Characterization of Aspartic Protease Enzyme from Fungi and Bacteria and Its Potential Application for Cheese Production

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Addis Ababa University


Cheese is a dairy product processed through milk clotting using rennet enzyme (chymosin) (EC Chymosin is a complex enzyme produced by animals, plants or microorganisms. Traditionally, it is extracted from the fourth stomach (abomasum) of young ruminants. However, only 20-30% of the world demand for milk-clotting enzymes is covered by calf rennet, indicating that the milk-clotting enzymes derived from animals are not sufficient to cover worldwide cheese production. This necessitates the search for alternative sources for calf rennet substitutes such as microbial aspartic proteases. In this study, a total of 237 (188 fungal and 49 bacterial) isolates were tested for milk-clotting enzyme production in primary and secondary screening techniques. After the secondary screening, 17 potential fungal and 14 bacterial isolates were successfully identified using, a combination of phenotypic and molecular techniques. The physicochemical parameter and media composition for potential fungus (Aspergillus oryzae DRDFS13) under SSF were optimized by one-factor-at-a-time and Response Surface Methodology (RSM) whereas the culture profile of the potential bacterium (Bacillus subtilis SMDFS 2B) was studied under partially optimized conditions. The enzyme from A. oryzae DRDFS13 was characterized molecularly and biochemically after purification by size-exclusion (SEC) and ion-exchange (IEC) chromatography. The enzyme from B. subtilis SMDFS 2B was characterized biochemically after partial purification by ii dialysis. Furthermore, the aspartic protease gene from A. oryzae DRDFS13 was characterized by cloning into Pichia pastoris using pGAPZαA as a vector and E. coli K12 for gene amplification. Finally a partially purified enzyme from A. oryzae DRDFS13 and B. subtilis SMDFS 2B used for Danbo cheese production using commercial rennet as a control. The Danbo cheeses produced using fungal enzyme (E1), bacterial enzyme (E2) and commercial rennet (C) were analyzed for body property, organoleptic characteristics, proximate and mineral composition when fresh and after 2 months of ripening. Seventeen fungi isolates were identified into different strains under the genera Aspergillus, Fusarium, and Pleurotus, whereas all the 14 bacterial isolates were identified to different strains under genus Bacillus. Moreover, A. oryzae DRDFS13 and B. subtilis SMDFS2B that showed enhanced MCA were selected for further study. The result from optimization and culture profile determination for A. oryzae DRDFS13 and B. subtilis SMDFS2B increased the MCA by 2.7 and 5.3 fold, respectively. The purified enzyme from A. oryzae DRDFS 13, IEC fraction A8 was exhibited a purification fold, specific activity, and yield of 6.20, 183.50 U/mg and 9.2%, respectively. The molecular weight of IEC A8 was 40 kDa, however, its MW was decreased to 30 KDa upon deglycosylation assay which infers that the protein is glycosylated. Inhibition study of IEC A8 with pepstatin A caused a 94 % inhibition on MCA. The dialyzed enzyme from A.oryzae DRDFS13 was shown maximum MCA at 60 oC and pH 5.0 with stability at pH 4.5-6.5 and temperature 35-45 0C. The partial purification of the crude enzyme from B. subtilis SMDFS 2B was increased its MCA by 2.0 fold. The dialyzed enzyme showed the highest MCA at 55 oC and pH 5.5 with stability at pH 4-6 and temperature 35 oC- 40 oC. The enzyme also showed the lowest residual MCA in the presence of EDTA (7.94%) and pepstatin-A (26.71%). The aspartic protease gene cloned into pGAPZαA (later pMKAP) was iii successfully expressed in P. pastoris and showed the highest MCA (190.47 U/mL) at pH 5 on the 6th day of incubation time. The recombinant protein has a MW between 32-46 kDa. Furthermore, the overall organoleptic characteristics, proximate composition, and mineral composition obtained from Danbo cheese made of A. oryzae DRDFS13 were closer to the control cheese produced using commercial rennet as compared to the bacterial enzyme. Therefore, the results from the present study confirmed that the enzyme from A. oryzae DRDFS 13 purified by an ion-exchange chromatography is an aspartic protease and could be used as a substitute for rennet enzyme in cheese production. This may open the way for applications of the enzymes in the food and dairy industries. Finally, the results from cheese production revealed that the fungal enzyme from A. oryzae DRDFS 13 is more appropriate for Danbo cheese production than the bacterial enzyme from Bacillus subtilis SMDFS 2B.



Aspergillus, Bacillus, Danbo Cheese, Milk-Clotting Activity, Milk-Clotting Protease, Minerals, Solid-State Fermentation, Submerged Fermentation