Abstract
Objective
We aimed to characterize a novel SGNH (Ser-Gly-Asn-His) family hydrolase from the annotated genome of marine bacteria with new features.
Results
A novel esterase Ali5 from Altererythrobacter ishigakiensis has been identified and classified into SGNH family. Ali5 presented a novel GNSL (Gly-Asn-Ser-Leu(X)) motif that differs from the classic GDSL (Gly-Asp-Ser-Leu(X)) motif of SGNH family. The enzyme has esterase and thioesterase activity and exhibited apparent temperature and pH optima of 40 °C and pH 7.5 (in phosphate buffer), respectively. Ali5 was found to be halotolerant and thermostable, and exhibited strong resistance to several organic solvents and metal ions. The residue Tyr196 has a great influence on the catalytic activity, which was proved by site-directed mutagenesis and subsequent kinetic characterization.
Conclusion
The esterase Ali5 with esterase and thioesterase activities, salt and metal ions resistance and unique structural features was identified, which holds promise for research on the SGNH family of hydrolases.
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Acknowledgements
This work was supported by Grants from the National Key R&D Program of China (2018YFC0310704), the National Natural Science Foundation of China (31770004, 41506183) and the Natural Science Foundation of Zhejiang Province (No. LR17D060001).
Supporting information
Supplementary Fig. 1—Kinetics plots of Ali5, N55D and Y196G for substrates p-NP butyrate (p-NPC4). a Michaelis–Menten plot of Ali5 for p-NPC4. b Michaelis–Menten plot of N55D for p-NPC4. c Michaelis–Menten plot of Y196G for p-NPC4. All values are the average of three independent assays.
Supplementary Fig. 2—Effects of substrate concentrations on thioesterase activity of Ali5, N55D and Y196G, with palmitoyl-CoA (C16-CoA) as the substrate. a Effects of C16-CoA concentrations on thioesterase activity of Ali5. b Effects of C16-CoA concentrations on thioesterase activity of N55D. c Effects of C16-CoA) concentrations on thioesterase activity of Y196G. All values are the average of three independent assays.
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Hong, LG., Jian, SL., Huo, YY. et al. A novel SGNH family hydrolase Ali5 with thioesterase activity and a GNSL motif but without a classic GDSL motif from Altererythrobacter ishigakiensis. Biotechnol Lett 41, 591–604 (2019). https://doi.org/10.1007/s10529-019-02662-w
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DOI: https://doi.org/10.1007/s10529-019-02662-w