Research Of Extreme Microbiology Sources And Cloning And Analysis Of Some Novel Genes Encoding Glycoside Hydrolase

The main subject of the thesis is exploitation for beneficial extreme microbiology sources and biochemical and mutational analyses of glycoside hydrolase gene. The thesis includes two parts. The first part is the diversity of culturable halophilic microorganisms and polyphasic taxonomy analysis of the halophilic archaea from hypersaline water in Daying city in Sichuan. The second part is cloning and expression and biochemical analysis of some novel genes encoding glycoside hydrolase from Caldicellulosiruptor besci.The highly promising microbial resources, halophiles have been paid extensive attention in recent years under novel research because of the unique physiological structure, hereditary property and metabolic mechanism of halophilic microorganisms. Exploitation of new halophilic microorganisms is beneficial for revealing the mechanisms of salt tolerance, getting new salt tolerance related genes, improving saline lands and so on. Therefore, the isolation of more unknown halophiles including extremely halophiles archaeon and moderately halophile bacterium is a necessary prerequisite and key for development of microganism resources. The halophilic microorganisms can grow up to the high salt concentrations, such as salt lakes, soda lakes, solar salterns, hypersaline soils, marine environments, etc, and they often have strong impact on the ecosystems in which the thrive. The property of microbial adaptation to high salt environments is mainly relation with enzymes and other bio-active substance. Our country with vast territory and long coastline has large saline lands and a lot of salt lake, which provide convenient condition for the isolation and genealogical classificationof halophilic microorganisms.In this study, we sampled the hypersaline water in Daying city in Sichuan and did research about the diversity of culturable halophilic microorganisms and polyphasic taxonomy analysis of the halophilic archaea. One hundred and twenty bacterial isolates were obtained from the hypersaline pond. And we found that there are12kinds of bacteria in the halophilic water by ARDRA analysis of16S rRNA gene sequences of bacterial isolates from DSL. Of these isolates,47were selected and examined by phylogenetic analysis of16S rRNA gene sequences and by tests of salt tolerance. The phylogenetic analysis placed the47bacterial isolates in Bacillaceae (phylum Firmicutes), Halomonadaceae and Idiomarinaceae (class Gammaproteobacteria). All tested isolates were either halophilic or halotolerant and several were capable of growth in the presence of30%(w/v) NaCl. Moreover we got that all the halophilic Archaea isolates belong to genus Halorubrum and genus Haloferax by tests of salt tolerance, the Utilization of carbon source and phylogenetic analysis of16S rRNA gene sequences. In this paper, we describe the morphological, physiological, chemotaxonomic and genetic analyses of the strain AS2-1which is a new species candidate of the genus Halorubrum to identify AS2-lwas a novel species.Thermophilic cellulases and hemicellulases are of significant interest to the biofuel industry due to their perceived advantages over their mesophilic counterparts. Caldicellulosiruptor bescii which is growing up to83℃and optimally75℃at pH7.1-7.3is Anaerobic, cellulolytic bacterium isolating from hot spring. As one of the highly secreted proteins of C. bescii, the enzyme is likelyto be critical to nutrient acquisition by the bacterium. We collected two gene clusters from Caldicellulosiruptor bescii to do cloning,expression and biochemical and mutational analyses.As result we got four encoding novel themohilic endoglucose genes Cb1953WT, Cb1953TM2, Cb1954TM3and Cb1954TM4, and one new encoding themohilic endomanose gene Cb1953TM1. The optimal temperature of Cb1953WT, Cb1953TM1, Cb1953TM2, Cb1954TM3and Cb1954TM4are90℃,85℃and95℃respectively. Cb1954TM3eanzyme, Cb1953TM1eanzyme and Cb1953TM2eanzyme include a modular protein composed of three carbohydratebinding modules flanked at the N terminus and the C terminus by a glycoside hydrolase family9(GH9) module and a GH5module, respectively. So we named them CbCel9A, CbMan5A and CbCel5A respectively.Then we did biochemical analyses of the enzymes from GH5and found some deference between CbMan5A and CbCel5A. Meanwhile we checked synergistic effects of CbMan5A/Cel5A-TM2with the endoglucanases from C. bescii and the coefficient of synergistic effects is1.2.