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Identification and Characterization of a Novel Carboxylesterase Belonging to Family VIII with Promiscuous Acyltransferase Activity Toward Cyanidin-3-O-Glucoside from a Soil Metagenomic Library

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Abstract

An alkaline esterase, designated as EstXT1, was identified through functional screening from a metagenomic library. Sequence analysis revealed that EstXT1 belonged to the family VIII carboxylesterases and contained a characteristic conserved S-x-x-K motif and a deduced catalytic triad Ser56-Lys59-Tyr165. EstXT1 exhibited the strongest activity toward methyl ferulate at pH 8.0 and temperature 55°C and retained over 80% of its original activity after incubation in the pH range of 7.0–10.6 buffers. Biochemical characterization of the recombinant enzyme showed that it was activated by Zn2+ and Co2+ metal ion, while inhibited by Cu2+ and CTAB. EstXT1 exhibited significant promiscuous acyltransferase activity preferred to the acylation of benzyl alcohol acceptor using short-chain pNP-esters (C2-C8) as acyl-donors. A structure–function analysis indicated that a WAG motif is essential to acyltransferase activity. This is the first report example that WAG motif plays a pivotal role in acyltransferase activity in family VIII carboxylesterases beside WGG motif. Further experiment indicated that EstXT1 successfully acylated cyanidin-3-O-glucoside in aqueous solution. The results from the current investigation provided new insights for the family VIII carboxylesterase and lay a foundation for the potential applications of EstXT1 in food and biotechnology fields.

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Funding

This research was supported by the Key Research and Development Program of Jiangsu Province (BE2017374-2) and special funds of agroproduct quality safety risk assessment of the Ministry of Agriculture of the People’s Republic of China (GJFP201701505; GJFP2019019).

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  1. Key Laboratory of Food Processing and Quality Control, College of Food Science and Technology, Nanjing Agricultural University, Nanjing, 210095, People’s Republic of China

    Yueqi Zhang, Liping Ding, Zhenzhen Yan, Dandan Zhou, Junwei Jiang, Jiarong Qiu & Zhihong Xin

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YZ and ZX conceived and designed research. YZ and LD conducted experiments. ZY, DZ, JJ, and JQ contributed new reagents or analytical tools. YZ and LD analyzed data and wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to Zhihong Xin.

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Zhang, Y., Ding, L., Yan, Z. et al. Identification and Characterization of a Novel Carboxylesterase Belonging to Family VIII with Promiscuous Acyltransferase Activity Toward Cyanidin-3-O-Glucoside from a Soil Metagenomic Library. Appl Biochem Biotechnol 195, 2432–2450 (2023). https://doi.org/10.1007/s12010-021-03614-9

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