Purification and characterisation of proteases A and D from Debaryomyces hansenii.
The proteases A (PrA; EC. 3.4.23.25) and D (PrD; EC. 3.4.24.37) of Debaryomyces hansenii CECT 12487 were characterised after their isolation by fractionation with protamine sulfate followed by three chromatographic separations, which included two anion exchange and one gel filtration chromatographic steps. The whole procedures for PrA and PrD resulted in 1349 and 2560 purification-fold with a recovery yield of 1.4 and 1.3%, respectively. PrA was active at acidic-neutral pH with an optimum pH between 5.0 and 6.0. PrD was active at neutral-basic pH with an optimum pH between 7.0 and 8.0. The molecular mass of the native PrA was 55 kDa and (being) 42 kDa in denaturing conditions. Polyclonal-antibodies raised against PrA from Saccharomyces cerevisiae cross-reacted with the corresponding PrA from D. hansenii. PrD showed a native molecular mass of 68 kDa and 65 kDa in denaturing conditions. PrA was an aspartic protease effectively inhibited by pesptatin A while PrD was classified as a metallo protease inhibited by 1,10-phenantroline and affected by some divalent cations such as zinc, cadmium and magnesium. The homology of the PrA to the lisosomal cathepsin D suggests its possible participation in the ripening of fermented meat products.
Bolumar
Tomás
T
Instituto de Agroquímica y Tecnología de Alimentos, Consejo Superior de Investigaciones Cientificas, Burjassot, Valencia, Spain.
Sanz
Yolanda
Y
Aristoy
M-Concepción
MC
Toldrá
Fidel
F
D000906:Antibodies; D016282:Aspartic Acid Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D003429:Cross Reactions; D005285:Fermentation; D005516:Food Microbiology; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D007610:Kallikreins / Q000302:isolation & purification* / Q000378:metabolism; D008461:Meat Products / Q000382:microbiology*; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D004718:Saccharomycetales / Q000201:enzymology*
D000906:Antibodies; D007527:Isoenzymes; D011480:Protease Inhibitors; D007610:Kallikreins; C083039:proteinase D; D016282:Aspartic Acid Endopeptidases; C022442:aspergillopepsin II