Cloning, expression and biochemical characterization of lignin-degrading DyP-type peroxidase from Bacillus sp. Strain BL5

https://doi.org/10.1016/j.enzmictec.2021.109917Get rights and content

Highlights

  • DyP-type peroxidase from Bacillus sp. was over expressed and biochemically characterized.

  • DyPBL5 showed significantly higher catalytic efficiency against ABTS as compared to the counterparts in literature.

  • Asn 244, Arg 339, Asp 383 and Thr 389 residues at active site are taking part in oxidation of ABTS.

  • DyP-type peroxidase from Bacillus sp. strain BL5 degraded polymeric lignin quite efficiently.

Abstract

Lignin is a major byproduct of pulp and paper industries, which is resistant to depolymerization due to its heterogeneous structure. The enzymes peroxidases can be utilized as potent bio-catalysts to degrade lignin. In the current study, an Efeb gene of 1251bp encoding DyP-type peroxidase from Bacillus sp. strain BL5 (DyPBL5) was amplified, cloned into a pET-28a (+) vector and expressed in Escherichia coli BL21 (DE3) cells. A 46 kDa protein of DyPBL5 was purified through ion-exchange chromatography. 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 5.0. Effects of different chemicals on DyPBL5 were determined. The enzyme activity was strongly inhibited by SDS, DDT and β-mercaptoethanol, whereas stimulated in the presence of organic solvents such as methanol and ethanol. The kinetic parameters were determined and Km, Vmax and Kcat values were 1.06 mM, 519.75 μmol/min/mg and 395 S̶ 1, respectively. Docking of DyPBL5 with ABTS revealed that, Asn 244, Arg 339, Asp 383 and Thr 389 are putative amino acids, taking part in the oxidation of ABTS. The recombinant DyPBL5 resulted in the reduction of lignin contents up to 26.04 %. The SEM and FT-IR analysis of test samples gave some indications about degradation of lignin by DyPBL5. Various low molecular weight lignin degradation products were detected by analyzing the samples through gas chromatography mass spectrometry. High catalytic efficiency and lignin degradation rate make DyPBL5 an ideal bio-catalyst for remediation of lignin-contaminated sites.

Introduction

Lignin is a heterogenous polymer comprising of several linked phenylpropane units i.e. p-coumaryl alcohol, guaiacyl alcohol and syringyl alcohol. The β-O-4-ether linkage is the most prevalent intermolecular bond in lignin [1,2]. Rapidly growing cellulosic ethanol [3] and pulp & paper industries are producing large amounts of lignin residues as by product annually [4]. Majority of the lignin residues either burned as low-cost fuel to provide heat and power to industries [5] or released in the aquatic resources without any treatment. The release of compounds accounts for undesirable coloration of water resources along with deterioration of aquatic flora and fauna [6] and only 2% of the available lignin residues is utilized for sustainable technologies for production of biofuels, chemicals and bio-based materials such as bio-plastics [7]. Most sustainable technologies focus on cellulose and hemicellulose valorization, whereas lignin is usually considered to be a low value product. Decomposition and sustainable use of lignin represents the major challenge due to heterogeneous structure and recalcitrant nature of the lignin, considerable effort has been devoted to understanding the major natural pathways involved in lignin degradation [8].

Several strategies have been implemented to degrade lignin, such as membrane filtration, sedimentation, chemical oxidation, ozonation and biological treatment [9,10]. Among these strategies, biological treatment involving the use of ligninolytic enzymes is considered as one of the promising routes, due to targeted oxidation of substrate, mild reaction conditions, low energy requirements, and less production of toxic waste [8,11]. Lignin modifying enzymes are known as ligninases or ligninolytic enzymes, produced by various fungus and bacterial species. The enzymes are grouped into laccases (Lac, EC 1.10.3.2) and heme-containing peroxidases, such as lignin peroxidase (LiP, EC 1.11.1.14), manganese peroxidase (MnP, 1.11.1.13), dye-decolorizing peroxidase (DyP, EC 1.11.1.19) [12,13]. Lignin degradation has mainly been studied in white-rot fungus Phanaerochaete chrysosporium, which produces extracellular heme-containing lignin peroxidase (LiP) and manganese peroxidase (MnP), which is able to catalyze lignin model compounds [[14], [15], [16]]. Due to inherent problems in fungal genetics and protein expression, these studies have not implemented to a commercial process for depolymerization of lignin.

