Biotechnology Letters

, Volume 41, Issue 1, pp 137–146 | Cite as

Identification and engineering of the key residues at the crevice-like binding site of lipases responsible for activity and substrate specificity

  • Xu Ding
  • Xiao-Ling Tang
  • Ren-Chao Zheng
  • Yu-Guo Zheng
Original Research Paper



Rational engineering of the crevice-like binding site of lipases for improvement of lipases’ catalytic properties.


The residues located at the crevice-like binding site of four representative lipases including Thermomyces lanuginosus lipases (TLL and Lip), Rhizopus oryzae lipase (ROL), and Rhizomucor miehei lipase (RML) were identified through structural analysis. The residues at the bottom of the crevice-like binding site recognizing the substrates with short/medium carbon chain length and those located at the right-hand wall of the surface crevice region affecting the product release were changed by site-directed mutagenesis. The corresponding double mutants exhibited ~ 5 to 14-fold higher activity towards p-nitrophenyl esters than their wild types, and their substrate preference shifted to acyl moiety with shorter carbon chain length. In addition, the mutations led to an increase of B-factor, resulting in decrease of their optimum temperature by 10–20 °C.


The key residues located at the crevice-like binding site play important roles in determining lipase activity, substrate preference and optimum temperature, which offers a useful new paradigm for facilitating rational design of lipases.


Crevice-like binding site Lipase Molecular docking Substrate preference 



This work was financially supported by the National High Technology Research and Development Programs of China (No. 2012AA022201), the Key Scientific and Technology Programs of Zhejiang Province (No. 2012C03005-2) and the Natural Science Foundation of Zhejiang Province (No. Z4090612).

Supporting information

Supplementary Table 1—Primers for site-directed mutagenesis of Lip, TLL, ROL and RML.

Supplementary Fig. 1—Sequence alignment of TTL and homologous proteins.

Supplementary Fig. 2—SDS-PAGE analysis of the purified lipases.

Supplementary Fig. 3—Far-UV CD spectra of wild-type lipase and their mutants plotted by scanning 190-400 nm at 35 °C.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

10529_2018_2620_MOESM1_ESM.docx (2 mb)
Supplementary material 1 (DOCX 2057 kb)


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Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  • Xu Ding
    • 1
    • 2
  • Xiao-Ling Tang
    • 1
    • 2
  • Ren-Chao Zheng
    • 1
    • 2
  • Yu-Guo Zheng
    • 1
    • 2
  1. 1.Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and BioengineeringZhejiang University of TechnologyHangzhouPeople’s Republic of China
  2. 2.Engineering Research Center of Bioconversion and Biopurification of Ministry of EducationZhejiang University of TechnologyHangzhouPeople’s Republic of China

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