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

  • Dhruv Agrawal
  • Neha Basotra
  • Venkatesh Balan
  • Adrian Tsang
  • Bhupinder Singh ChadhaEmail author
Article
  • 39 Downloads

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.

Keywords

Thermophilic fungi Heterologous expression Lytic polysaccharide monooxygenases (LPMOs) H2O2 Hydrolysis 

Notes

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.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no competing interests.

Supplementary material

12010_2019_3198_MOESM1_ESM.doc (896 kb)
ESM 1(DOC 896 kb)

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Authors and Affiliations

  1. 1.Department of MicrobiologyGuru Nanak Dev UniversityAmritsarIndia
  2. 2.Department Engineering Technology, Biotechnology Program, College of TechnologyUniversity of HoustonHoustonUSA
  3. 3.Center for Structural and Functional GenomicsConcordia UniversityQuebecCanada

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