Optimization Of Culture Medium For Genetic Engineering Bacteria To Produce Neutral Cellulase And Development Of The Enzyme Product

The cellulose is the largest carbohydrates in the world, at present in the nature only some small parts have been used. The production of cellulase is transformed into human need energy, food, chemical raw materials, to solve the problem of energy crisis, the food shortage, environment pollution has the significant practical significance.The laboratory selected a high yield from the neutral cellulose in Bacillus subtitles, produce cloned neutral cellulose (endo-glucanase) gene (Genbank accession number: DQ782954), and Bacillus megaterium be expressed successfully, build a high-yield and stable production of neutral cellulose engineering bacteria Bacillus megaterium WH320-pHIS1525-G7.In order to WH320～pHIS1525-G7 engineered bacteria used in industrial production, the study of the engineering strain through medium optimization and fermentation conditions in 5 L fermentor study, and the crude enzyme product research process for exploration, the results are as follows:1, Through carries on the optimization to the WH320～pHIS1525-G7 project fungus's culture medium, had determined take the wheat bran as the carbon source, the corn thick liquid is suitable for the nitrogen source for the industrial production culture medium (wheat bran 2.5%, corn thick liquid 1.5%, KH2PO4 0.75%, MgSO4 0.04%, NaCl 0.25%). This culture medium and the original LB-CMC culture medium compared to, its enzyme enhanced 6.7% exactly, in this culture medium the essential component wheat bran, the corn thick liquid raw material origin is widespread, the price is cheap, is suitable in the industrialization production.2, In the optimized medium, based on on WH320～pHIS1525-G7 engineered bacteria in 5 L fermentation tank fermentation conditions to determine the conditions for fermentation were:temperature 37℃, pH 7.0, tank pressure 0.03-0.05 Mpa, speed 600 r/min, liquid volume of 3 L, inoculum 10%, after 4 h culture 0.25 g/L/h flow rate of xylose fed 10 h,16 h after the fermentation, dissolved oxygen by way of constant fed-batch 8 h, a total of up feeding 320 mL. Fermentation process control dissolved oxygen≥30%. Frozen after the end of fermentation broth by centrifugation and measured activity in the supernatant, the activity reached 3846.48 U/mL, was 4.1 times the shaking flask.3, This study established a crude enzyme product of cellulose production process: fermentation, centrifuged to remove bacteria obtained crude enzyme solution, and then add a final concentration of mannitol was 15 mg/mL, with fine rice bran and starch (9:1) as the vector, and the fermentation broth 1:1 (W:V) mixed granulation,40℃constant temperature drying to 5% water, then spray carrier concentration of 10% NaCl,40℃constant temperature drying to 5% water, with carrier concentration 0.5% of chitosan-coated, air heat treatment at 80℃0.5 h, the measured residual activity of 95.4% was obtained for the 5～8 mm diameter, the yellow crude enzyme particles.