A fast and sensitive activity assay for lytic polysaccharide monooxygenase

Abstract

BackgroundLytic polysaccharide monooxygenases (LPMO) release a spectrum of cleavage products from their polymeric substrates cellulose, hemicellulose, or chitin. The correct identification and quantitation of these released products is the basis of MS/HPLC-based detection methods for LPMO activity. The duration, effort, and intricate analysis allow only specialized laboratories to measure LPMO activity in day-to-day work. A spectrophotometric assay will simplify the screening for LPMO in culture supernatants, help monitor recombinant LPMO expression and purification, and support enzyme characterization.ResultsBased on a newly discovered peroxidase activity of LPMO, we propose a fast, robust, and sensitive spectrophotometric activity assay using 2,6-dimethoxyphenol (2,6-DMP) and H2O2. The fast enzymatic assay (300 s) consists of 1 mM 2,6-DMP as chromogenic substrate, 100 µM H2O2 as cosubstrate, and an adequate activity of LPMO in a suitable buffer. The high molar absorption coefficient of the formed product coerulignone (ε469 = 53,200 M−1 cm−1) makes the assay sensitive and allows reliable activity measurements of LPMO in concentrations of approx. 0.5–50 mg L−1.ConclusionsThe activity assay based on 2,6-DMP detects a novel peroxidase activity of LPMO. This activity can be accurately measured and used for enzyme screening, production, and purification, and can also be applied to study binding constants or thermal stability. However, the assay has to be used with care in crude extracts, because other enzymes such as laccase or peroxidase will interfere with the assay. We also want to stress that the peroxidase activity is a homogeneous reaction with soluble substrates and should not be correlated to heterogeneous LPMO activity on polymeric substrates.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1063-6) contains supplementary material, which is available to authorized users.

Detection and Characterization of a Novel Copper‐Dependent Intermediate in a Lytic Polysaccharide Monooxygenase

“…LPMO activity assay using 2,6‐dimethoxyphenol (2,6‐DMP) was performed as described by Breslmayr et al. (2018) . The standard reaction mixture for the LPMO activity assay contained 100 μ m H 2 O 2 , 1.0 m m 2,6‐DMP and 4 μ m of TaLPMO9A.…”

Lignocellulose degradation: An overview of fungi and fungal enzymes involved in lignocellulose degradation

“…All these substrates show a common feature, namely the β-1,4 linkages connecting the single sugar moieties in the backbone. A fast and sensitive spectrophotometric (using 2,6-dimethoxyphenol as chromogenic substrate and H 2 O 2 as cosubstrate) assay for LPMO activity determination was developed to follow the production and purification as well as to study enzyme-binding constants or thermal stability [85]. α-1,4 bonds).…”

Biochemical, structural insights of newly isolated AA16 family of Lytic Polysaccharide Monooxygenase (LPMO) fromAspergillus fumigatusand investigation of its synergistic effect using biomass

“…Enzyme kinetics using 2,6-DMP as substrate (Breslmayr et al 2018) confirmed the oxidative activity of the enzyme. So the biochemical and structural information and the efficiency in lignocellulosic biomass conversion suggested that AfLPMO16 could be an important member of the Cellulase cocktail of industrial use.…”

“…2,6 DMP (2,6-dimethoxyphenol) was used as a substrate for AfLPMO16 in this study (Breslmayr et al 2018). The reaction was done in phosphate buffer (100mM pH 6.0) containing 10mM DMP, 5μM hydrogen peroxide, and 50μg of purified AfLPMO16 at 30 .…”

“…3b). The activity of the protein was also verified by using 2,6 DMP as substrate (Breslmayr et al 2018). The expressed recombinant AfLPMO16 band appeared at approximately 32kDa position in SDS-PAGE ( Fig.…”