Production, characterization, and bio-ethanologenic potential of a novel tripartite raw starch-digesting amylase from Priestia flexa UCCM 00132.

The biological conversion of agro-waste biomass into value-added metabolites is one of the trendy biotechnological research areas in recent times. One of the major drawbacks of the bioprocess is the saccharification potential of the amylolytic enzyme that releases reducing sugar from complex biomass to serve as substrate for fermentation. The present study reports the production of a novel tripartite raw starch-digesting amylase (RSDA) by an indigenous Priestia flexa strain with α-, β-, and gluco-amylolytic activities and its potential for bioethanol production. Response surface statistics was employed to develop a suitable medium for improved production of the tripartite enzyme by submerged fermentation. The bioprocess selected raw starch (4.36%) Ca²⁺(2.71 g/L) and Zn²⁺ (0.0177 g/L) as significant variables which demonstrated a total RSDA activity of 7208.23 U/mL in a 5-L batch bioreactor. SDS/Native-PAGE determined the molecular weights of the 27-fold purified product as 25.2 kDa, 57.3 kDa, and 90.1 kDa for α-, β-, and gluco-amylases, respectively. Optimum temperature and pH for enzyme activity were respectively broad at 30-70 °C and 4-11. The enzyme mixture demonstrated digestibility above 90% against a variety of raw starches and simultaneous fermentation of digestate with Saccharomyces cerevisiae generated 71.69 g/L of bioethanol within 24 h suggesting great potential for bioethanologenesis.