碩士 / 大葉大學 / 食品工程研究所 / 87 / The fermentation broth of the protease bacterium Bacillus subtilis Y-108 , was isolated from soil in the northern Taiwan. Under the optimized culture condition that the culture was shaken in 250 mL Erlenmeyer flasks with 100 mL medium （7％shrimp and crab shell powder（SCSP）, 0.1％K2HPO4, 0.05％MgSO4, 1.0％arabinose, 1.5％NaNO3 , and 1.5％CaCl2 , pH 6.0 adjusted by phosphate buffer solution）at 30℃for 3 days, the protease activity of Bacillus subtilis Y-108 was as high as 8.64U/mL. It was about fifty fold of the activity （0.17U/mL）of Bacillus subtilis Y-108 under the unoptimized basal culture condition.
The protease of Bacillus subtilis Y-108 in the optimum produce condition was used to test as protein cleaner. In liquid state culture, the efficiency of deproteinization of NSS（natural shrimp shell）、crab、lobster and squid was 88％、73%、84% and 75%, then after acid treated the NSS、crab and lobster was up to 76％、62% and 59%. In comparison with Pseudomonas maltophilia and Bacillus subtilis（CCRC10029、11602、16048）, the efficiency of protein cleaning of marine waste for Bacillus subtilis Y-108 are the highest one.
The protease of Bacillus subtilis Y-108, produced under the optimized culture condition, the first step was precipitated and dialyzed by using ammonium sulfate. The further purification and separation procedures of the protease were processed by the use of DEAE-Sephacel ionic exchange chromatography、Sephacryl S-200 gel permeation chromatography and chromatofocusing. Purification was 37-fold with the crude enzyme solution. After purification and separation, the activity of the protease was still stable at 40℃and pH 6∼9, while the optimal temperature and pH for the enzyme reaction were at 50℃and pH 8, respectively. By SDS-PAGE electrophoresis and gel permeation chromatography, the molecular weight of the protease was identified as 44KDa.
The protease produced from Bacillus subtilis Y-108 can covalently linked to the supporting AS-L（hydroxypropyl methyl cellulose acetate succinate）polymers. Therefore, the crude solution of protease was used for enzyme immobilization with the AS-L polymer carriers. The efficiency of enzyme activity immobilization was 81％（Protease 80%、Ficin 78%、Bromelain 49%、Papain 34%）, and the protein immobilization was 59%（Protease 7%、Ficin 53%、Bromelain 32%、Papain 42%）. The pH- and heat-stability ranges of the immobilized enzyme were at pH 6∼7 and 50℃, respectively. The optimal enzyme reaction for the immobilized protease activity were under 50℃and pH 8.
| Identifer | oai:union.ndltd.org:TW/087DYU00250021 |
| Date | January 1999 |
| Creators | Yang J. G., 楊政國 |
| Contributors | Wang San-Lang, 王三郎 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 135 |
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