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Published on: March 25, 2010
Purification and characterization of an acid proteinase from mesophilic Mucor sp. solid-state cultures
H M Fernandez-Lahore1, R M Auday, E R Fraile
1Cátedra de Microbiología Industrial y Biotecnología, Universidad de Buenos Aires, Argentina.
Abstract:
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.
Insights
A novel Mucor sp. enzyme exhibits high milk-clotting activity, suitable for cheese production. This microbial rennet shows potential as an alternative to traditional enzymes, though with lower heat stability.
Area of Science:
- Enzymology
- Food Science
- Microbiology
Background:
- Microbial rennets are crucial in cheese manufacturing.
- Identifying novel enzymes with desirable properties is essential for the dairy industry.
Purpose of the Study:
- To characterize a milk-clotting proteinase from a mesophilic Mucor sp. (M-105) strain.
- To evaluate its potential for cheese production.
Main Methods:
- Solid-state culture, ultrafiltration, ion-exchange chromatography, size-exclusion chromatography.
- High-performance liquid chromatography (HPLC), SDS-PAGE, N-terminal sequencing for enzyme characterization.
- Assay of milk-clotting and proteolytic activities, heat stability, and inhibition studies.
Main Results:
- The purified enzyme displayed high milk-clotting activity with a favorable clotting/proteolytic ratio.
- The proteinase has a molecular weight of 33 kDa, pI of 4.21, and optimal activity at pH 3.0-3.5.
- The enzyme showed lower heat stability compared to thermophilic enzymes and bovine chymosin, and was inhibited by pepstatin A.
Conclusions:
- The Mucor sp. (M-105) proteinase is a promising candidate for cheese making due to its high milk-clotting ability.
- Further research is needed to improve its heat stability for broader industrial application.
