Determination of effective diffusion coefficient of substrate in gel particles with immobilized biocatalyst

A method of determining of the effective diffusion coefficient of substrate in a particle, where the diffusion and consumption of substrate by biocatalytic reaction are present simultaneously, was designed and experimentally verified. The method is based on measuring the overall rate of heterogeneous biocatalytic reaction in particles of varying diameter. The effective diffusion coefficient, D_e, was determined by fitting the measured reaction rates with the solution of the reaction-diffusion equation. The method is tailored for cases where the enzyme reaction is governed by the Michaelis-Menten kinetics. The value of K_m required for the solution of the mathematical model was adopted from the measurement of the kinetics of free cells, whereas the rate parameter, k₂, was optimized together with D_e. As an experimental model, the sucrose hydrolysis catalyzed by Ca-alginate-entrapped yeast cells was examined. The particle diameter varied in the range of 1.2–3.9 mm and the initial reaction rates were measured in a batch-stirred reactor at a sucrose concentration of 100 m . The D_e of sucrose at 30°C was found to be 2.9 · 10⁻¹⁰ m²s⁻¹.     

Continuous immobilized cell reactor for amide hydrolysis

Summary This article deals with continuous hydrolysis of acrylamide into acrylic acid using the wild-typeBrevibacterium sp. R312 which can hydrolyze all water-soluble amides into their corresponding acids. Biotransformation has been carried out in a fluidized bed reactor specially designed to obtain good contact conditions between cells entrapped into small calcium alginate beads (2–3 mm) and low-concentration acrylamide solutions (10–40g·l^–1). Different flow rates, biocatalyst loads and substrate concentrations have been investigated. Kinetic constants for the immobilized enzyme have been identified. It appears that the Michaelis constant does not change with operating conditions and remains roughly equal to the value obtained for free cells. In contrast, the maximum rate of hydrolysis is considerably decreased, as if only cells on the outskirts of beads were involved in the transformation. On the whole it is proved that corynebacteria cells could be usefully used for the bioconversion of amides in a continuous immobilized cell reactor; the higher the solid hold-up and/or the smaller the beads, the more efficient the biological transformation.     

左旋麻黄素半生物合成的研究固定化酵母细胞生物合成L-PAC(L-苯基乙酰基甲醇)

本文对影响酵母菌生物合成L-PAC的主要因素进行了研究.结果表明酵母菌株Sc-5按2g鲜细胞/3.2%50ml海藻酸钠凝胶固定化后,所获固定化凝胶珠置于装有4倍体积反应液的容器中,在振荡频率220转/分、温度28-30℃、添加0.2%的Vc时,枇次生物合成L-PAC产量最高,达2.0g/L.     

Preparation of high activity whole cell biocatalyst by permeabilization of recombinant flocculent yeast with alcohol

Flocculent yeast Saccharomyces cerevisiae YF234 (MATa ura3–52 trp1Δ2 his ade 2–1 can1–100 sta1 FLO8) cells overexpressing glyoxalase I and having strong flocculation ability were permeabilized with isopropyl alcohol and ethanol under various conditions. The treatment with 40% isopropyl alcohol significantly improves the initial reaction rates of recombinant flocculent yeast cells. Moreover, the reactivity of permeabilized flocculent yeast cells was similar to that of dispersed cells with EDTA. On the other hand, the flocculation ability of yeast cells was not affected by the treatment with alcohol solutions of various concentrations and treatment time length. Therefore, the recombinant flocculent yeast cells permeabilized with alcohol are very effective whole cell biocatalysts.     

Flow dynamics within a bioreactor for tissue engineering by residence time distribution analysis combined with fluorescence and magnetic resonance imaging to investigate forced permeability and apparent diffusion coefficient in a perfusion cell culture chamber

This study reveals that residence time distribution (RTD) analysis with pH monitoring after acid bolus injection can be used to globally study the flow dynamics of a perfusion bioreactor, while fluorescence microscopy and magnetic resonance imaging (MRI) were used to locally investigate mass transport within a hydrogel scaffold seeded or not with cells. The bioreactor used in this study is a close‐loop tubular reactor. A dispersion model in one dimension has been used to describe the non‐ideal behavior of the reactor. From open‐loop experiments (single‐cycle analysis), the presence of stagnant zones and back mixing were observed. The impact of the flow rate, the compliance chamber volume and mixing were investigated. Intermediate flows (30, 45, 60, and 90 mL min⁻¹) had no effect over RTD function expressed in reduced time (θ). Lower flow rates (5 and 15 mL min⁻¹) were associated to smaller extent of dispersion. The compliance chamber volume greatly affected the dynamics of the RTD function, while the effects of mixing and flow were small to non‐significant. An empirical equation has been proposed to localize minima of the RTD function and to predict Pe_r. Finally, cells seeded in a gelatin gel at a density of 800,000 cells mL⁻¹ had no effect over the permeability and the apparent diffusion coefficient, as revealed by fluorescent microscopy and MRI experiments. Biotechnol. Bioeng. 2011;108: 2488–2498. © 2011 Wiley Periodicals, Inc.