Purification and Characterization of Mannanase from Aspergillus awamori for Fruit Juice Clarification

Background: Fruit juice clarification is a challenging aspect of beverage industry which needs to be addressed for economical and hygienic production of fruit juices. Objective: Current study is focused on the complete purification, characterization and thermodynamic analysis of an efficient mannanase enzyme to analyze its applicability in biological clarification fruit juice. Methods: Mannanase production using Aspergillus awamori IIB037 in a 25 L stirred fermenter at pre optimized reaction conditions was carried out. Enzyme purification was carried out via series of steps. Characterization of enzyme along with kinetics and thermodynamic studies was conducted. Purified and characterized enzyme was assessed for its applicability in fruit juice clarification through clarification experiments on fresh apple juice. Results: Purification fold of 3.98 was obtained along with 86.80% purification yield of mannanase with specific activity of 158.16 U/mg. The molecular size of purified enzyme was determined as 66 kDa. The enzyme depicted 56% residual activity at 60°C after 8 hrs. Thermodynamic studies of an enzyme revealed enthalpy of activation (ΔH) and activation energy (Ea) as 30.53KJ/mol, 27.76KJ/mol, respectively. The enzyme activity increased in the presence of ß-mercaptoethanol surprisingly. On the other hand, methyl alcohol, ethanol, Hg²⁺ and Cu²⁺ inhibited enzyme activity. The enzyme showed Km and Vmax values of 11.07 mM and 19.08 μM min-1 for Locust Bean Gum (LBG) under optimal conditions. Juice treated with mannanase showed decrease in absorbance and increase in reducing sugar content. Conclusion: The current study demonstrated that mannanase from Aspergillus awamori in its purified form has significant characteristics to be employed industrially for juice clarification.