Unlocking the potential of Pleurotus ostreatus NRC 620 laccase isoenzymes in juice processing: Purification, kinetics, and biochemical characterization.

Abstract

Two laccase isoenzymes, Lacc A and Lacc B, were purified and characterized from Pleurotus ostreatus NRC 620, and Lacc A was subsequently evaluated for apple juice treatment. The two isoenzymes exhibited distinct thermal profiles, where after 120 min, Lacc B retained about 92% and 82% of its activity at 40 and 50 °C, respectively, whereas Lacc A retained at least 95% at 30 °C but lost most of its activity within 60 min at 50 °C and within 15 min at 60-70 °C. pH stability profiling revealed a complementary pattern, with Lacc A showing greater acid tolerance (70% residual activity at pH 3.0) and Lacc B exhibiting pronounced alkaline stability (85-90% residual activity at pH 9.0). Kinetic analysis using ABTS as substrate showed that Lacc A had higher substrate affinity (Km, 0.2728 mM) than Lacc B (Km, 0.7431 mM), while Lacc B displayed a higher maximum catalytic rate (Vmax, 0.3845 μmol·min-1·mL-1) than Lacc A (0.1734 μmol·min-1·mL-1). Metal ion sensitivity assays further differentiated the two isoenzymes, where Lacc A was activated by CuSO₄ (170%), MgSO₄, and CoSO₄, whereas Lacc B was unaffected by these ions and was completely inhibited by ZnSO₄. Application of purified Lacc A to apple juice induced compound-specific phenolic modifications; chlorogenic acid, the predominant phenolic compound, was only slightly reduced, whereas gallic acid, caffeic acid, vanillic acid, and catechin decreased substantially, with several becoming undetectable after storage. HPLC/GC analysis identified 27 volatile compounds, with laccase treatment enhancing key aroma-active esters, including ethyl acetate and ethyl butyrate. Sensory evaluation confirmed acceptable product quality over two weeks of refrigerated storage, supporting the potential of Lacc A as a biocatalyst for enzymatic apple juice processing.