Abstract
A novel esterase (EstKa) from marine Klebsiella aerogenes was characterized with hydrolytic activity against p-nitrophenyl caprylate (pNPC, C8) under optimum conditions (50 °C and pH 8.5). After two rounds of mutagenesis, two highly potential mutants (I6E9 and L7B11) were obtained with prominent activity, substrate affinity and thermostability. I6E9 (L90Q/P96T) and L7B11 (A37S/Q100L/S133G/R138C/Q156R) were 1.56- and 1.65-fold higher than EstKa in relative catalytic efficiency. The influence of each amino acid on enzyme activity was explored by site-directed mutation. The mutants Pro96Thr and Gln156Arg showed 1.29- and 1.48-fold increase in catalytic efficiency (Kcat/Km) and 54.4 and 36.2% decrease in substrate affinity (Km), respectively. The compound mutant Pro96Thr/Gln156Arg exhibited 68.9% decrease in Km and 1.41-fold increase in Kcat/Km relative to EstKa. Homology model structure analysis revealed that the replacement of Gln by hydrophilic Arg on the esterase surface improved the microenvironment stability and the activity. The replacement of Pro by Thr enabled the esterase enzyme to retain 90% relative activity after 3 h incubation at 45 °C. Structural analysis confirmed that the formation of a hydrogen bond leads to a notable increase of catalytic efficiency under high temperature conditions.
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Abbreviations
- E. coli :
-
Escherichia coli
- C2 :
-
p-Nitrophenyl acetate (pNPA)
- C4 :
-
p-Nitrophenyl butyrate (pNPB)
- C6 :
-
p-Nitrophenyl hexanoate (pNPH)
- C8 :
-
p-Nitrophenyl caprylate (pNPC)
- C12 :
-
p-Nitrophenyl laurate (pNPL)
- C16 :
-
p-Nitrophenyl palmitate (pNPP)
- IPTG:
-
Isopropyl β-D-thiogalactopyranoside
- PBS:
-
Phosphate buffered saline
- GST:
-
Glutathione S-transferase
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Acknowledgements
This work was supported by grants from The Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture and Top-notch Academic Programs Project of Jiangsu Higher Education Institutions
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GHF and ZRT equally designed and performed the experiments and drafted the manuscript. LZL contributed to protein expression and purification; WWY performed the characterization; LZD contributed to revision of the manuscript. HN is the corresponding author, who conceived and supervised the experiments. All authors have read and approved the final manuscript.
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Gao, H., Zhu, R., Li, Z. et al. Improving the catalytic efficiency and substrate affinity of a novel esterase from marine Klebsiella aerogenes by random and site-directed mutation. World J Microbiol Biotechnol 37, 106 (2021). https://doi.org/10.1007/s11274-021-03069-4
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DOI: https://doi.org/10.1007/s11274-021-03069-4