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: Km = 0.38 mM, Vmax = 71.42 U ml−1; MnP: Km = 0.17 mM, Vmax = 106.38 U ml−1) compared to NITW715076_1 (lac: Km = 3.97 mM, Vmax = 148.8 U ml−1; MnP: Km = 0.90 mM, Vmax = 114.67 U ml−1) and NITW715076 (lac: Km = 0.46 mM, Vmax = 23.42 U ml−1; MnP: Km = 0.19 mM, Vmax = 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.
Highlights
Laccases (EC 1.10.3.2; benzenediol: oxygen oxidoreductases) belong to a group of copper containing glycoproteins with molecular weight between 60–100 kDa and are widely distributed among plants, fungi and bacteria.[1]
The activities of crude laccase and Manganese peroxidase (MnP) from Klebsiella pneumoniae strains were examined at different temperatures with ABTS and MnSO4 as a substrate respectively and the enzymes were found to be active in a temperature range of 30–70 C
Laccase and MnP secreted from Klebsiella pneumoniae strains were puri ed and characterized
Summary
Laccases (lac) (EC 1.10.3.2; benzenediol: oxygen oxidoreductases) belong to a group of copper containing glycoproteins with molecular weight between 60–100 kDa and are widely distributed among plants, fungi and bacteria.[1]. In the past few decades both lac and MnPs have received considerable scienti c attention for their versatile substrate speci city and ability to oxidise a wide range of phenols and polyphenols such as ortho and para-diphenols, methoxy-substituted phenols, phenolic acids and several other compounds. They are capable of reducing molecular oxygen to water with one electron oxidation mechanism. Lac and MnP have been found to be widely distributed among plants and fungi but known laccase-like multi copper oxidases and peroxidases (MnPs) have been detected in the genome of many bacterial species. Ligninolytic bacteria can withstand high temperature and pH making them highly desirable candidate for environmental bioremediation, biofuel production, bio-pulping and textile industries and as biocatalysts for synthesis of value added products or structural modi cations.[4,5] Bacillus subtilis SF, B. halodurans, B. pumilus, B. subtilis WPI, Serratia marcescens, K
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