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Discovery and Expression of Thermostable LPMOs from Thermophilic Fungi for Producing Efficient Lignocellulolytic Enzyme Cocktails

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Abstract

In this study, two novel thermostable lytic polysaccharide monooxygenases (LPMOs) were cloned from thermophilic fungus Scytalidium thermophilum (PMO9D_SCYTH) and Malbranchea cinnamomea (PMO9D_MALCI) and expressed in the methylotrophic yeast Pichia pastoris X33. The purified PMO9D_SCYTH was active at 60 °C (t1/2 = 60.58 h, pH 7.0), whereas, PMO9D_MALCI was optimally active at 50 °C (t1/2 = 144 h, pH 7.0). The respective catalytic efficiency (kcat/Km) of PMO9D_SCYTH and PMO9D_MALCI determined against avicel in presence of H2O2 was (6.58 × 10-3 and 1.79 × 10-3 mg-1 ml min-1) and carboxy-methylcellulose (CMC) (1.52 × 10-1 and 2.62 × 10-2 mg-1 ml min-1). The HRMS analysis of products obtained after hydrolysis of avicel and CMC showed the presence of both C1 and C4 oxidized oligosaccharides, in addition to phylogenetic tree constructed with other characterized type 1 and 3 LPMOs demonstrated that both LPMOs belongs to type-3 family of AA9s. The release of sugars during saccharification of acid/alkali pretreated sugarcane bagasse and rice straw was enhanced upon replacing one part of commercial enzyme Cellic CTec2 with these LPMOs.

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Acknowledgments

This research was supported by the Department of Biotechnology, India. Projects (BT/PR15271/PBD/26/509/2015) entitled “Bioprospecting for novel lignocellulolytic gylcosyl hydrolases and auxiliary enzymes from diverse thermophilic fungal strains using proteome based approaches” and (BT/PR31115/PBD/26/766/2019) entitled “Novel concepts for developing efficient cellulolytic cocktail for hydrolysis of bio-refinery relevant pre-treated lignocellulosics” are highly acknowledged. Dr. Balan would like to thank the University of Houston for small equipment grant and State of Texas for his startup funds.

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  1. Department of Microbiology, Guru Nanak Dev University, Amritsar, Punjab, 143005, India

    Dhruv Agrawal, Neha Basotra & Bhupinder Singh Chadha

  2. Department Engineering Technology, Biotechnology Program, College of Technology, University of Houston, Houston, TX, 77004, USA

    Venkatesh Balan

  3. Center for Structural and Functional Genomics, Concordia University, 7141 Sherbrooke Street West, Montreal, Quebec, H4B 1R6, Canada

    Adrian Tsang

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Agrawal, D., Basotra, N., Balan, V. et al. Discovery and Expression of Thermostable LPMOs from Thermophilic Fungi for Producing Efficient Lignocellulolytic Enzyme Cocktails. Appl Biochem Biotechnol 191, 463–481 (2020). https://doi.org/10.1007/s12010-019-03198-5

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