutant H206S and K167E of Cel5B alkaline shifted from pH 5.0 to pH 5.5, which indicated that the characterists of amino acid residues around the active center could influence its property, such as the charged character, the ionization state and the hydrogen-bonding net, etc. |
PDF Full Text Request