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The new thermostable fungal aspartic protease was produced through solid-state fermentation from Rhizopus azygosporus (MTCC 10195). The protease was purified by a three-tandem steps of ammonium sulfate precipitation (25-65% saturation), ion exchange (DEAE sepharose CL 6B) chromatography, and gel filtration (Sephacryl S 200) chromatography. The optimum pH and temperature for protease activity were 4 and 52 ± 1.8 °C, respectively. The enzyme was unusually thermostable, as it retained 50% of its activity beyond 85 °C after 20 min of incubation. The apparent molecular weight was 33.2 ± 2.3 kDa with a final specific activity of 86.6 U/mg. The enzyme was rich in β sheets (63%) and was inhibited by pepstatin A, indicating that it is an aspartic protease. MS/MS analysis revealed the enzyme is an endopeptidase cleaving the C-terminus of Phe, Leu, Lys, Val, His, Glu, Met, and Tyr. Amino-terminal sequencing, coupled with in-gel trypsin digestion and MS analysis revealed that the enzyme was being reported for the first time with “experimental evidence at protein-level”.

Galactosidases, ubiquitous in nature, are complex carbohydrate-active enzymes and find extensive applications in food, pharma, and biotechnology industries. The present study deals with the production of galactosidases from fungi by solid-state fermentation. Fifteen fungi were screened and Aspergillus awamori (MTCC 548), exhibited the highest α and β-galactosidase activities of 75.11±0.29 U/g and 155.34±1.26 U/g, respectively. 30 g of wheat bran substituted with 6% defatted soy flour, at 28°C, pH 5.0 for 120 h, was established as the optimum production conditions by one-factor approach. The enzyme was purified to homogeneity with an apparent mass of 118 ± 2 kDa by ammonium sulfate precipitation (50–80%), ion exchange and hydrophobic interaction chromatography. Specific activities for α and β-galactosidase were 22 and 74 U/mg, respectively. Optimum temperature and pH ranges for enzyme activities were 55–60 °C, 5.0–5.5, respectively. The thermal inactivation mid-point was 65 °C. The purified enzyme not only exhibited α and β-galactosidase activities, but also exhibited β-xylosidase and β-glucosidase activities, indicating the enzyme has broad substrate specificity. Sequence analysis by in-gel digestion and tandem mass spectrometry (MS/MS) revealed that the enzyme was a probable β-galactosidase A, belonging to glycoside hydrolase 35 family, and is being reported for the first time.