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Production and characterization of recombinant cyprosin B in Saccharomyces cerevisiae (W303-1A) strain

Paper ID Volume ID Publish Year Pages File Format Full-Text
21720 43237 2008 8 PDF Available
Title
Production and characterization of recombinant cyprosin B in Saccharomyces cerevisiae (W303-1A) strain
Abstract

The Saccharomyces cerevisiae W303-1A strain transformed with a centromeric plasmid containing CYPRO11, which codifies the aspartic protease cyprosin B, was grown in a 3 l bioreactor under aerobic conditions. Expression of cyprosin B is directly dependent on the concentration of galactose used as the inducer and carbon source in 1% yeast extract, 2% bactopeptone, and 4% galactose in culture medium. For 4% of galactose, 209 mg·l−1 total protein, and 1036 U·ml−1 recombinant cyprosin B activity were obtained from 6.1 g dcw·l−1 biomass. The recombinant cyprosin B, purified by two consecutive anion-exchange chromatographies (diethyl amino-ethyl [DEAE]-Sepharose and Q-Sepharose XK-16 columns), shows a specific activity of 62×103 U·mg−1, corresponding to a purification degree of 12.5-fold and a recovery yield of 25.6% relative to that in fermentation broth. The proteolytic activity of recombinant cyprosin B is optimal at 42°C and pH 4.5. The recombinant cyprosin B activity is 95% inhibited by pepstatin A, which confirms its aspartic protease nature. The pure recombinant cyprosin B is composed of two subunits, one with 14 and the other with 32 kDa. It exhibits clotting activity, similar to that of the natural enzyme from Cynara cardunculus flowers. The results reported here show that recombinant cyprosin B, the first clotting protease of plant origin produced in a bioreactor, can now be produced in large scale and may constitute a new and efficient alternative to enzymes of animal or fungal origin that are widely used in cheese making.

Keywords
Cynara cardunculus; cyprosin B; aspartic protease; fermentation; milk clotting; Saccharomyces cerevisiae
First Page Preview
Production and characterization of recombinant cyprosin B in Saccharomyces cerevisiae (W303-1A) strain
Publisher
Database: Elsevier - ScienceDirect
Journal: Journal of Bioscience and Bioengineering - Volume 105, Issue 4, April 2008, Pages 305–312
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering