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Purification and characterization of trypsin from an entomopathogen,Nomuraea rileyi NRRL 13755

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Abstract

Nomuraea rileyi isolate NRRL-13755 produced a large amount of trypsin enzyme when cultured on basal salt medium containing 1% (w/v) gelatin. The trypsin was purified nearly 60-fold, with a recovery of about 13% of the initial activity from the culture supernatant. This protease exhibited a remarkably high specific activity of nearly 370,000 IU/mg protein. The native molecular weight was estimated by gel permeation chromatography to be 30 kDa, and the subunit molecular weight was determined to be about 30 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The pH and temperature optima were determined to be 8.5 and 35°C, respectively. With a relative trypsin activity of 100%, this purified preparation showed about 10% chymoelastase and nearly 50% chymotrypsin activity. Metal-chelating agents such as EDTA and EGTA at 2mm inhibited the enzyme activity by 40%, whereas N-carbobenzoxy-glycyl-l-phenylalaninamide (CBZ-gly-phe-NH2) (2mm) and DTT (2mm) had no effect on activity. Trypsin inhibitor from turkey egg-white at 100 μg/ml strongly inhibited the enzyme activity.

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Author notes

  1. Subhash C. Gupta

    Present address: Animal and Plant Health Inspection Service, USDA, 6505 Belcrest Road, 20782, Hyattsville, MD, USA

Authors and Affiliations

  1. Biopolymer Research Unit, National Center for Agricultural Utilization Research, U.S. Department of Agriculture, Agricultural Research Service, 61604, Peoria, Illinois, USA

    Subhash C. Gupta &  Timothy D. Leathers

  2. Biological Control of Insects Research Laboratory, USDA, ARS, Columbia, Missouri, USA

    Galal N. El-Sayed & Carlo M. Ignoffo

Authors
  1. Subhash C. Gupta
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  2. Timothy D. Leathers
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  3. Galal N. El-Sayed
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  4. Carlo M. Ignoffo
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Gupta, S.C., Leathers, T.D., El-Sayed, G.N. et al. Purification and characterization of trypsin from an entomopathogen,Nomuraea rileyi NRRL 13755. Current Microbiology 27, 103–107 (1993). https://doi.org/10.1007/BF01570866

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