Abstract
The industrial demand for proteolytic enzymes is stimulating the search for new enzyme sources. Fungal enzymes are preferred over bacterial enzymes, and more effective and easier to extract. The aim of this work was to evaluate the potential of protease production by solid state fermentation (SSF) of Mucor subtilissimus UCP 1262, evaluate different specific activities, purify and partially characterize the enzyme in terms of biochemical as to the optimal pH and temperature. Initially, the enzyme crude extract was screened for 3 different proteolytic activities, collagenolytic (161.4 U/mL), keratinolytic (39.6 U/mL) and fibrinolytic (26.1 U/mL) in addition to conventional proteinase activity. After ammonium sulfate precipitation, the active fractions with fibrinolytic activity were dialyzed in 15 mM Tris-HCl buffer, pH 8, loaded onto DEAE-Sephadex A50 ion-exchange column and gel filtrated through Superdex 75 HR10/300. The enzyme showed a fibrinolytic maximum activity at 40 C and pH 9,0. The purified enzyme showed activity against a chromogenic chymotrypsin substrate, SDS-PAGE showing a molecular mass of approximately 70 kDa and, the specific activity of 25.93 U/mg. These characteristics suggest that the enzyme could be and efficiently produced in a simple and low-cost way using Mucor subtilissimus UCP 1262 in SSF.
Highlights
Proteases are one of the most important groups of enzymes found in all living organisms from bacteria to mammals (Shamsi et al 2018)
Evaluation of enzyme activities In previous study by Nascimento et al (2015) we found that a wheat bran amount of 3g, a moisture content of 50%, a temperature of 30°C and a fermentation time of 72 hours were the best conditions for protease production by M. subtilissimus UCP 1262 they were used in these experiments for enzyme production
Another study by Shirasaka et al (2012) about protease production by Aspergillus oryzae KSK-3 isolated from commercial rice-koji for miso brewing, showed a fibrinolytic activity of 21.8 U/mL, less activity than that found in our work (26.1 U/mL)
Summary
Proteases are one of the most important groups of enzymes found in all living organisms from bacteria to mammals (Shamsi et al 2018) This class of enzymes belongs to the peptidylpeptide hydrolases, which occupy a essential position with respect to their application, wide applicability in the medical field and industrial, representing approximately 60% of the enzymes sold worldwide (Suryia-Prabha et al 2015, AlDhabi et al 2020, Adetunji & Olaniran 2020). After addition of 7.5 mL of 245 mM sodium phosphate buffer, pH 7, per g of substrate, flasks were placed in an orbital shaker (Model 430 –RD, Ethiktechnology, São Paulo, Brazil) at 150 rpm for 90 min at room temperature After this period, the suspension was centrifuged (Frontier 5000 Multi Pro, Rio de Janeiro, Brazil) at 3,500 rpm for 10 min, and the supernatant used for determination of different enzyme activities
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