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High specific immobilization of His-tagged recombinant Microbacterium esterase by Ni-NTA magnetic chitosan microspheres for efficient synthesis of key chiral intermediate of d-biotin

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Abstract

The novel Ni-NTA-functionalized magnetic chitosan microspheres (MCS-NTA-Ni) were prepared via amino functionalization of MCS with epichlorohydrin and ethylenediamine, followed by the introduction of the aldehyde groups and NTA in turn, and nickel (II) ions were chelated in the end. MCS-NTA-Ni contained numerous long-armed NTA-Ni surface groups, ensuring high enzyme loading and providing more space and flexibility to attach enzymes and maintain their activity. This microsphere can have highly selective adsorption of his-tagged recombinant protein. The his-tagged recombinant Microbacterium esterase of E. coli BL21 (DE3)/pET21a-EstSIT01 was first immobilized on MCS-NTA-Ni by affinity fixation, giving high immobilization yield (90.1%) and enzyme loading (120 mg/g). Compared with free esterase, the immobilized esterase was found to exhibit higher pH stability and thermal stability. In addition, the immobilized esterase had excellent reusability for the synthesis of key chiral intermediate of d-biotin and the substrate conversion could still keep 100% after 8 cycles continuously.

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Abbreviations

CS:

Chitosan

ECH:

Epichlorohydrin

NTA:

Nα, Nα-bis (carboxymethyl)-l-lysine

MCS:

Magnetic chitosan microspheres

MCS-ECH:

Magnetic chitosan microspheres functionalized by ECH

MCS-NH2 :

Amino-functionalized magnetic chitosan microspheres

MCS-CHO:

Aldehyde-functionalized magnetic chitosan microspheres

MCS-NTA:

NTA-functionalized magnetic chitosan microspheres

MCS-NTA-Ni:

Ni-NTA-functionalized magnetic chitosan microspheres

FTIR:

Fourier-transform infrared spectrometer

EDS:

Energy-dispersive X-ray spectroscopy

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

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Acknowledgements

This work was supported by the Shanghai Committee of Science and Technology (No. 13430503400), the Science Foundation of Shanghai Institute of Technology (YJ2010-04), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (No. ZX2012-05), Innovation Program of Shanghai Municipal Education Commission (No. 11YZ227), and the Science Foundation of Shanghai Institute of Technology (ZQ2018-2).

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  1. School of Chemistry and Environmental Engineering, Shanghai Institute of Technology, 100 Haiquan Road, Shanghai, 201418, China

    Song He, Xiaomei Wu, Baodi Ma & Yi Xu

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He, S., Wu, X., Ma, B. et al. High specific immobilization of His-tagged recombinant Microbacterium esterase by Ni-NTA magnetic chitosan microspheres for efficient synthesis of key chiral intermediate of d-biotin. Bioprocess Biosyst Eng 44, 2193–2204 (2021). https://doi.org/10.1007/s00449-021-02595-7

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