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Impact of Metal Ions on Catalytic Kinetics, Stability, and Reactivation of Purified Tannase from Aspergillus niger

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Abstract

This study explores the purification and impact of metal ions on Aspergillus niger tannase, a metalloenzyme. Gel permeation chromatography achieved a 22.03-fold purification, yielding a specific activity of 106 U/mg. The enzyme, confirmed as a homodimer (100 kDa native, 50 kDa SDS PAGE), exhibited enhanced activity in the presence of calcium ions, reaching 163.46 U/mg, a 1.42-fold increase. UV absorption with 6 µM Ca ions indicated structural stability. Kinetic studies revealed a sigmoidal Michaelis–Menten graph with calcium ions, suggesting allosteric effects. The Lineweaver–Burk plot showed increased Km (14.04 to 36.39 mM) with mercuric ions, indicating competitive inhibition, while Vmax remained stable. Reactivation studies with DTT and different metal ions showed maximum reactivation with Hg (88.8%) and Ag (81.4%), implicating binding with cysteine residue in inactivation. EDTA-induced reactivation displayed maximum reactivation with Cu ions (67.6%) and minimum with Hg (22.5%), indicating Cu ions form complexes with amino acid groups. In contrast, Hg forms a coordination sphere with the thiol group of a cysteine residue. This comprehensive examination provides insights for optimizing tannase-based processes in various industries and expanding its biotechnological applications.

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Abbreviations

U :

Units

Mg:

Milligrams

kDa :

Kilodalton

SDS :

Sodium dodecyl sulfate

PAGE :

Polyacrylamide gel electrophoresis

UV :

Ultraviolet

Km :

Michaelis constant

Vmax :

Maximum velocity

mM :

Millimolar

µM :

Micromolar

EDTA :

Ethylenediamine tetraacetic acid

EGTA :

Ethylene glycol-bis (β-aminoethyl ether)-N, N, N′, N′-tetraacetic acid

DTT :

Dithiothreitol

Ca :

Calcium

Hg :

Mercury

Cu :

Copper

Cys :

Cysteine

3-D :

Three-dimensional

TEMED :

N, N, N’, N’- Tetramethylethylenediamine

BSA :

Bovine serum albumin

NCCS :

National Centre for Cell Science

NCBI :

National Center for Biotechnology Information

SmF :

Submerged fermentation

rpm :

Revolutions per minute

NaCl :

Sodium chloride

µl :

Microliter

M :

Molar

nm :

Nanometre

DEAE :

Diethyl aminoethyl

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Acknowledgements

The authors are highly grateful to the Department of Biotechnology, Himachal Pradesh University, Shimla, India, for providing the laboratory and chemical facilities during the study. The authors also gratefully acknowledge ICMR, New Delhi, India for providing the financial assistance as SRF (JRF-2019/ HRD-008, 20546) and necessary facilities for the completion of this research work.

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Authors and Affiliations

  1. Department of Biotechnology, Reseach Laboratory-III, Himachal Pradesh University, Shimla, 171005, Himachal Pradesh, India

    Kumari Alka, Lalita Kaushal,  Arti, Ekta Arya & Duni Chand

  2. Faculty of Applied Sciences and Biotechnology, Shoolini University of Biotechnology and Management Sciences, Solan, Himachal Pradesh, India

    Pradeep Kumar

  3. Department of Forensic Science, Himachal Pradesh University, Shimla, Himachal Pradesh, India

    Pradeep Kumar

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  1. Kumari Alka
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  2. Lalita Kaushal
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Correspondence to Duni Chand.

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Highlights

1. Examining the impact of various metal ions, including alkali, alkali earth, and transition metals, on the structural attributes of purified tannase sourced from Aspergillus niger.

2. Performing kinetic analyses to understand the mechanisms that activate or inhibit tannase function.

3. Exploring methods to reactivate inhibited tannase by employing metal ion chelators.

4. Suggesting a potential regulatory role of Ca ions and identifying tannase as an allosteric enzyme.

5. Investigating the impact of metal ion inactivation on tannase using EDTA and DTT, highlighting interactions with specific thiol and non-thiol groups within the enzyme.

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Alka, K., Kaushal, L., Arti et al. Impact of Metal Ions on Catalytic Kinetics, Stability, and Reactivation of Purified Tannase from Aspergillus niger. Catal Lett (2024). https://doi.org/10.1007/s10562-024-04664-4

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