Crude enzyme extracts were obtained from the digestive glands of Paci®c white shrimp, Litopenaeus vannamei (Boone), reared in oligotrophic well water and eutrophic shrimp pond water to compare digestive enzyme activity between the two groups. Speci®c activities of serine protease, collagenase, amylase, cellulase, lipase and acid phosphatase were signi®cantly higher (P < 0.01) in pond water-reared shrimp (PW shrimp) than in well water rearedshrimp (WW shrimp). For most enzymes assayed, speci®c activity was more than two times higher in PW shrimp, and cellulase activity was over six times higher. In contrast, chitinase activity was signi®cantly higher (P < 0.001) in WW shrimp. Higher speci®c activity of most digestive enzymes in PW shrimp was probably due to natural productivity in the pond water that served as a source of organic substrates, and this increased activity may contribute to the growth-enhancing effect of shrimp pond water.

To alleviate plasmid instability and to prolong the production phase of subtilisin, integrable plasmid and spore mutants are used. Compared with batch-type shake flask cultures, spore mutants' ability to produce subtilisin can be well pronounced in fed-batch and continuous cultures. Hence, the two culture methods make it possible to identify the peculiar characteristics of the spore mutants unobtainable in batch culture. Spore mutants can enhance subtilisin productivity and prolong subtilisin production time in fed-batch culture as well as enable us to use very low dilution rates (<0.1 h(-1)) without losing productivity in continuous culture, thereby improving the conversion yield of the nitrogen source. At 0.05 h(-1) the spollG mutant of Bacillus subtilis DB104 (Deltanpr Deltaapr) (Em(r)) spollG (Bim(r)):: pMK101 (Cm(r)) showed a subtilisin yield about ten times higher than that from wild-type DB104 (Deltanpr Deltaapr)::pMK101 (Cm(r)). (c) 1995 John Wiley & Sons, Inc.