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Enhancing the atypical esterase promiscuity of the γ-lactamase Sspg from Sulfolobus solfataricus by substrate screening

  • Jianjun Wang
  • Hongtao Zhao
  • Guogang Zhao
  • Dunfu Chen
  • Yong Tao
  • Sheng WuEmail author
Biotechnologically relevant enzymes and proteins
  • 69 Downloads

Abstract

Promiscuous enzymes can be modified by protein engineering, which enables the catalysis of non-native substrates. γ-lactamase Sspg from Sulfolobus solfataricus is an enzyme with high activity, high stability, and pronounced tolerance of high concentrations of the γ-lactam substrate. These characteristics suggest Sspg as a robust enzymatic catalyst for the preparation of optically pure γ-lactam. This study investigated the modification of this enzyme to expand its application toward resolving chiral esters. γ-Lactamase-esterase conversion was performed by employing a three-step method: initial sequence alignment, followed by substrate screening, and protein engineering based on the obtained substrate-enzyme docking results. This process of fine-tuning of chemical groups on substrates has been termed “substrate screening.” Steric hindrance and chemical reactivity of the substrate are major concerns during this step, since both are determining factors for the enzyme-substrate interaction. By employing this three-step method, γ-lactamase Sspg was successfully converted into an esterase with high enantioselectivity towards phenylglycidate substrates (E value > 300). However, since both wild-type Sspg and Sspg mutants did not hydrolyze para-nitrophenyl substrates (pNPs), this esterase activity was termed “atypical esterase activity.” The γ-lactamase activity and stability of the Sspg mutants were not severely compromised. The proposed method can be applied to find novel multi-functional enzyme catalysts within existing enzyme pools.

Keywords

γ-lactamase Promiscuity Phenylglycidate Substrate screening 

Notes

Funding

This study was funded by a grant from the National Natural Science Foundation of China (No. 31570077 to SW).

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2019_9758_MOESM1_ESM.pdf (357 kb)
ESM 1 (PDF 357 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Microbial Resources, Institute of MicrobiologyChinese Academy of SciencesBeijingPeople’s Republic of China
  2. 2.Medivir ABStockholmSweden
  3. 3.The College of Life SciencesHebei Agricultural UniversityBaodingPeople’s Republic of China
  4. 4.Medical CollegeYanbian UniversityJilinPeople’s Republic of China

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