Identification and characterization of a novel AA9-type lytic polysaccharide monooxygenase from a bagasse metagenome
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BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Identification and characterization of a novel AA9-type lytic polysaccharide monooxygenase from a bagasse metagenome Benjarat Bunterngsook 1 & Wuttichai Mhuantong 1 & Pattanop Kanokratana 1 & Yu Iseki 2 & Takashi Watanabe 2 & Verawat Champreda 1 Received: 27 May 2020 / Revised: 20 October 2020 / Accepted: 3 November 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Lytic polysaccharide monooxygenases (LPMOs) are auxiliary enzymes catalyzing oxidative cleavages of cellulose chains in crystalline regions, resulting in their increasing accessibility to the hydrolytic enzyme counterparts and hence higher released sugars from biomass saccharification. In this study, a novel auxiliary protein family 9 LPMO (BgAA9) was identified from a metagenomic library derived from a thermophilic microbial community in bagasse collection site where diverse AA9 and AA10 putative sequences were annotated. The enzyme showed highest similarity to a glycoside hydrolase family 61 from Chaetomium thermophilum. Recombinant BgAA9 expressed in Pichia pastoris cleaved cellohexaose (DP6) into shorter cellooligosaccharides (DP2, DP3, and DP4). Supplementation BgAA9 to a commercial cellulase, Accellerase® 1500 showed strong synergistic effect on saccharification of Avicel® PH101, decrystallized cellulose, filter paper, and alkaline-pretreated sugarcane bagasse, resulting in 63–93% increase in the total reducing sugar yield after incubation at 50 °C for 72 h. Strong synergism was shown between BgAA9 and the cellulase with the highest total fermentable sugar yield obtained from 75:25% of Accellerase®1500:BgAA9 which released 39 mg glucose/FPU (filter paper unit) equivalent to 38.7% higher than Accellerase®1500 alone at the same total protein dosage of 5 mg/g substrate according to the mixture design study. The enzyme represented the first characterized LPMO from environmental metagenome and a potent auxiliary component for biomass saccharification. Key points • BgAA9 represents the first characterized LPMO from metagenome. • 12 AA families were annotated in thermophilic bagasse fosmid library by NGS. • BgAA9 showed homology to Cel61 in Chaetomium thermophilum. • BgAA9 oxidized cellohexaose and PASC to DP2, DP4, and DP6. • BgAA9 showed strong synergism to Accellerase on bagasse hydrolysis. Keywords Biorefinery . Lytic polysaccharide monooxygenase . Lignocellulose . Saccharification . Synergy
Introduction Lignocellulosic agricultural biomass can be converted to biofuels, biochemicals, and biomaterials by biorefinery, * Verawat Champreda [email protected] 1
Enzyme Technology Laboratory, Biorefinery and Bioproduct Technology Research Group, National Center for Genetic Engineering and Biotechnology, 113 Thailand Science Park, Phahonyothin Road, Khlong Luang, Pathumthani 12120, Thailand
2
Laboratory of Biomass Conversion, Research Institute for Sustainable Humanosphere (RISH), Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
which is a promising sustainable alternative
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