Protease B from Debaryomyces hansenii: purification and biochemical properties.
The protease B (PrB; EC. 3.4.21.48) of Debaryomyces hansenii CECT 12487 was purified by selective fractionation with protamine sulfate followed by three chromatographic separations. The whole procedure resulted in 324-fold purification with a recovery yield of 1.0%. PrB was active at neutral-basic pH ranging from 6.0 to 12.0 with an optimum at pH 8.0. The molecular mass of the denatured enzyme was 30 kDa. Polyclonal-antibodies raised against PrB from Saccharomyces cerevisiae cross-reacted with the corresponding 30-kDa protein from D. hansenii. The serine protease inhibitor 3,4-DCI and sulphydryl group reagents markedly reduced the enzyme activity. The Km against N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin was 1.79 mM. The presence of endogenous inhibitor for PrB was detected in cell-free extracts of D. hansenii although their inhibitory effect was lost after incubation at 25 degrees C for 20 h. PrB was able to hydrolyze muscle sarcoplasmic proteins by in vitro assays. This is the first endopeptidase purified and characterized from the yeast D. hansenii, whose possible contributions to meat fermentation processes are discussed.
Bolumar
Tomás
T
Instituto de Agroquímica y Tecnología de Alimentos (C.S.I.C.), Apartado de Correos 73, 46100 Burjassot, Valencia, Spain.
Sanz
Yolanda
Y
Aristoy
M-Concepción
MC
Toldrá
Fidel
F
D000906:Antibodies; D003429:Cross Reactions; D005285:Fermentation; D005516:Food Microbiology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008461:Meat Products / Q000382:microbiology*; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000276:immunology; D004718:Saccharomycetales / Q000201:enzymology* / Q000378:metabolism; D012697:Serine Endopeptidases / Q000302:isolation & purification*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D013696:Temperature; D029701:Saccharomyces cerevisiae Proteins
D000906:Antibodies; D012697:Serine Endopeptidases; D015842:Serine Proteinase Inhibitors; C023302:yeast proteinase B; D029701:Saccharomyces cerevisiae Proteins