Enhanced bioconversion rate and released substrate inhibition in (R)-phenylephrine whole-cell bioconversion via partial acetone treatment.

An approach was developed to enhance the efficiency for the bioconversion of 1-(3-hydroxyphenyl)-2-(methyamino)-ethanone to (R)-phenylephrine. The strain Serratia marcescens N10612, giving the benefit of 99% enantiomeric excess in (R)-PE conversion, was used. The fermentation was devised to harvest cells with high hydrophobic prodigiosin content inside the cells. Then, the partial acetone extraction was applied to remove prodigiosin from the cells. The treatment was found to increase the cells conversion rate without loss of the cells NADPH redox system. When using 50% (v/v) acetone for 5min, the processed cells can give a specific conversion rate of 16.03µmol/h/g-cells. As compared the treated cells with cells under the basal medium, the maximum reaction rate (Vmax) increased from 6.69 to 10.27 (µmol/h/g-cells), the dissociation constant (Km) decreased from 0.236 to 0.167mM and the substrate inhibition constant (KSi) increased from 0.073 to 1.521mM. The 20-fold increase in substrate inhibition constant referred to a great release from the substrate inhibition for the use of S. marcescens N10612 in the bioconversion, which would greatly benefit the bioconversion to be industrialized.

Ultrasound-assisted (R)-phenylephrine whole-cell bioconversion by S. marcescens N10612.

The strain Serratia marcescens N10612 is used to perform the bioconversion of 1-(3-hydroxyphenyl)-2-(methyamino)-ethanone (HPMAE) to (R)-phenylephrine ((R)-PE), which is an ephedrine drug substitute. The use of an ultrasound approach is found to improve the efficiency of the (R)-PE bioconversion. The optimization of the (R)-PE bioconversion is carried out by means of statistical experiment design. The optimal conditions obtained are 1.0mM HPMAE, 18.68 g/L glucose and ultrasound power of 120 W, where the predicted specific rate of the (R)-PE bioconversion is 31.46 ± 2.22 (ìmol/h/g-cells) and the experimental specific rate is 33.27 ± 1.46 (ìmol/h/g-cells), which is 3-fold higher than for the operation under ultrasound power of 200 W (11.11 ìmol/h/g-cells) and 4.3-fold higher than for the shaking operation (7.69 ìmol/h/g-cells). The kinetics study of the bioconversion also shows that under the ultrasound operation, the optimal rate (Vmax) of the (R)-PE bioconversion increases from 7.69 to 11.11 (µmol/h/g-cells) and the substrate inhibition constant (KSi) increases from 1.063 mM for the shaking operation to 1.490 mM for ultrasound operation.

Production of D-hydantoinase via surface display and self-cleavage system.

In this study, the cell surface expression system was the first time used to directly produce extracellular enzyme. In the plasmid construction, the truncated ice nucleation protein (INP) was fused with intein (INT) and target protein, D-hydantoinase (DHTase), to form the INP-INT-DHTase gene. The plasmid containing this gene was transformed into Escherichia coli ER2566 cells. The gene construct enables the expression of INP-INT-DHTase fusion protein, which might anchor on cell membrane surface. The induction conditions were studied and optimal conditions were as follows: E. coli ER2566 was incubated at 37°C and 200 rpm till OD600 reached 0.6. Then, 0.05 mM IPTG was added and the induction was conducted at 15°C for 24 h. The cell was harvested and resuspended in the cleavage buffer (50 mM Tris-HCl buffer, pH 6). The cleavage reaction was carried out at 25°C, and 100 rpm for 24 h. The DHTase with an activity of 0.225 U/ml and a purity of 63.2% was obtained via centrifugation. This study demonstrated the feasibility of direct extracellular enzyme production using E. coli via only two steps of centrifugation.