Optimization of Reynoutria sachalinensis leaf extract preparation for determination of shikimate dehydrogenase activity

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Abstract

Introduction. Reynoutria sachalinensis is a promising subject for studying the mechanisms of tannin synthesis in plants due to the high proanthocyanidins content.

This study aimed to optimize the preparation of R. sachalinensis leaf extracts to determine the activity of shikimate dehydrogenase, a key enzyme in the shikimate pathway involved in tannin biosynthesis.

Material and methods. Aqueous extraction followed by protein precipitation using 60 or 80% (v/v) acetone, with or without glycerol, was carried out. The effect of freezing at –80 °C on shikimate dehydrogenase activity was also assessed.

Results. It was found that freezing both crude and acetone-purified extracts did not decrease shikimate dehydrogenase activity, while increasing the acetone concentration to 80% (v/v) led to an increase in enzyme activity compared to 60%(v/v) acetone. Glycerol had no significant effect on enzyme activity.

Conclusion. Thus, the optimized extraction using 80% (v/v) acetone precipitation and low-temperature storage allows for efficient extraction of proteins and enzymes, particularly shikimate dehydrogenase, from R. sachalinensis leaves for subsequent biochemical studies.

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About the authors

S. A. Bondarev

All-Russian Scientific Research Institute of Medicinal and Aromatic Plants

Author for correspondence.
Email: xcsergey@mail.ru
ORCID iD: 0009-0009-7039-2939
SPIN-code: 9186-5325

Junior Researcher, Laboratory of Genomics and Biochemistry

Russian Federation, 7 Green str., Moscow, 117216

A. A. Sereda

All-Russian Scientific Research Institute of Medicinal and Aromatic Plants

Email: nensi.sereda@mail.ru
ORCID iD: 0009-0007-1891-4846
SPIN-code: 6292-5126

Junior Researcher, Laboratory of Genomics and Biochemistry of Medicinal Plants

Russian Federation, 7 Green str., Moscow, 117216

D. N. Baleev

All-Russian Scientific Research Institute of Medicinal and Aromatic Plants

Email: dbaleev@gmail.com
ORCID iD: 0000-0002-1228-0594

Ph.D. (Agrical.), Leading Researcher, Laboratory of Atomic-Molecular Bioregulation and Selection

Russian Federation, 7 Green str., Moscow, 117216

E. R. Nikonorova

All-Russian Scientific Research Institute of Medicinal and Aromatic Plants

Email: gatiatulinaer@gmail.com
ORCID iD: 0000-0002-6360-2194
SPIN-code: 5392-5170

Ph.D. (Med.), Leading Researcher, Laboratory of Genomics and Biochemistry of Medicinal Plants

Russian Federation, 7 Green str., Moscow, 117216

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