Protease produced by Pseudomonas aeruginosa K-187 and its application in the deproteinization of shrimp and crab shell wastes

In addition to chitinase/lysozyme, Pseudomonas aeruginosa K-187 also produc ed a protease useful for the deproteinization of shrimp and crab shell wast es. The optimal culture conditions for P. aeruginosa K-187 to attain the hi ghest protease activity were investigated and discussed. The highest protea se activity was as high as 21.2 U/ml, 10-fold that (2.2 U/ml) obtained prio r to optimization. The protease of P. aeruginosa K-187, produced under the optimal culture conditions, was tested for crustacean waste deproteinizatio n. The percent of protein removal for shrimp and crab shell powder (SCSP) a fter 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 pro tease produced under pre-optimization conditions, the percent of protein re moval for SCSP, NSS, and acid-treated SCSP was 48%, 55%, and 40%, respectiv ely. For comparison, three other protease-producing microbes were tested fo r crustacean waste deproteinization. However, they were shown to be less ef ficient in deproteinization than P. aeruginosa K-187. The crude protease pr oduced 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 bet ween 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 protea se (8 days). The utilization of the immobilized enzyme for the deproteiniza tion of SCSP has resulted in a 67% protein removal. By contrast, SCSP prote in removal by using free enzymes was 72%. The protease was further purified and characterized. The purification steps included ammonium sulfate precip itation, DEAE-Sepharose CL-GB ion-exchange chromatography, and Sephacryl S- 200 gel-permeation chromatography. The enzyme had a molecular weight estima ted to be 58.8 kDa by using sodium dodecyl sulfate-polyacrylamide gel elect rophoresis. The purified enzyme was active from pH 7 to 9 and its optimal p H was 8. (C) 2000 Elsevier Science Inc. All rights reserved.