| Presently , waste and pollution of high-fiber roughage resource arising from seriously. The efficiency of roughage utilization is still at low level. One of the effective approaches to improve the efficiency of roughage utilization is to search the bacterium which can degrade the cellulose and then be able to get lots of cellulase. This research was designed to find a hot-resistant cellulose degrading bacterium in geothermal resources of YangIing·ShaanXi, then manipulate the cellulase gene through gene engineering technology and optimize cultivation conditions, to maximize the production of cellulase.The main results of this research are:1. One strain of bacterium which can degrade cellulose was screened out from tested water using Congo red stain. A single colony of this strain around which there is a clear halo was obtained after repeated selection. Cellulase activity was tested in fermentation liquid by the DNS method. The bacterium was gram-positive. The conservative sequence 16SrDNA was amplified through PCR, then sequenced and analyzed. The homology of the conservative sequence was 100% same with that of other Bacillus subtilis'. According to the morphology of the bacterium, it was named as B. subtilis DR2. The optimized conditions were established by the combining experiments of the 6 factors,each of them can affect the growth of bacterium and the product of cellulase, and was tested by 6 unattached single factor experiments. Screening test results for optimum growth condition of the bacterium are pH6.5 and temperature 37-41℃; the optimum condition for enzyme production was 37-41℃, pH was 6-7 and 36h cultivation; Under the conditions when CMC-Na was used as carbon source and typetone as nitrogen source.3. The cellulase was obtained by cultivating the bacterium in large quantity and properties of this enzyme were analyzed. The results are: the cellulase of B. subtilis DR is an endo-glucanase, belonging to hot-resistant cellulase. The optimum temperature of the reaction with substrate was 50℃, the optimum pH was 6.5. The enzyme is tolerant to high temperature: at 70℃for 30min, 70% of cellulase activities still remained.4. A DNA fragment (about 1.5Kb) was amplified from B. subtilis DR genome DNA by specific primers, which was designed according to the reported cellulase gene of Bacillus subtilis in GenBank. This fragment was ligated to vector and sequenced. The homology of this gene's partial sequences and other reported cellulase gene reached 97% through blast online, and the whole sequence of B. subtilis DR cellulase gene has never been reported in GenBank. This cellulase gene consisted of 1524 base pairs and was an integral open reading frame. The start codon was ATG and the terminal codon was TAG, coding 508 amino acids continuously.5. Two restriction enzyme sites BamHI and NotI were introduced in the 5'end of the second primers. The cellulase gene was amplified by PCR, the product of PCR was ligated to pET-28a expression system and transformed into E. coli, and then the cellulase gene was expressed in E. coli. When the E. coli was cultivated at 37℃, induced for 5~6 hours, the cellulase activity of E. coli was 0.9411U/ml, which is three times higher than that of wide type bacterium. The cellulase protein was nearly 56kD tested by using SDS-PAGE, which is in accordance with the result obtained previously.
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