Abstract
Purpose
We purified and characterized a novel ene-reductase (KaDBR1) from Kazachstania exigua HSC6 for the synthesis of dihydro-β-ionone from β-ionone.
Methods
KaDBR1 was purified to homogeneity by ammonium sulfate precipitation and phenyl-Sepharose Fast Flow and Q-Sepharose chromatography. The purified enzyme was characterized by measuring the amount of dihydro-β-ionone from β-ionone with LC–MS analysis method.
Results
The molecular mass of KaDBR1 was estimated to be 45 kDa by SDS-PAGE. The purified KaDBR1 enzyme had optimal activity at 60 °C and pH 6.0. The addition of 5 mM Mg2+, Ca2+, Al3+, Na+, and dithiothreitol increased the activity of KaDBR1 by 25%, 18%, 34%, 20%, and 23%, respectively. KaDBR1 favored NADH over NADPH as a cofactor, and its catalytic efficiency (kcat/Km) toward β-ionone using NADH was 8.1-fold greater than when using NADPH.
Conclusion
Owing to its unique properties, KaDBR1 is a potential candidate for the enzymatic biotransformation of β-ionone to dihydro-β-ionone in biotechnology applications.
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Supporting information
Supplementary Fig. 1—Mass spectrometry analysis of the digested KaDBR1. The amino acid sequences of the ene-reductase from Kazachstania exigua (Accession No. AEP22543.1) and those of the eight fragments of purified KaDBR1 determined by MALDI-TOF-MS are shown.
Supplementary Fig. 2—HPLC analysis of the catalytic product of purified KaDBR1. (A) authentic β-ionone, (B) authentic dihydro-β-ionone, (C) HPLC analysis of the reaction products.
Supplementary Fig. 3—In vitro kinetic analysis of purified KaDBR1 with β-ionone as substrate. Reaction velocity (mmol/min/L) as a function of substrate concentration is plotted for 0–55 μM NADH (A), 0–80 μM NADPH (B) and 0–10 mM β-ionone (C).
Funding
This work was supported by grants from the Zhongyuan Science and Technology Innovation Leading Talent Project (224200510017), and the Science and Technology Project of China Guangxi Tobacco Industry Co. Ltd. (2020450000340027).
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Long, Z., Li, K., Xue, Y. et al. Purification and biochemical characterization of a novel ene- reductase from Kazachstania exigua HSC6 for dihydro-β-ionone from β-ionone. Biotechnol Lett 45, 499–508 (2023). https://doi.org/10.1007/s10529-023-03355-1
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DOI: https://doi.org/10.1007/s10529-023-03355-1