Several researchers have reported bacteria from soil that are able to metabolize lignin and lignin model compounds [17,18], such as Streptomyces viridosporus reported to metabolize lignin model compounds through the production of extracellular peroxidase but enzymology of bacterial lignin depolymerization is not well characterized [19,20]. A dye degrading peroxidase (DyPB) has been identified in Rhodococcus jostii RHA1 and used to oxidise β-aryl ether lignin model compound, and this enzyme can catalyze lignin in the presence of Mn2+ [21], later on the catalytic efficiency of R. jostii DyPB has been enhanced through site-directed mutagenesis [22]. Another DyP from Amycolatopsis sp. has been identified with higher activity towards Mn2+ oxidation, and showed peroxidase activity against phenols, azo dyes, and anthraquinone dyes [23]. DyP from Thermobifida fusca can oxidase lignin model compounds, such as guaiacol and 2, 6-dimethoxyphenol [24]. Recently, DyP-type peroxidase has been characterized from Pseudomonas fluorescens Pf-5 [8]. YfeX and EfeB from Escherichia coli belongs to DyP-type peroxidase family, exhibits modest guaiacol peroxidase activity [24,25]. The bacterial dye-decolorizing peroxidase (Dyp) family, therefore, represent an interesting bio-catalysts for lignin depolymerization.

In current study, newly identified DyP-type peroxidase from Bacillus sp. strain BL5 isolated from black liquor was cloned, expressed, and biochemically characterized. The recombinant DyPBL5 showed significantly higher ABTS oxidation as compared to other counter parts in the literature. The 3D structure of the protein was determined and docked into model substrate ABTS to reveal putative amino acid residues taking part in oxidation of lignin. The observations can help to rational design DyPBL5 for further efficient oxidation of ligninolytic compound. The newly identified DyP-type peroxidase is capable of lignin degradation, and this is the first study in which DyP-type peroxidase from Bacillus sp. was used to degrade polymeric lignin, hence characterized as bacterial lignin peroxidase.

Section snippets

Chemicals, bacterial strains and plasmids

Alkali lignin and ABTS (2, 2′-Azino-bis [3-ethylbenzthiazoline-6-sulfonic acid) were procured from Sigma-Aldrich (St. Louis, MO, USA). DNA purification and plasmid isolation kits, Taq polymerase, gel extraction kit, T4 DNA ligase, NcoI and HindIII restriction endonucleases were purchased from Promega (Madison, WI, USA). pGEM-T Easy vector (Promega) and E. coli strain JM101 (NEB) were used for cloning. pET-28a (+) vector (Novagen) and E. coli strain BL21 (DE3) (NEB) were used for protein

Isolation and characterization of DyP-type peroxidase producing bacteria

The black liquor samples were collected from pulping unit of paper industry containing high amount of lignin for the isolation of ligninolytic bacteria. Several researchers have reported ligninolytic bacteria from lignin contaminated sites because these bacteria thrive naturally developed tolerance against lignin and its derivatives [[30], [31], [32]]. Five bacterial strains designated as BL1-BL5 were isolated from black liquor on MSM agar plates containing lignin as a sole carbon source. The

Conclusion

In this study, a highly active DyP-type peroxidase enzyme DyPBL5 from indigenously isolated Bacillus sp. strain BL5, was cloned and over-expressed in E. coli BL21 (DE3) cells, and biochemically characterized. This is the first detailed characterization of lignin degrading DyP-type peroxidase from Bacillus sp. Lignin released from pulp and paper mills carries alkaline pH 7–8, and the activity of DyPBL5 at pH 3.0–8.0 makes it a prime candidate to be utilized for lignin degradation. The

Declaration of Competing Interest

All the authors have no financial as well as commercial conflict of interest.

Acknowledgements and Funding Information

This work was supported by Office of Scientific Research Projects of Karadeniz Technical University (Project number: FBA-2018-7504) and Research Mobility Grant of Higher Education Commission, Pakistan (Grant No. 9-5(Ph-1-MG-1)/Pak-Turk/R&D/HEC/2017).

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