Screening and genetic identification of acidic and neutral protease-producing yeasts strains by 26S rRNA gene sequencing

Protease enzymes (proteases), particularly those produced by microorganisms, play very important roles in industry, due to their diverse applications. Considering the richness of microbial diversity in nature, a good chance always exists that proteases more suitable, with better properties for commercial application, may be discovered while screening novel microorganisms from local environments. In this study, 94 yeasts were isolated from different natural sources collected from the Abha region, Kingdom of Saudi Arabia, to determine extracellular protease production and activity. Among them, 23 isolates (24.46%) showed protease activity using a casein hydrolysis test. Of these, five isolates (21.74%) were selected and identified as the best protease producers by exhibiting the largest clearance zones around colonies. A 26S rRNA gene D1/D2 domain sequence alignment, comparison, and phylogenetic analysis of our study yeasts to published D1/D2 domain rRNA gene sequences from GenBank, identifies the isolates as Rhodotorula mucilaginosa KKU-M12c, Cryptococcus albidus KKU-M13c, Pichia membranifaciens KKU-M18c, Hanseniaspora uvarum KKU-M19c, and Candida californica KKU-M20c. The influence of varying pH (4.0–9.0) on the yield and activity of the proteases was investigated using 0.5% (w/v) casein as a substrate, to detect optimum pH values for yeast extracellular protease production. Enzyme activity was measured using qualitative and quantitative assays. Results show all of the study yeasts secreting protease enzyme at all tested pH levels, with the exception of pH 9.0. This indicates that none of the five yeasts are alkaline protease producers. Maximum protease activity (187 U/mL) was observed in strain H. uvarum KKU-M19c at pH 6.0 (only), indicating that strain KKU-M19c only produces neutral protease. The other four yeast isolates, R. mucilaginosa KKU-M12c, C. albidus KKU-M13c, P. membranifaciens KKU-M18c, and C. californica KKU-M20c, produced both acidic (at pH 4.0) and neutral (at pH 6.0 and 7.0) proteases. Strain C. californica KKU-M20c was found to be the best acidic and neutral protease producer (138 U/mL at pH 4.0, and 185 U/mL at pH 7.0). This is the first report of the discovery and isolation of local, powerful yeasts producing acidic and neutral protease enzymes from the Abha region, Kingdom of Saudi Arabia.