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Structure-based engineering of a pectate lyase with improved specific activity for ramie degumming

  • Biotechnologically relevant enzymes and proteins
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Abstract

Biotechnological applications of microbial pectate lyases (Pels) in plant fiber processing are promising, eco-friendly substitutes for conventional chemical degumming processes. However, to potentiate the enzymes’ use for industrial applications, resolving the molecular structure to elucidate catalytic mechanisms becomes necessary. In this manuscript, we report the high resolution (1.45 Å) crystal structure of pectate lyase (pelN) from Paenibacillus sp. 0602 in apo form. Through sequence alignment and structural superposition with other members of the polysaccharide lyase (PL) family 1 (PL1), we determined that pelN shares the characteristic right-handed β-helix and is structurally similar to other members of the PL1 family, while exhibiting key differences in terms of catalytic and substrate binding residues. Then, based on information from structure alignments with other PLs, we engineered a novel pelN. Our rational design yielded a pelN mutant with a temperature for enzymatic activity optimally shifted from 67.5 to 60 °C. Most importantly, this pelN mutant displayed both higher specific activity and ramie fiber degumming ability when compared with the wild-type enzyme. Altogether, our rational design method shows great potential for industrial applications. Moreover, we expect the reported high-resolution crystal structure to provide a solid foundation for future rational, structure-based engineering of genetically enhanced pelNs.

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Acknowledgements

The authors thank Dr. Yuzhong Zhang for his kind help during data collection and Jie Zhang for assistance with structure determination. This work was supported, in part, by grants from the Hundred Talents Program of the Chinese Academy of Sciences (CAS), the Knowledge Innovation Program of CAS (KSCX2-EW-G-8), and the Tianjin Municipal Science & Technology Commission (10ZCKFSY05600). T.M. L and A.L. would like to thank the Isaac Newton Institute for Mathematical Sciences for support through EPSRC Grant No. EP/K032208/1. J.S. is a recipient of the Hundred Talents Program of CAS. J.L. is an NHMRC Senior Research Fellow.

This article does not contain any studies with human participants or animals.

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Authors and Affiliations

  1. National Engineering Laboratory for Industrial Enzymes and Key Laboratory of Systems Microbial Biotechnology, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China

    Zhanping Zhou, Yang Liu, Zhenying Chang, Yanhe Ma & Jiangning Song

  2. Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China

    Huilin Wang

  3. Isaac Newton Institute for Mathematical Sciences, University of Cambridge, Cambridge, UK

    André Leier & Tatiana T. Marquez-Lago

  4. Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Melbourne, VIC, 3800, Australia

    Jian Li

  5. Infection and Immunity Program, Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, Monash University, Melbourne, VIC, 3800, Australia

    Jiangning Song

  6. Monash Centre for Data Science, Faculty of Information Technology, Monash University, Melbourne, VIC, 3800, Australia

    Jiangning Song

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  1. Zhanping Zhou
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  2. Yang Liu
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  3. Zhenying Chang
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Correspondence to Jiangning Song.

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Zhou, Z., Liu, Y., Chang, Z. et al. Structure-based engineering of a pectate lyase with improved specific activity for ramie degumming. Appl Microbiol Biotechnol 101, 2919–2929 (2017). https://doi.org/10.1007/s00253-016-7994-6

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  • DOI: https://doi.org/10.1007/s00253-016-7994-6

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