Applied Microbiology and Biotechnology

, Volume 96, Issue 2, pp 443–450 | Cite as

Enhanced activity of Rhizomucor miehei lipase by directed evolution with simultaneous evolution of the propeptide

  • Jue Wang
  • Dan Wang
  • Bo Wang
  • Zhuo-hang Mei
  • Ji Liu
  • Hong-wei Yu
Applied genetics and molecular biotechnology
First Online:
Received:
Revised:
Accepted:

Abstract

Propeptides are short sequences that facilitate the folding of their associated proteins. The present study found that the propeptide of Rhizomucor miehei lipase (RML) was not proteolytically removed in Escherichia coli. Moreover, RML was not expressed if the propeptide was removed artificially during the cloning process in E. coli. This behavior in E. coli permitted the application of directed evolution to full-length RML, which included both propeptide and catalytic domain, to explore the role played by the propeptide in governing enzyme activity. The catalytic rate constant, k cat, of the most active mutant RML protein (Q5) was increased from 10.63 ± 0.80 to 71.44 ± 3.20 min−1 after four rounds of screening. Sequence analysis of the mutant displayed three mutations in the propeptide (L57V, S65A, and V67A) and two mutations in the functional region (I111T and S168P). This result showed that improved activity was obtained with essential involvement by mutations in the propeptide, meaning that the majority of mutants with enhanced activity had simultaneous mutations in propeptide and catalytic domains. This observation leads to the hypothesis that directed evolution has simultaneous and synergistic effects on both functional and propeptide domains that arise from the role played by the propeptide in the folding and maturation of the enzyme. We suggest that directed evolution of full-length proteins including their propeptides is a strategy with general validity for extending the range of conformations available to proteins, leading to the enhancement of the catalytic rates of the enzymes.

Keywords

RML Propeptide Protein folding Directed evolution Activity 
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Notes

Acknowledgments

This work was financially supported by the National High-tech R&D Program (2010AA101502), the Natural Science Foundation of China (21176215 and 21176102) and the Outstanding Young Scholar of Zhejiang Province (R4110092).

Supplementary material

253_2012_4049_MOESM1_ESM.pdf (237 kb)
ESM 1 (PDF 237 kb)

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Jue Wang
    • 1
  • Dan Wang
    • 1
  • Bo Wang
    • 1
  • Zhuo-hang Mei
    • 1
  • Ji Liu
    • 1
  • Hong-wei Yu
    • 1
  1. 1.Institute of Bioengineering, Department of Chemical and Biological EngineeringZhejiang UniversityHangzhouPeople’s Republic of China

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