| Elastase is a kind of protease that can specially degrade elastin. Elastase comes from animal pancreas and also from microbe culture. Elastase production by fermentation was more promising when its activity beyond 155 U/mL. For its special enzymatic characteristics, it has broad applications in medical therapy, in food processing, in functional cosmetics and so on.In this study, using high yield elastase-producing strain Bacillus sp. EL 31410 which preserved by our lab and its product—elastase, the effect factors of cultivation on batch fermentation course in 5 L bioactor were fully discussed. Pilot scale was carried out in 70 L bioactor, elastase was purified by serials purification steps and its characteristics were determined. Complex cryoprotectant was screened and optimized by orthogonal experimental design. Storage period of elastase preparations was evaluated by accelerated storage test. Aqueous two- phase system was researched on elastase purification from broth. Ability of elastase hydrolysis on silk fibroin was investigated. The results were as follows:1) Batch cultivation process was studied in 5 L bioactor. Based on characteristics of cultivation course and analysis to dynamic data, the optimum cultivation strategy was suggested. The elastase produced by Bacillus sp. EL31410 was affected by temperature significantly. Cultivation temperature of 37°C was more suitable to cell growth at earlier cultivation stage while of 30 °C was benefit to get higher elastase activity, when compared to the results of batch cultivation at four kinds of cultivation mode, the conclusion was drawn that cultivation at 30 °C was appropriate for elastase produce. pH is one of important factors which affected fermentation course. It was discussed that pH-stat batch cultivation in 5 L bioactor was controlled at constant pH 6.0, 6.5, 7.0, 7.5, respectively. Cell growth was better at fermentation broth pH controlling at stable pH 6.5 or 7.0 than at others. However, the elastase activity was improved when cultivation at single pH 7.0. Analysis to the cultivation courses and dynamic data, two-stage pH- controlled strategy was adopted that broth pH wascontrolled at constant 6.5 from the beginning of cultivation to 24 h and was shifted to constant 7.0 form 24 h to the end. The two-stage pH strategy was carried out and the result was satisfied that the optimum elastase activity was achieved 699 U/mL when cultivated 48 h. When stirred speed was increased from 300 rpm to 400 rpm and the other cultivation condition was operated at 30 °C with two-stage pH controlled strategy in 5 L fermentor, cell grew well and elastase produced well, too. And the cultivation time to reach maximal elastaseactivity advanced 6 h.2) Pilot test was carried out in 70 L bioactor. Cultivation parameters were adjustedaccording to those in 5 L except temperature and pH. After cultivation 33 h, the maximal elastase activity was 645 U/mL which close to that of in 5 L fermentor. A simple model was proposed using Logistic equation for growth, the Luedeking-Piret equation for elastase production and Luedeking-Piret-like equation for glucose consumption. The models were as follows:3) The broth collected after cultivation for hours was centrifuged at 3000 rpm and the upper was salted out using (NH4)2SO4. Purified elastase was obtained after the two steps chromatography purification with DEAE Saphadex A 50 and Saphadex G 100 later. One clear band was shown by SDS-PAGE and the relative molecular weight was 31820 Da. Some enzymatic characteristics of purified elastase were evaluated: the optimum temperature for elastase hydrolysis action on elastin was 50 °C and it was very stable at 30-50 °C. The optimum pH for elastase hydrolysis action on elastin was pH 7.4 and pH 8.2 in boric acid buffer and in Tris-HCl buffer, respectively. Elastase behaved stable at pH 7-8. Some ions in low concentration such as Ca2+ and K+ showed active action on elastase while others such as inactive elastase activity to some degree. Elastase catalyzed hydrolysis action on solubleelastin more str... |
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