Applied Microbiology and Biotechnology

, Volume 103, Issue 10, pp 4077–4087 | Cite as

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


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.


γ-lactamase Promiscuity Phenylglycidate Substrate screening 



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)


  1. Aharoni A, Gaidukov L, Khersonsky O, Gould SM, Roodveldt C, Tawfik DS (2005) The ‘evolvability’ of promiscuous protein functions. Nat Genet 37(1):73–76. Google Scholar
  2. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215(3):403–410. Google Scholar
  3. Arpigny JL, Jaeger KE (1999) Bacterial lipolytic enzymes: classification and properties. Biochem J 343(1):177–183Google Scholar
  4. Bornscheuer UT, Kazlauskas RJ (2004) Catalytic promiscuity in biocatalysis: using old enzymes to form new bonds and follow new pathways. Angew Chem Int Ed Eng 43(45):6032–6040. Google Scholar
  5. Chen CS, Wu SH, Girdaukas G, Sih CJ (1987) Quantitative analyses of biochemical kinetic resolution of enantiomers. 2. Enzyme-catalyzed esterifications in water-organic solvent biphasic systems. ChemInform 109(36):2812–2817Google Scholar
  6. Cilia E, Fabbri A, Uriani M, Scialdone GG, Ammendola S (2005) The signature amidase from Sulfolobus solfataricus belongs to the CX3C subgroup of enzymes cleaving both amides and nitriles. Ser195 and Cys145 are predicted to be the active site nucleophiles. FEBS J 272(18):4716–4724. Google Scholar
  7. Gao P, Wu S, Praveen P, Loh KC, Li Z (2016) Enhancing productivity for cascade biotransformation of styrene to (S)-vicinal diol with biphasic system in hollow fiber membrane bioreactor. Appl Microbiol Biotechnol 101:1857–1868. Google Scholar
  8. Hogrefe HH, Cline J, Youngblood GL, Allen RM (2002) Creating randomized amino acid libraries with the QuikChange (R) Multi Site-Directed Mutagenesis Kit. Biotechniques 33(5):1158–1165Google Scholar
  9. Holmquist M (2000) Alpha/Beta-hydrolase fold enzymes: structures, functions and mechanisms. Curr Protein Pept Sci 1(2):209–235Google Scholar
  10. Hult K, Berglund P (2007) Enzyme promiscuity: mechanism and applications. Trends Biotechnol 25(5):231–238. Google Scholar
  11. Kazlauskas RJ (2005) Enhancing catalytic promiscuity for biocatalysis. Curr Opin Chem Biol 9(2):195–201. Google Scholar
  12. Khersonsky O, Tawfik DS (2010) Enzyme promiscuity: a mechanistic and evolutionary perspective. Annu Rev Biochem 79:471–505. Google Scholar
  13. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685Google Scholar
  14. Li H, Chen C, Cao X (2015) Essential oils-oriented chiral esters as potential pesticides: asymmetric syntheses, characterization and bio-evaluation. Ind Crop Prod 76:432–436. Google Scholar
  15. Line K, Isupov MN, Littlechild JA (2004) The crystal structure of a (-) gamma-lactamase from an Aureobacterium species reveals a tetrahedral intermediate in the active site. J Mol Biol 338(3):519–532. Google Scholar
  16. Mitsukura K, Shimizu M, Matsushita K, Yoshida T, Nagasawa T (2010) Characteristics and function of Alcaligenes sp. NBRC 14130 esterase catalysing the stereo-selective hydrolysis of ethyl chrysanthemate. J Appl Microbiol 108(4):1263–1270. Google Scholar
  17. Nishizawa M, Gomi H, Kishimoto F (1993) Purification and some properties of carboxylesterase from Arthrobacter globiformis; stereoselective hydrolysis of ethyl chrysanthemate. Agric Biol Chem 57(4):594–598Google Scholar
