Versatile peroxidase degradation of humic substances: use of isothermal titration calorimetry to assess kinetics, and applications to industrial wastes

Khawar Sohail Siddiqui; Haluk Ertan; Timothy Charlton; Anne Poljak; A K Daud Khaled; Xuexia Yang; Gavin Marshall; Ricardo Cavicchioli

doi:10.1016/j.jbiotec.2014.03.002

Versatile peroxidase degradation of humic substances: use of isothermal titration calorimetry to assess kinetics, and applications to industrial wastes

J Biotechnol. 2014 May 20:178:1-11. doi: 10.1016/j.jbiotec.2014.03.002. Epub 2014 Mar 11.

Authors

Khawar Sohail Siddiqui¹, Haluk Ertan², Timothy Charlton³, Anne Poljak⁴, A K Daud Khaled³, Xuexia Yang⁵, Gavin Marshall⁶, Ricardo Cavicchioli⁷

Affiliations

¹ School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia. Electronic address: [email protected].
² School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia; Department of Molecular Biology and Genetics, Istanbul University, Turkey.
³ School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia.
⁴ Bioanalytical Mass Spectrometry Facility, The University of New South Wales, Sydney, NSW 2052, Australia; School of Medical Sciences, The University of New South Wales, Sydney, NSW 2052, Australia.
⁵ School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia; College of Chemistry, Chemical Engineering and Biotechnology, Donghua University, North Renmin Road 2999, Shanghai 201620, PR China.
⁶ School of Chemical and Biomolecular Engineering, University of Sydney, Sydney, NSW 2006, Australia.
⁷ School of Biotechnology and Biomolecular Sciences, The University of New South Wales, Sydney, NSW 2052, Australia. Electronic address: [email protected].

PMID: 24631722
DOI: 10.1016/j.jbiotec.2014.03.002

Abstract

The kinetic constants of a hybrid versatile-peroxidase (VP) which oxidizes complex polymeric humic substances (HS) derived from lignin (humic and fulvic acids) and industrial wastes were determined for the first time using isothermal titration calorimetry (iTC). The reaction conditions were manipulated to enable manganese-peroxidase (MnP) and/or lignin-peroxidase (LiP) activities to be evaluated. The peroxidase reactions exhibited varying degrees of product inhibition or activation; properties which have not previously been reported for VP enzymes. In contrast to previous work (Ertan et al., 2012) on small non-polymeric substrates (MnSO4, veratryl alcohol and dyes), all kinetic plots for polymeric HS were sigmoidal, lacked Michaelis-Menten characteristics, and were indicative of positive cooperativity. Under conditions when both LiP and MnP were active, the kinetic data fitted to a novel biphasic Hill Equation, and the rate of enzymatic reaction was significantly greater than the sum of individual LiP plus MnP activities implying synergistic activation. By employing size-exclusion chromatography and electrospray ionization mass spectrometry, the characteristics of the oxidative degradation products of the HS were also monitored. Our study showed that the allosteric behaviour of the VP enzyme promotes a high level of regulation of activity during the breakdown of model and industrial ligninolytic substrates. The work was extended to examine the kinetics of breakdown of industrial wastes (effluent from a pulp and paper plant, and fouled membrane solids extracted from a ground water treatment membrane) revealing unique, VP-mediated, kinetic responses. This work demonstrates that iTC can be successfully employed to study the kinetic properties of VP enzymes in order to devise reaction conditions optimized for oxidative degradation of HS present in materials used in a wide range of industries.

Keywords: Allosteric regulation; Biphasic sigmoidal Hill's plot; Environmental biotechnology; Enzyme kinetics; Lignin-manganese-peroxidase.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Allosteric Regulation
Biodegradation, Environmental
Biotechnology
Calorimetry / methods*
Coriolaceae / enzymology
Humic Substances / analysis*
Industrial Waste / analysis*
Kinetics
Lignin
Manganese
Oxidation-Reduction
Peroxidase / chemistry
Peroxidase / metabolism*

Substances

Humic Substances
Industrial Waste
Manganese
Lignin
Peroxidase