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Abstract:
When Mucor hiemalis NRRL 3103 was grown in soybean medium, only a small fraction of the proteinase produced by the organism appeared in the culture filtrate, whereas the bulk of the enzyme was bound to the mycelial surface. Optimal pH of the proteinase ranged from 3.0 to 3.5. Inclusion of sodium chloride or other ionizable salts in the growth medium, however, resulted in the liberation from the mycelium of the loosely bound enzyme as it was formed. Maximal release of proteinase was achieved at a sodium chloride concentration of 0.5 m. The loosely bound proteinase was eluted also from intact resting mycelium by ionizable salts but not by water or by nonionizable substances. The amount of enzyme eluted from the mycelium depended upon the concentration of sodium chloride up to 0.3 m. Since liberation took place rapidly even at 0 C, a loose ionic linkage must exist rather than a biochemical binding of the enzyme to the mycelium. The recovery of proteolytic activity from repeated salt extractions was greater than that originally detected in the intact mycelium, possibly owing to unmasking of more active enzymes or functional groups. Further proteinase activity was released when salt-extracted mycelium was ruptured. Part of the proteinase thus observed was firmly attached to the cell fraction, and part of it appeared in the supernatant fluid. These conditions implied the presence of intracellular or firmly attached proteinase which could be partially released.
Insights
Most proteinase produced by Mucor hiemalis remains bound to its mycelium. Ionizable salts effectively release this bound enzyme, indicating a loose ionic linkage, not biochemical binding.
Area of Science:
- Microbiology
- Enzymology
- Biochemistry
Background:
- Mucor hiemalis NRRL 3103 produces proteinase, with most remaining bound to the mycelium rather than secreted.
- Understanding the localization and release of microbial enzymes is crucial for biotechnological applications.
Purpose of the Study:
- To investigate the binding of proteinase to Mucor hiemalis mycelium.
- To determine methods for releasing the bound proteinase.
- To characterize the nature of the enzyme-mycelium linkage.
Main Methods:
- Culturing Mucor hiemalis in soybean medium.
- Assessing proteinase activity in culture filtrate and mycelial fractions.
- Treating mycelium with varying concentrations of sodium chloride and other salts.
- Eluting proteinase from intact and ruptured mycelium.
- Testing elution with water and nonionizable substances.
Main Results:
- The majority of proteinase produced by Mucor hiemalis binds to the mycelial surface.
- Ionizable salts, particularly sodium chloride (optimal at 0.5 M), effectively liberate the loosely bound proteinase.
- Elution occurs rapidly even at low temperatures, suggesting a loose ionic linkage.
- Salt extraction recovered more activity than initially detected, and further activity was released from ruptured mycelium, indicating both loosely bound and firmly attached/intracellular proteinases.
Conclusions:
- Mucor hiemalis proteinase is primarily cell-bound, with a significant portion loosely attached via ionic interactions.
- Ionizable salts are effective agents for releasing this bound enzyme.
- The findings suggest potential for optimizing enzyme recovery and purification from microbial sources.