Purification and characterization of an acid proteinase from mesophilic Mucor sp. solid-state cultures.
The fourth-day extract of a solid-state culture of the mesophilic Mucor sp. (M-105) strain showed a high milk-clotting activity and a clotting/proteolytic activity ratio similar to that of commercial preparations from microbial origin used in cheese manufacture. After ultrafiltration of the crude extract, the milk-clotting proteinase was purified in two steps: ion-exchange followed by size-exclusion chromatography. Enzyme homogeneity was assessed by HPLC, SDS-PAGE and N-terminal residue determination. A pI value of 4.21 was obtained and a molecular weight of 33 kDa was calculated from size-exclusion chromatography and SDS-PAGE data. The optimum pH for proteolytic activity towards dimethylcasein was in the 3.0-3.5 range. The proteinase retained 26 and 13% of its proteolytic activity after a 30-min incubation period, at pH 5.0 and 50 and 60 degrees C, respectively. This evidenced a lower heat stability than that of the thermophilic enzymes currently used in the cheese industry and also than that of bovine chymosin. The enzyme was fully inhibited by pepstatin A and no effect was observed with PMSF, p-CMPS or EDTA. The N-terminal amino acid sequence: GTGTVPVTDDGNLNEYYXTVTVGXP was compared with those from other fungal enzymes.
Fernandez-Lahore
H M
HM
Cátedra de Microbiología Industrial y Biotecnología, Universidad de Buenos Aires, Argentina.
Auday
R M
RM
Fraile
E R
ER
Biscoglio de Jimenez Bonino
M
M
Pirpignani
L
L
Machalinski
C
C
Cascone
O
O
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D004795:Enzyme Stability; D005285:Fermentation; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008892:Milk / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009089:Mucor / Q000201:enzymology*; D010436:Pepstatins / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid
D010436:Pepstatins; D011480:Protease Inhibitors; C014195:Streptomyces pepsin inhibitor; D010450:Endopeptidases; D016282:Aspartic Acid Endopeptidases; C031375:pepstatin