Enzymatic Degradation of Lignin by Bacteria Isolated from Pulp and Paper Mill Effluent

Abstract

Lignin is one of the key by-product of the paper industries, which is polymeric in nature and shows resistance to depolymerization. The ligninolytic enzymes can be apply to degrade lignin. The present study was designed to evaluate the lignin degradation by lignin degrading peroxidase (LDP) and laccase (Lac) from bacteria isolated from pulp and paper mill effluent. The total 55 bacterial strains were isolated from effluent and investigated for lignin degradation efficiency and ligninolytic enzymes production, strain BL5 and SL7 showed maximum lignin degradation efficiency 35% and 44%, respectively, and were selected for further experiments. On the basis of morphological depictions, biochemical characterization and 16S rRNA sequencing, strain BL5 and SL7 were identified as Bacillus sp. and Bacillus altitudinis, respectively. Effects of physiochemical factors were investigated for production of LDP from Bacillus sp. BL5 and Lac from Bacillus altitudinis SL7. The optimum production of LDP and Lac was attained at 30°C and pH 8.0 and 7.0, respectively, in lignin amended minimal salt medium (L-MSM). The best carbon and nitrogen source for production of LDP and Lac were found to be (glucose, peptone) and (starch, yeast extract), respectively. The concentration of nutritional factors was optimized by Central Composite Design, and highest LDP and Lac production under optimized condition were found to be 56.9 U/mg and 29.5 U/mg, respectively, after 72 h of fermentation. The native LDP and Lac was purified by acetone and gel filtration chromatography, and SDS-PAGE analysis revealed their molecular weights as 45 kDa and 55 kDa, respectively. The Efeb gene of 1251bp encoding DyP-type lignin degrading peroxidase from Bacillus sp. BL5 (DyPBL5) and laccase gene of 1500 bp encoding laccase from Bacillus altitudinis SL7 was amplified in the current study and subsequently cloned into a pET-28a (+) vector and expressed in E. coli BL21 (DE3) cells. Expressed DyPBL5 and LacSL7 were purified though ion-exchange chromatography and molecular weight was determined as 46 kDa and 56 kDa, respectively, which was almost same as native enzymes. High level of enzyme activity produced by recombinant Bacillus sp. BL5 and Bacillus altitudinis SL7 enzymes. The specific activity of purified DyPBL5 and LacSL7 was up to 1319 and 2530 IU/mg with 37.9 and 33 fold purity, respectively. The kinetic parameters of DyPBL5 and LacSL7 were determined and Km, Vmax, and Kcat values were (1.06, 0.28 mM,), (519.75, 2073 μmol/min/mg) and (395, 3878 S 1), respectively. DyPBL5 showed the highest sequence similarity with DyP-type peroxidase of Bacillus subtilis (WP148982369). While viii LacSL7 showed the highest sequence similarity with the laccase of Bacillus altitudinis (APP17947). Purified DyPBL5 was active at wide temperature (25-50°C) and pH (3.0-8.0) range with optimum activity at 35°C and pH 4.0. While LacSL7 is thermostable enzyme and was active at high temperature, ranges from 25-80°C and pH 3.0-8.0 with optimum activity at 55°C and pH 5.0. DyPBL5 and LacSL7 were stable in the presence of low concentration of metals and higher concentration caused inhibitory effect on it. DDT and -mercaptoethanol caused strong inhibition of both the enzymes, whereas SDS inhibited DyPBL5 only, and LacSL7 showed resistance to SDS. DyPBL5 and LacSL7 activity was enhanced in the presence of organic solvents such as methanol and ethanol. The recombinant DyPBL5 and LacSL7 resulted in the reduction of lignin contents up to 27.04% and 31.2%, respectively, and combination of both the enzymes degraded lignin up to 63.14%. The lignin degradation was further confirmed by SEM and FT-IR analysis of enzymatically treated samples. The smooth surface of lignin was completely eroded, deformed and particle size was reduced during the course of treatment. FT-IR analysis indicated significant changes in composition of functional groups of degraded lignin such as carboxyl (-COOH), carbonyl (C=O), amines (-NH2), alkene (C=C) and sulfonic (-SO3). Further, various low molecular weight compounds were detected such as vanillin, 2-methyoxyhenol, 3-methyl phenol, oxalic acid, and ferulic acid, suggested the degradation of coniferyl and sinapyl groups of lignin. Lignin degradation rate of DyPBL5 and LacSL7 suggested that both enzymes can be ideal bio-catalysts for remediation of lignin containing effluent.