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Heterologous expression, refolding, and characterization of a calcium-independent phospholipase A1 from Streptomyces albidoflavus

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Abstract

Phospholipase A1 (PLA1) is a member of the hydrolase family with applications in various fields, especially in the food industry. A calcium-independent PLA1 from Streptomyces albidoflavus was expressed in E. coli BL21 (DE3) in this study. The results indicated that the soluble expression of PLA1 was at low level, which was possibly due to the toxicity of PLA1 to the host. In contrast, the expression of the enzyme as inclusion bodies exhibited a high-level expression and 0.3 mg inclusion bodies protein could be derived from 1 mL culture medium. Furthermore, the renaturation of PLA1 was achieved through a direct dilution method, yielding 29.6 U/mL PLA1 activity after 16 h of renaturation at 4 °C. For improving the efficiency of the dilution refolding process, a continuous refolding strategy was established, and 155 U/mL PLA1 activity was derived from the continuous refolding process. With soybean lecithin as the substrate, the specific activity of purified renatured PLA1 was 1380 U/mg and the optimal temperature and pH was found to be 60 °C and 6.5. In addition, the renatured PLA1 was observed with better activity towards phosphatidyl inositol, whilst lipase activity was detected when the catalyzing temperature was below 55 °C. Overall, this study provides a possible solution to obtain calcium-independent PLA1 with high yield by heterologous expression in E. coli and hence to promote its further application in the field of food industry.

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Abbreviations

PLA1 :

Phospholipase A1

saPLA1 :

Phospholipase A1 from Streptomyces albidoflavus

FFA:

Free fatty acid

DLPC:

1,2-Dilauryl-sn-glycero-3-phosphocholine

DMPC:

1,2-Ditetradecanoyl-sn-glycero-3-phosphocholine

DPPC:

1,2-Dipalmitoyl-sn-glycero-3-phosphocholine

DSPC:

1,2-Distearoyl-sn-glycero-3-phosphocholine

DPPA:

1,2-Dipalmitoyl-sn-glycero-3-phosphate

DPPS:

1,2-Dipalmitoyl-sn-glycero-3-phospho-L-serine

DPPE:

1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine

DPPG:

1,2-Dipalmitoyl-sn-glycero-3-phospho-(1'-rac-glycerol)

PI:

L-α-phosphatidylinositol

POPC:

1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

GSSG:

Oxidized glutathione

GSH:

Reduced glutathione

IPTG:

Isopropyl β-D-thiogalactopyranoside

PelB:

Pectate lyase B

SDS-PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

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Funding

This work was supported by the National Key Research & Developmental Program of China (2021YFC2100303, 2018YFA0900300).

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Authors and Affiliations

  1. National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi, 214122, Jiangsu, People’s Republic of China

    Shi Cheng, Zitao Guo, Chaojuan Liang, Yi Shi, Peng Geng, Yu Xin, Zhenghua Gu & Liang Zhang

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  1. Shi Cheng
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Contributions

SC, ZG, YX, and LZ conceived and designed the research. SC, CL, and PG conducted the experiments. SC, ZG, and YS analyzed the data and wrote the manuscript. ZG, YX, and LZ reviewed the manuscript. All authors have read and agreed to the published version of the manuscript.

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Correspondence to Liang Zhang.

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On behalf of all the authors, the corresponding author states that there is no conflict of interest.

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Cheng, S., Guo, Z., Liang, C. et al. Heterologous expression, refolding, and characterization of a calcium-independent phospholipase A1 from Streptomyces albidoflavus. Syst Microbiol and Biomanuf 2, 487–497 (2022). https://doi.org/10.1007/s43393-021-00070-8

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