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Refolding with Simultaneous Purification of Recombinant Serratia marcescens Lipase by One-Step Ultrasonication Process

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Abstract

A new lipase from Serratia marcescens SRICI-01 (Trx-SmL) was successfully overexpressed in Escherichia coli with thioredoxin (Trx) fusion tag. Intriguingly, the concentration of potassium phosphate buffer (KPB) showed significant impact on the aggregation state of Trx-SmL during ultrasonic disruption. The proportion of inclusion bodies increased dramatically with the increase of KPB concentration from almost completely soluble in 10 mM KPB to insoluble in 200 mM KPB. Based on this new finding, a novel method for refolding and purification of recombinant Trx-SmL was developed by one-step ultrasonication. The Trx-SmL was firstly precipitated in 200 mM KPB, washed for three times, and subsequently subjected to ultrasonic process in 10 mM KPB where refolding and purification occurred simultaneously. This established method was proved to be a straightforward, economical, and efficient purification approach to facilely obtain recombinant Trx-SmL protein with high purity (> 90%) and activity recovery yield (> 80%) from cell lysates. The application potential of the purified fusion Trx-SmL was further demonstrated by kinetic bioresolution of (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM] producing optically pure (−)-MPGM, a key intermediate for diltiazem, with an overall yield of 41.5% and ee of 99%.

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Abbreviations

MPGM:

3-(4′-Methoxyphenyl)glycidic acid methyl ester

SmL:

Serratia marcescens lipase

Trx-SmL:

Serratia marcescens lipase fused with thioredoxin

KPB:

Potassium phosphate buffer

IBs:

Inclusion bodies

pNPA:

p-Nitrophenyl acetate

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Acknowledgments

Thanks are due to Prof. Xu Yi, School of Chemical and Environmental Engineering Shanghai Institute of Technology, for providing the organism Serratia marcescens.

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Correspondence to Yue-Cai Yin or Xin-Sen Wu.

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Yin, YC., Li, HQ. & Wu, XS. Refolding with Simultaneous Purification of Recombinant Serratia marcescens Lipase by One-Step Ultrasonication Process. Appl Biochem Biotechnol 191, 1670–1683 (2020). https://doi.org/10.1007/s12010-019-03172-1

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