A sensitive, accurate, and high-throughput gluco-oligosaccharide oxidase-based HRP colorimetric method for assaying lytic polysaccharide monooxygenase activity

Abstract

Background The AA9 (auxiliary activities) family of lytic polysaccharide monooxygenases (AA9 LPMOs) is a ubiquitous and diverse group of enzymes in the fungal kingdom. They catalyse the oxidative cleavage of glycosidic bonds in lignocellulose and exhibit great potential for biorefinery applications. Robust, high-throughput and direct methods for assaying AA9 LPMO activity, which are prerequisites for screening LPMOs with excellent properties, are still lacking. Here, we present a gluco-oligosaccharide oxidase (GOOX)-based horseradish peroxidase (HRP) colorimetric method for assaying AA9 LPMO activity. Results We cloned and expressed a GOOX gene from Sarocladium strictum in Trichoderma reesei, purified the recombinant SsGOOX, validated its properties, and developed an SsGOOX-based HRP colorimetric method for assaying cellobiose concentrations. Then, we expressed two AA9 LPMOs from Thielavia terrestris, TtAA9F and TtAA9G, in T. reesei, purified the recombinant proteins, and analysed their product profiles and regioselectivity towards phosphoric acid swollen cellulose (PASC). TtAA9F was characterized as a C1-type (class 1) LPMO, while TtAA9G was characterized as a C4-type (class 2) LPMO. Finally, the SsGOOX-based HRP colorimetric method was used to quantify the total concentration of reducing lytic products from the LPMO reaction, and the activities of both the C1- and C4-type LPMOs were analysed. These LPMOs could be effectively analysed with limits of detection (LoDs) less than 30 nmol/L, and standard curves between the A515 and LPMO concentrations with determination coefficients greater than 0.994 were obtained. Conclusions A novel, sensitive and accurate assay method that directly targets the main activity of both C1- and C4-type AA9 LPMOs was established. This method is easy to use and could be performed on a microtiter plate for high-throughput screening of AA9 LPMOs with desirable properties.

Analysis of lytic polysaccharide monooxygenase activity in thermophilic fungi by high-performance liquid chromatography–refractive index detector

“…An accurate and comprehensive method for the determination of LPMO activity can screen out highly active LPMOs, as well as better study their properties and catalytic mechanisms ( Kittl et al, 2012 ; Wu et al, 2022 ). The previous methods for activity assay mainly focus on the side activity of LPMO, mostly by quantifying the formation of H 2 O 2 to assay the activity of LPMO ( Kittl et al, 2012 ; Wang et al, 2018 ; Wu et al, 2022 ), but H 2 O 2 is unstable under high-temperature conditions. Furthermore, it is difficult to pool all the different oligosaccharides produced by LPMO for assaying by the existing methods ( Breslmayr et al, 2018 , 2019 ; Brander et al, 2021 ; Wang et al, 2021 ).…”

“…This method directly targets LPMO activity but can only detect LPMO with stereoselectivity for C1 oxidation, not for C4 oxidation. Recently, a horseradish peroxidase (HRP) colorimetric method was developed based on gluco-oligosaccharide oxidase (GOOX) to determine AA9 LPMO activity ( Wu et al, 2022 ). However, the use of the HRP colourimetric method can be somewhat limited if the sample is contaminated with endoglucanase.…”

Characterization of a thermostable cellobiose dehydrogenase from Myceliophthora thermophila and its function of electron‐donating for lytic polysaccharide monooxygenase

“…A recombinant C4‐type AA9 family LPMO from Thielavia terrestris ( Tt AA9G) was used in the LPMO reaction. The protein was expressed and purified as described previously 32 . Lysing enzyme from Trichodema harzianum was purchased from Sigma (Saint Louis, MO, USA); mouse anti‐6 × His monoclonal antibody and HRP‐labeled rabbit anti‐mouse IgG were purchased from Yeasen Biotechnology (Shanghai, China); Taq DNA polymerase and ClonExpress™ II were purchased from Vazyme (Nanjing, China).…”

“…The protein was expressed and purified as described previously. 32 Lysing enzyme from Trichodema harzianum was purchased from Sigma (Saint Louis, MO, USA); mouse anti-6 × His monoclonal antibody and HRP-labeled rabbit anti-mouse IgG were purchased from Yeasen Biotechnology (Shanghai, China); Taq DNA polymerase and ClonExpress™ II were purchased from Vazyme (Nanjing, China). The fungal genomic DNA extraction kit (B518259-0100) was purchased from Sangon Biotech (Shanghai, China), and standard protein markers (Thermo Scientific PageRuler Prestained Protein Ladder) were purchased from Thermo Fisher Scientific (Waltham, MA, USA).…”

“…Western blotting was performed using an anti-6× His-tag mouse monoclonal antibody and horseradish peroxidase-conjugated rabbit antimouse IgG (Yeasen Biotechnology). Recombinant proteins were purified as described by Wu et al 32 For the mass spectrum analysis of the recombinant proteins, gel pieces containing the target protein bands were cut, destained, dehydrated and digested with trypsin. After digestion, peptides were extracted and processed for matrix-assisted laser desorption ionization-time of flight (MALDI TOF/ TOF) MS analysis (ABI 4800 MALDI-TOF/TOF proteomics analyzer mass spectrometer, AB SCIEX).…”