# Characterization of vitamin D3 biotransformation by the cell lysate of Actinomyces hyovaginalis CCASU-A11-2

## TL;DR

Researchers used a cell-free lysate to convert vitamin D3 into calcitriol in just 6 hours, a significant improvement over previous methods.

## Contribution

The study demonstrates a 10-fold faster and more efficient bioconversion of vitamin D3 into calcitriol using cell-free lysate.

## Key findings

- Calcitriol production reached 31 µg/10 mL in 6 hours using cell-free lysate, compared to 32.8 µg/100 mL in 5 days with a fermenter.
- Optimal conditions were pH 7.8, 28°C, and 6 hours of reaction time for maximum calcitriol yield.
- Cell-free lysate is a viable and efficient method for industrial-scale vitamin D3 bioconversion.

## Abstract

A former work conducted in our Lab, lead to in a effective scale up of vitamin D3 bioconversion into calcitriol by Actinomyces (A.) hyovaginalis isolate CCASU-A11-2 in Lab fermenter (14 L) resulting in 32.8 µg/100 mL of calcitriol. However, the time needed for such a bioconversion process was up to 5 days. Therefore, the objective of this study was to shorten the bioconversion time by using cell-free lysate and studying different factors influencing bioconversion. The crude cell lysate was prepared, freeze-dried, and primarily fractionated into nine fractions, of which, only three fractions, 50, 100, and 150 mM NaCl elution buffers showed 22, 12, and 2 µg/10 mL, calcitriol production, respectively. Ammonium sulfate was used for protein precipitation, and it did not affect the bioconversion process except at a concentration of 10%w/v. Secondary fractionation was carried out using 80 mL of the 50 mM NaCl elution buffer and the results showed the 80 mL eluent volume was enough for the complete elution of the active protein. The pH 7.8, temperature 28 °C, and 6 h reaction time were optimum for maximum calcitriol production (31 µg/10 mL). In conclusion, the transformation of vitamin D3 into calcitriol was successfully carried out within 6 h and at pH 7.8 and 28 °C using fractionated cell lysate. This process resulted in a 10-fold increase in calcitriol as compared to that produced in our previous study using a 14 L fermenter (32.8 µg/100 mL). Therefore, cell-free lysate should be considered for industrial and scaling up vitamin D3 bioconversion into calcitriol.

Biotransformation of vitamin D into calcitriol has been achieved within 6 h using a cell-free lysate of A. hyovaginalis isolate CCASU-A11-2.pH 7.8 and incubation temperatures 28 °C were optimum for maximum calcitriol production using the cell-free lysate.This study results in a 10-fold (31 µL/10 mL) to that obtained using a 14 L Lab fermenter.Bioconversion using cell-free lysate should be considered for industrial production of calcitriol.

Biotransformation of vitamin D into calcitriol has been achieved within 6 h using a cell-free lysate of A. hyovaginalis isolate CCASU-A11-2.

pH 7.8 and incubation temperatures 28 °C were optimum for maximum calcitriol production using the cell-free lysate.

This study results in a 10-fold (31 µL/10 mL) to that obtained using a 14 L Lab fermenter.

Bioconversion using cell-free lysate should be considered for industrial production of calcitriol.