  18. O’Brien PJ, Herschlag D (1999) Catalytic promiscuity and the evolution of new enzymatic activities. Chem Biol 6(4):R91–R105. Google Scholar
  19. Shin S, Lee TH, Ha NC, Koo HM, Kim SY, Lee HS, Kim YS, Oh BH (2002) Structure of malonamidase E2 reveals a novel Ser-cisSer-Lys catalytic triad in a new serine hydrolase fold that is prevalent in nature. EMBO J 21(11):2509–2516. Google Scholar
  20. Singh R, Vince R (2012) 2-Azabicyclo[2.2.1]hept-5-en-3-one: chemical profile of a versatile synthetic building block and its impact on the development of therapeutics. Chem Rev 112(8):4642–4686. Google Scholar
  21. Sun Y, Zhao H, Wang J, Zhu J, Wu S (2015) Identification and regulation of the catalytic promiscuity of (-)-gamma-lactamase from Microbacterium hydrocarbonoxydans. Appl Microbiol Biotechnol 99(18):7559–7568. Google Scholar
  22. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22(22):4673–4680Google Scholar
  23. Torres LL, Schliessmann A, Schmidt M, Silva-Martin N, Hermoso JA, Berenguer J, Bornscheuer UT, Hidalgo A (2012) Promiscuous enantioselective (-)-gamma-lactamase activity in the Pseudomonas fluorescens esterase I. Org Biomol Chem 10(17):3388–3392. Google Scholar
  24. Wang J, Zhang X, Min C, Wu S, Zheng G (2011) Single-step purification and immobilization of γ-lactamase and on-column transformation of 2-azabicyclo [2.2.1] hept-5-en-3-one. Process Biochem 46(1):81–87Google Scholar
  25. Wang J, Zhu J, Wu S (2015) Immobilization on macroporous resin makes E. coli RutB a robust catalyst for production of (-) Vince lactam. Appl Microbiol Biotechnol 99(11):4691–4700. Google Scholar
  26. Wei C, Ling J, Shen H, Zhu Q (2014) Bioresolution production of (2R,3S)-ethyl-3-phenylglycidate for chemoenzymatic synthesis of the Taxol C-13 side chain by Galactomyces geotrichum ZJUTZQ200, a new epoxide-hydrolase-producing strain. Molecules 19(6):8067–8079. Google Scholar
  27. Xue S-S, Zhao M, Lan J-X, Ye R-R, Li Y, Ji L-N, Mao Z-W (2016) Enantioselective hydrolysis of amino acid esters by non-chiral copper complexes equipped with bis (β-cyclodextrin)s. J Mol Catal A Chem 424:297–303. Google Scholar
  28. Yan X, Wang J, Sun Y, Zhu J, Wu S (2016) Facilitating the evolution of esterase activity from a promiscuous enzyme (Mhg) with catalytic functions of amide hydrolysis and carboxylic acid perhydrolysis by engineering the substrate entrance tunnel. Appl Environ Microbiol 82(22):6748–6756. Google Scholar
  29. Yin JG, Gong Y, Zhang XY, Zheng GW, Xu JH (2016) Green access to chiral Vince lactam in a buffer-free aqueous system using a newly identified substrate-tolerant (-)-gamma-lactamase. Catal Sci Technol 6(16):6305–6310. Google Scholar
  30. Zhao H, Caflisch A (2014) Discovery of dual ZAP70 and Syk kinases inhibitors by docking into a rare C-helix-out conformation of Syk. Bioorg Med Chem Lett 24(6):1523–1527Google Scholar
  31. Zheng G, Yuan Q, Yang L, Zhang X, Wang J, Sun W (2006) Preparation of (2S, 3R)-methyl-3-phenylglycidate using whole cells of Pseudomonas putida. J Mol Catal B Enzym 43(1–4):133–136. Google Scholar
  32. Zhou D, Pan J, Yu H, Zheng G, XU J (2013) Target-oriented discovery of a new esterase-producing strain Enterobacter sp. ECU1107 for whole cell-catalyzed production of (2S,3R)-3-phenylglycidate as a chiral synthon of Taxol. Appl Microbiol Biotechnol 97(14):6293–6300. Google Scholar

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

Personalised recommendations