Activity and substrate specificity of lytic polysaccharide monooxygenases: An ATR FTIR-based sensitive assay tested on a novel species from Pseudomonas putida

TY - JOUR

T1 - Activity and substrate specificity of lytic polysaccharide monooxygenases

T2 - An ATR FTIR-based sensitive assay tested on a novel species from Pseudomonas putida

AU - Serra, Ilenia

AU - Piccinini, Daniele

AU - Paradisi, Alessandro

AU - Ciano, Luisa

AU - Bellei, Marzia

AU - Bortolotti, Carlo Augusto

AU - Battistuzzi, Gianantonio

AU - Sola, Marco

AU - Walton, Paul H.

AU - Di Rocco, Giulia

N1 - Funding Information: This work was supported by the University of Modena and Reggio Emilia Finanziamento FARDSV 2019. Luisa Ciano and Paul H. Walton gratefully acknowledge the support of the UK's Biotechnology and Biological Sciences Research Council (BBSRC) through grants BB/L001926/1 and BB/L021633/1. Alessandro Paradisi and Paul H. Walton gratefully thank the Lesley Wild Scholarship for funding. Publisher Copyright: © 2021 The Protein Society.

PY - 2021/12/11

Y1 - 2021/12/11

N2 - Pseudomonas putida W619 is a soil Gram-negative bacterium commonly used in environmental studies thanks to its ability in degrading many aromatic compounds. Its genome contains several putative carbohydrate-active enzymes such as glycoside hydrolases and lytic polysaccharide monooxygenases (PMOs). In this study, we have heterologously produced in Escherichia coli and characterized a new enzyme belonging to the AA10 family, named PpAA10 (Uniprot: B1J2U9), which contains a chitin-binding type-4 module and showed activity toward β-chitin. The active form of the enzyme was produced in E. coli exploiting the addition of a cleavable N-terminal His tag which ensured the presence of the copper-coordinating His as the first residue. Electron paramagnetic resonance spectroscopy showed signal signatures similar to those observed for the copper-binding site of chitin-cleaving PMOs. The protein was used to develop a versatile, highly sensitive, cost-effective and easy-to-apply method to detect PMO's activity exploiting attenuated total reflection-Fourier transform infrared spectroscopy and able to easily discriminate between different substrates.

AB - Pseudomonas putida W619 is a soil Gram-negative bacterium commonly used in environmental studies thanks to its ability in degrading many aromatic compounds. Its genome contains several putative carbohydrate-active enzymes such as glycoside hydrolases and lytic polysaccharide monooxygenases (PMOs). In this study, we have heterologously produced in Escherichia coli and characterized a new enzyme belonging to the AA10 family, named PpAA10 (Uniprot: B1J2U9), which contains a chitin-binding type-4 module and showed activity toward β-chitin. The active form of the enzyme was produced in E. coli exploiting the addition of a cleavable N-terminal His tag which ensured the presence of the copper-coordinating His as the first residue. Electron paramagnetic resonance spectroscopy showed signal signatures similar to those observed for the copper-binding site of chitin-cleaving PMOs. The protein was used to develop a versatile, highly sensitive, cost-effective and easy-to-apply method to detect PMO's activity exploiting attenuated total reflection-Fourier transform infrared spectroscopy and able to easily discriminate between different substrates.

KW - activity

KW - ATR FTIR spectroscopy

KW - chitin

KW - EPR

KW - lytic polysaccharide monooxygenase

UR - http://www.scopus.com/inward/record.url?scp=85121435202&partnerID=8YFLogxK

U2 - 10.1002/pro.4255

DO - 10.1002/pro.4255

M3 - Article

AN - SCOPUS:85121435202

SN - 0961-8368

JO - Protein science

JF - Protein science

ER -