| Soil microbial growth is the main place, rich in all the nutrients their growth, is the headquarters of microbial growth. Different nutrient conditions suitable for the growth of different microorganisms. Structure and diversity of soil microbial communities in extreme environments is necessary to understand the soil ecosystem conditions. The degradation of cellulose hydrolysis by cellulase were broken down into simple sugars glucose and cellobiose and oligosaccharides produce ethanol and other bio-energy substances again. The product thus obtained lignin nature of maximum use to prevent the waste of resources. From soil sampling, extraction of DNA, microbial diversity analysis, optimization of cellulase-producing bacteria isolated and screened, mixed fermentation Cellulase enzymatic properties, mixed fermentation conditions, the cellulase gene cloning and research and other aspects of expression to make the theoretical basis for the extreme environments of cellulase-producing strain of a series of studies. Experimental studies are as follows:1 Collection Turpan area of soil, different soil microbial total DNA extraction methods, wherein the resulting high salt and Martin Law Improvement Act of DNA concentration and purity were lower than those of the test improved method and kit method, referred to in the present DNA test improved methods the maximum concentration of 63.6 ng / ul, strong Microbial DNA Isolation kit DNA purity referred to a maximum of A260 / A280 = 1.82. The resulting DNA can be used both soil microbial community structure and diversity of research and analysis.2 Strains isolation and counting the collected samples, determination of physical and chemical properties of soil, the number of bacteria was 3.03*103. Soil water salt for 6.93g/kg, pH value of 7.81, belonging to the saline alkali soil. Using culture technology directly extracted from soil total DNA of bacteria and plate culture bacterial DNA and were constructed by bacterial universal primers were used to amplify 16 S rDNA gene clone library based on, by limiting enzyme of library clones were identified by enzyme digestion analysis, by constructing two bacterial library system phylogenetic tree, and analysis of main plant communities in composition,suggesting that the region of the culturable bacteria diversity was low, main taxa of genus Bacillus and Pseudomonas sp., which may related to the soil matrix and the environment. Enzyme digestion and identification analysis of the total soil bacterial clone library, high polymorphism of restriction enzyme fragment, and the vast majority of which are not to be cultured.3 From soil 11 cellulase producing strain through primary and secondaryscreening Screening. Enzyme activity as measured by a maximum strain C-6, through physiological and biochemical and molecular identification of the strain Bacillus Bacillus tequilensis strain C-6. Fermentation test time was 4d, the enzyme solution of the components were the highest enzyme activity, the enzyme optimum temperature is50 ℃, optimum pH of 7 is neutral enzyme produced by high-temperature enzymes.Provide a theoretical basis for the use of industrial enzymes.4 Cellulase enzyme is a composite induced hydrolysis of the intermediate product will cellulase enzymes from the feedback inhibition effect, the test will produce enzymes and yeast strains mix fermentation, enzymatic properties were studied. The results show that: the yeast optimal access time of 24 h, 4d access yeast fermentation time, β-G reaches maximum activity, when mixed fermentation 5d FPase and CMCase activity reached maximum. Best mixed culture temperature is 32 ℃, the best culture PH 5, with a higher concentration of enzyme activity is 2% to 4% is moderately salt-tolerant enzymes. Glucose, glycerol and xylose on the activity was inhibited as the concentration is increased activity decreased. Some metal ions,surfactants and organic solvents on the enzyme activity inhibition and activation.Fermentation crude enzyme solution of(NH4) 2SO4 salting optimal concentration of80% acetone precipitation optimum ratio enzyme solution: acetone = 1:1.4, the purified enzyme was made SDS-PAGE discontinuous vertical slab electrophoresis,two measured size is 56 KD and 42 KD protein band, measured through the activity,respectively CMCase and β-G.5 Factors are mixed fermentation medium components and culture conditions,test by Plackett-Burman experimental design identify the impact of enzyme production from sodium carboxymethyl cellulose, peptone, yeast, fermentation temperature and the oxygen content of the five factors the main effect factor, and then determine the best combination by steepest ascent experiment factor sync long last means of response surface analysis optimum Box-Behnken central composite design test design Expert 8.0 software get the main effect factors, the most good conditions for enzyme production: sodium carboxymethyl cellulose 1.2g, yeast powder 0.9g, the optimum culture conditions were 32 ℃, 180 r / min shaking fermentation 5 days to get the best activity 368.44 U / ml, while in the non-optimized conditions mixed fermentation enzyme activity was 163.0U / mL, single fermentation activity was127.0U / ml, thus mixed fermentation enzyme activity under optimal fermentation conditions was 2.26 times mixed fermentation unoptimized before a single fermentation of 2.9 times.6 According to the GenBank enzyme digestion glucanase gene primers were designed according to the sequence, by PCR method, cloning which cellulase gene,through the construction of prokaryotic expression vector, transformed into Escherichia coli induced expression in induced H protein expression was the highest,poly acrylamide gel electrophoresis about 56 KD protein through cellulase.Determination of the enzyme activity reached 526.47U/ml, 3.57 times of original strain enzyme activity. |
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