Production and characterization of recombinant cyprosin B in Saccharomyces cerevisiae (W303-1A) strain.
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 x 10(3) 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 degrees 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.
Sampaio
Pedro N
PN
Unidade de Biologia Molecular e Biotecnologia de Plantas, Instituto de Ciência Aplicada e Tecnologia, Faculdade de Ciências, Universidade de Lisboa, 1749-016 Lisboa, Portugal. pnsampaio@gmail.com
Fortes
Ana Margarida
AM
Cabral
Joaquim M S
JM
Pais
Maria S
MS
Fonseca
Luís P
LP
D016282:Aspartic Acid Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D019149:Bioreactors / Q000382:microbiology; D002852:Chromatography, Ion Exchange; D031942:Cynara / Q000201:enzymology / Q000235:genetics; D005690:Galactose / Q000737:chemistry; D006358:Hot Temperature; D010436:Pepstatins / Q000737:chemistry; D010940:Plant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D021122:Protein Subunits / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development*
D010436:Pepstatins; D010940:Plant Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D016282:Aspartic Acid Endopeptidases; C087364:cyprosin; C031375:pepstatin; D005690:Galactose