Biochemical and kinetic characterization of laccase and manganese peroxidase from novel Klebsiella pneumoniae strains and their application in bioethanol production

Abstract

Laccase (lac) and manganese peroxidase (MnP) enzymes from the novel Klebsiella pneumoniae isolates, grown on lignin basic media (LBM) were purified by 80% ammonium sulphate fractionation, dialysis and DEAE-sepharose column chromatography. The optimum temperatures for laccase production were 60 °C, 50 °C and 50 °C and for MnP production were 50 °C, 70 °C and 60 °C from NITW715076_2, NITW715076_1 and NITW715076 isolates, respectively. The optimal pH for production was found to be 5 for production of both the enzymes from all the isolates. 2.8–3.5 fold enzyme purification was achieved retaining around 60–70% of the initial activity. SDS-PAGE revealed the molecular mass of laccase and MnP to be 66 kDa and 48 kDa, respectively. The substrate ABTS and MnSO₄ exhibited more specificity towards NITW715075_2 derived laccase and MnP (lac: K_m = 0.38 mM, V_max = 71.42 U ml⁻¹; MnP: K_m = 0.17 mM, V_max = 106.38 U ml⁻¹) compared to NITW715076_1 (lac: K_m = 3.97 mM, V_max = 148.8 U ml⁻¹; MnP: K_m = 0.90 mM, V_max = 114.67 U ml⁻¹) and NITW715076 (lac: K_m = 0.46 mM, V_max = 23.42 U ml⁻¹; MnP: K_m = 0.19 mM, V_max = 108.10 U ml⁻¹) derived. L-Cysteine and sodium azide imposed a strong inhibitory effect on the activities of both the enzymes. EDTA inhibited laccase and MnP activity at higher concentration. SDS strongly inhibited activity while for MnP it showed less inhibitory effect. The enzymes were employed for ethanol production from rice and wheat bran biomass which showed 39.29% improved production compared to control. After evaluating the applicability of these enzymes it can be suggested that the ligninolytic enzyme of Klebsiella pneumoniae isolates could be effectively employed in enhanced ethanol production and could be explored for other putative applications.