Production, characterization and applications of proteases produced by Bacillus licheniformis, Acinetobacter pittii and Aspergillus niger using neem seed oil cake as the substrate

Abstract

Alkaline proteases suitable for industrial applications were produced using two bacterial and one fungal strain with de-oiled neem seed cake as the substrate. Neem oil cakes contain proteins and carbohydrates and are renewable and sustainable sources available in large quantities at low cost. In this study, ability of Bacillus licheniformis, Acinetobacter pittii and Aspergillus niger to produce proteases at different culture conditions were studied. Culture conditions such as pH, temperature, incubation time were optimized to obtain highest yield possible. Further, various carbon and nitrogen sources were added as supplements to the cake and the changes in the enzyme production was determined. De-oiled neem seed cake showed potential for production of proteases with yield as high as 11–12 U/ml by both Bacillus licheniformis and Acinetobacter pittii without any additional inducers and basal media (nutrient broth). Addition to sucrose and fructose lead to substantial increase (up to 57 %) in enzyme activity for all the three microorganisms. Similarly, yeast and urea were more favourable to increase enzyme yield of up to 355 %. The enzymes were able to clot milk and precipitate casein and could be used in the food industry. Similarly, the enzymes obtained were able to remove the blood stains on clothes suggesting suitability for laundry applications. Additionally, the crude enzyme was able to hydrolyse the gelatine layer from used x-ray films enabling recycling and reuse.