Oh Y, Shih I, Tzeng Y, Wang S
Department of Food Engineering, Da-Yeh University, Chang-Hwa, Taiwan
Enzyme Microb Technol. 2000 Jul 1;27(1-2):3-10. doi: 10.1016/s0141-0229(99)00172-6.
In addition to chitinase/lysozyme, Pseudomonas aeruginosa K-187 also produced a protease useful for the deproteinization of shrimp and crab shell wastes. The optimal culture conditions for P. aeruginosa K-187 to attain the highest protease activity were investigated and discussed. The highest protease activity was as high as 21.2 U/ml, 10-fold that (2.2 U/ml) obtained prior to optimization. The protease of P. aeruginosa K-187, produced under the optimal culture conditions, was tested for crustacean waste deproteinization. The percent of protein removal for shrimp and crab shell powder (SCSP) after 7-day incubation was 72%, while that of natural shrimp shell (NSS) and acid-treated SCSP was 78% and 45%, respectively. In contrast, with the protease produced under pre-optimization conditions, the percent of protein removal for SCSP, NSS, and acid-treated SCSP was 48%, 55%, and 40%, respectively. For comparison, three other protease-producing microbes were tested for crustacean waste deproteinization. However, they were shown to be less efficient in deproteinization than P. aeruginosa K-187. The crude protease produced by P. aeruginosa K-187 can be covalently immobilized on a reversibly soluble polymeric support (hydroxypropyl methycellulose acetate succinate). The immobilized enzyme was soluble above pH 5.5 but insoluble below pH 4.5. Immobilization efficiency was 82%. The immobilized enzyme was stable between pH 6 and 9 and at temperatures below 60 degrees C. The optimum pH and temperature for the immobilized enzyme was pH 8 and 50 degrees C. The half-life of the immobilized enzyme was 12 days, longer than that of free protease (8 days). The utilization of the immobilized enzyme for the deproteinization of SCSP has resulted in a 67% protein removal. By contrast, SCSP protein removal by using free enzymes was 72%. The protease was further purified and characterized. The purification steps included ammonium sulfate precipitation, DEAE-Sepharose CL-6B ion-exchange chromatography, and Sephacryl S-200 gel-permeation chromatography. The enzyme had a molecular weight estimated to be 58.8 kDa by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was active from pH 7 to 9 and its optimal pH was 8.
除了几丁质酶/溶菌酶外，铜绿假单胞菌K-187还产生了一种蛋白酶，可用于虾蟹壳废料的脱蛋白。研究并讨论了铜绿假单胞菌K-187达到最高蛋白酶活性的最佳培养条件。最高蛋白酶活性高达21.2 U/ml，是优化前（2.2 U/ml）的10倍。对在最佳培养条件下产生的铜绿假单胞菌K-187蛋白酶进行了甲壳类废料脱蛋白测试。孵育7天后，虾蟹壳粉（SCSP）的蛋白质去除率为72%，而天然虾壳（NSS）和酸处理后的SCSP的蛋白质去除率分别为78%和45%。相比之下，对于在优化前条件下产生的蛋白酶，SCSP、NSS和酸处理后的SCSP的蛋白质去除率分别为48%、55%和40%。为作比较，测试了其他三种产蛋白酶的微生物对甲壳类废料的脱蛋白效果。然而，结果表明它们在脱蛋白方面不如铜绿假单胞菌K-187高效。铜绿假单胞菌K-187产生的粗蛋白酶可以共价固定在一种可逆溶性聚合物载体（羟丙基甲基纤维素乙酸琥珀酸酯）上。固定化酶在pH 5.5以上可溶，在pH 4.5以下不溶。固定化效率为82%。固定化酶在pH 6至9之间以及60摄氏度以下的温度下稳定。固定化酶的最佳pH和温度分别为pH 8和50摄氏度。固定化酶的半衰期为12天，比游离蛋白酶的半衰期（8天）更长。使用固定化酶对SCSP进行脱蛋白，蛋白质去除率为67%。相比之下，使用游离酶时SCSP的蛋白质去除率为72%。对该蛋白酶进行了进一步纯化和表征。纯化步骤包括硫酸铵沉淀、DEAE-琼脂糖CL-6B离子交换色谱和Sephacryl S-200凝胶渗透色谱。使用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳估计该酶的分子量为58.8 kDa。纯化后的酶在pH 7至9有活性，其最佳pH为8。
