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Production, Purification and Characterization of a Novel Thermostable Caffeine Dehydrogenase from Pichia manshurica Strain CD1 Isolated from Kombucha Tea

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Abstract

Kombucha tea is a consortium of yeasts and bacteria (predominantly acetic acid ones). One caffeine-degrading yeast was isolated from kombucha tea, identified using 18S rRNA sequencing and phylogenetic analysis, and classified as Pichia manshurica CD1 (GenBank accession number KY799109). The strain was found to able not only to degrade caffeine, but also to use it as a sole source of carbon and nitrogen. In such a caffeine medium the strain required about two days of incubation to reach maximum growth. An NBT-dependent (nitro blue tetrazolium chloride), caffeine dehydrogenase activity assay was established and the enzyme was purified. It was found that the caffeine dehydrogenase was a monomer of approximately 85 kDa and had optimal activity at pH 7.5. Interestingly, the enzyme showed high activity and stability over a wide range of temperatures, with almost 71% of activity being retained after incubation at 55°C for 20 min and 54% of its initial activity was retained even after incubation at 100°C. The purified caffeine dehydrogenase had high substrate specificity towards caffeine (Km 11.2 µM and Vmax 0.372 nmol/(mL min)) at 0.5 mM concentration. The enzyme activity was partially inhibited in the presence of Cr2+, Pb2+, Zn2+, Ni2+, Hg2+, and Mg2+ at 1 mM, and completely inhibited by Cu2+. The enzyme activity was also strongly suppressed by β‑mercaptoethanol, SDS, DTT and EDTA. These interesting industry-oriented properties of caffeine dehydrogenase isolated from Pichia manshurica CD1 may be useful for many biotechnological processes in the future.

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ACKNOWLEDGMENTS

We thank the State Government, Government of West Bengal for fellowship to RP.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Authors and Affiliations

  1. Jadavpur University, Department of Life Science and Biotechnology, Kolkata, 700032, West Bengal, India

    R. Parvin, S. Bhattacharya, S. Som Chaudhury & R. Gachhui

  2. Department of Environmental Studies, Budge Budge College, Kolkata, 700137, West Bengal, India

    U. Roy

  3. Jadavpur University, School of Environmental Studies, Kolkata, 700032, West Bengal, India

    J. Mukherjee

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  1. R. Parvin
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Contributions

R. Parvin#: conceptualization, investigation, methodology, formal analysis, data curation, drawing all the graphs, and writing-original draft.

S. Bhattacharya#: conceptualization, investigation, methodology, formal analysis, and writing-original draft.

S.S. Chaudhury: methodology, data curation.

U. Roy: Data curation.

J. Mukherjhee: conceptualization, writing-review and editing, validation, resources, formal analysis.

R. Gachhui: supervision, conceptualization, writing-review and editing, validation, resources, formal analysis.          #Equal contributions.

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Correspondence to R. Gachhui.

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Conflict of interest. The authors declare that they have no conflict of interest.

Statement on the welfare of animals. This article does not contain any experiments with human participants or animals performed by any of the authors.

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Parvin, R., Bhattacharya, S., Chaudhury, S.S. et al. Production, Purification and Characterization of a Novel Thermostable Caffeine Dehydrogenase from Pichia manshurica Strain CD1 Isolated from Kombucha Tea. Microbiology 92, 230–241 (2023). https://doi.org/10.1134/S0026261722601476

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