Abstract
BackgroundThe 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.ResultsWe 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.ConclusionsA 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.
Highlights
The Auxiliary activities family 9 (AA9) family of lytic polysaccharide monooxygenases (AA9 Lytic polysaccharide monooxygenases (LPMO)) is a ubiquitous and diverse group of enzymes in the fungal kingdom
AA9 LPMO activity assay protocol Based on the above results, we suggest the following protocol for assaying AA9 LPMO activity
We suggest that the SsGOOX-based horseradish peroxidase (HRP) colorimetric method for assaying AA9 LPMO activity presented here has the following advantages: (i) after the LPMO reaction, the whole assay process can be conveniently operated on a microtiter plate, and the assay results can be recorded with a microtiter plate reader; it is a highthroughput assay method; (ii) the assay directly targets the main activity of AA9 LPMOs rather than the side activities; (iii) the activities of both C1- and C4-type LPMOs can be assayed; (iv) the method is sensitive and accurate; and (v) except for a microplate reader, no other expensive instrument is needed; it is a cheap and easy-to-use method
Summary
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. High-throughput and direct methods for assaying AA9 LPMO activity, which are prerequisites for screening LPMOs with excellent properties, are still lacking. We present a gluco-oligosaccharide oxidase (GOOX)-based horseradish peroxidase (HRP) colorimetric method for assaying AA9 LPMO activity
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
