Taxadiene Synthase purification and Molecular Characterization in Podocarpus elongatus EFBL-NZM for an Enhanced Taxadiene biosynthesis, a Key Taxol Biosynthetic Intermediate | ||||
| Bulletin of Faculty of Science, Zagazig University | ||||
| Article 6, Volume 2024, Issue 2, July 2024, Page 54-63 PDF (954.67 K) | ||||
| Document Type: Original Article | ||||
| DOI: 10.21608/bfszu.2023.236547.1310 | ||||
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| Authors | ||||
Nabil Z. Mohamed  1; Lamis Shaaban2; Samia safan3
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| 1Botany and microbiology department, Faculty of Science, Zagazig University | ||||
| 2Botany and Microbiology department, Faculty of Science, Zagazig University | ||||
| 3Botany and Microbiology department, Faculty of Science, Zagazig University | ||||
| Abstract | ||||
| The cyclization of geranylgeranyl diphosphate (GGPP) to diterpene taxadiene is carried out by Taxadiene synthase (TDS), one of the most effective rate-limiting enzymes for the production of taxol. However, enzyme stability, catalytic efficiency, and silencing the expression of TDS in Taxol producers are the main challenges related to the reduction of the Taxol productivity. Podocarpus elongatus EFBL-NZM has been mentioned as a potent producer of Taxol. Consequently, the objective of this work is to characterize the TDS extracted from Podocarpus elongatus EFBL-NZM at the molecular, biochemical, and kinetic levels. P. elongatus displayed a high potency for its crude TDS activity (4.35 μg/mg/min). Upon utilizing of ion-exchange chromatography to purify TDS enzyme, the specific activity of TDS (280.86 μg/mg/min) increased over the corresponding TDS crude enzyme (4.35 μg/mg/min) by 2.15-purification fold with 78 and approximately 160 kDa by denaturing and non denaturating PAGE techniques respectively, ensuring the TDS enzyme consists of two identical subunits that make up the homodimeric identity of enzyme. The released taxadiene chemical identity provided a similar mass fragmentation pattern (272.2 m/z) conducted by GC-MS analyses, compared to authentic taxadiene. The rate-limiting tds gene was further amplified using PCR, and the results showed positive amplicons (approximately 700 bp), displaying a greater resemblance to the tds from Pacific yew; Taxus brevifolia. Podocarpus elongatus showed a maximum level of TDS activity at optimum temperature 37 °C. Upon the DTNB and MBTH addition to the P. elongatus TDS enzymatic reaction, a dramatic reduction of TDS relative activity | ||||
| Keywords | ||||
| Taxadiene synthase; Taxol; Enzyme purification; active site fingerprint; protein modeling | ||||
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