Scale-Down Studies of Protein Effects at the Gas-Liquid Interface

Abstract

The present study investigated the kinetic stability and activity of water-forming NAD(P)H oxidase (NOX) in a Liebig-type bubble column reactor, simulating industrial-scale conditions at laboratory scale. The study is divided into three parts, examining hydrodynamic parameters, NOX deactivation rate and protein concentration dynamics.
The first part investigated and characterized the hydrodynamic phenomena observed in the bubble column reactor at varying gas types (oxygen, air, nitrogen and helium), flow rates (0.05, 0.10 and 0.15 L∙min-1) and sparger diameters (2 mm, 1 mm and porous). Porous spargers with pore diameters less than 0.5 mm significantly enhanced gas holdup (ε), reaching ε=6.2% for oxygen at 0.15 L∙min-1, compared to 4.8% using a 2 mm sparger. The increase in gas holdup corresponded to a higher gas-liquid interfacial area, promoting more efficient mass transfer. The high aspect ratio (AR) defined between length and cross-sectional area of the bubble column (~23), promoted axial dispersion effects and prolonged homogeneous flow profile, increasing gas-liquid interfacial area and limiting bubble coalescing behavior, especially in the use of smaller diameter sparger. Increasing gas flow rate corresponded to high Reynolds bubble number (ReB), turbulence phenomena and low drag coefficient (CD), potentially contributing to the acceleration of NOX deactivation.
The second part investigated NOX kinetic activity and stability at the same conditions. Smaller sparger designs reduced NOX biocatalytic deactivation and improved kinetic stability, increasing NOX half-life from 18.4 to 21.4 hours when switching from 2 mm to porous sparger at 0.15 L∙min-1.
The third part examined NOX protein concentration changes using different gas types at varying gas flow rates and sparger diameters. NOX protein displayed higher concentrations across decreasing sparger dimensions at 0.15 L∙min-1 in oxygen bubbling, while increased flow rates in nitrogen and helium bubbling accelerated protein losses. A strong positive correlation (ρ=0.76) between kLa and the normalized NOX concentration decay rates indicated that mass transfer effects were potentially linked with the complex hydrodynamic phenomena participating in NOX protein losses.
This comprehensive investigation enhances understanding of NOX behavior at gas-liquid interfaces and contributes to optimizing biocatalytic processes in bubble column reactors.

Popular Abstract

Enzymes are nature’s catalysts, accelerating chemical reactions essential for life. They function by ‘consuming’ specific molecules that bind on specific locations of their structures, named substrates. In this study, a specific enzyme called NAD(P)H oxidase (NOX) was investigated. NOX is a type of enzyme that depends on the presence of complementary organic molecules or cofactors (NADPH cofactors), which bind on a specific site of NOX’s structure and enable substrate consumption. NAD(P)H-dependent enzymes are essential in the synthesis of pharmaceutical substances, necessary for the formulation of antibiotics, amino acids and other value-added chemicals. Nevertheless, conventional chemical processes employed in the synthesis of NAD(P)H are associated with high costs, and the generation of hazardous substances. A cost-efficient and environmentally-friendly strategy is enzymatic cofactor regeneration, where NOX and NAD(P)H in the presence of oxygen as substrate allow the synthesis of new cofactors of its kind.
Experiments were performed in a bubble column reactor, where gas is ejected from the bottom of the column and migrates through liquid, in a setup that mimics industrial conditions on a laboratory scale. However, a major limitation using bubble columns is loss of NOX performance over time. This occurs as NOX attaches to the gas bubbles, which burst at the surface of the liquid, leading to gradual changes in the enzyme structure. The goal was to gain a deep understanding of how NOX behaves when exposed to different gases and bubbling conditions, which is important for improving industrial biotechnology processes. The study was divided into three parts:
Part I: In this part it was examined how the complex movements and interactions of bubbles among different gases (oxygen, air, nitrogen and helium) affect the liquid flow and the overall bubble column reactor behavior, , by evaluating the quantity of gas held in the liquid, the bubble sizes, bubble shapes and the efficiency of gas transfer into the liquid.
Part II: Next, it was investigated how gas flow rate and spargers of varying orifice diameter affected NOX activity and stability over time. This assisted in understanding which conditions were optimal for the enzyme to function efficiently.
Part III: In the last part it was studied how the amount of NOX in the liquid changed under various conditions and explored whether there is a connection between gas transfer rates and enzyme stability.
This comprehensive research provided valuable information about how enzymes like NOX interact in gas-liquid interfaces. The findings could lead to more efficient and scalable enzymatic reactions in industrial biotechnology, potentially improving processes in areas such as pharmaceutical production.

• author: Pappas, Konstantinos
• supervisor: Rajni Hatti-Kaul; Carl Grey
• organization: Biotechnology (MSc); Biotechnology (M.Sc.Eng.)
• course: KBTM01 20241
• year: 2025
• type: H2 - Master's Degree (Two Years)
• subject: Technology and Engineering
• keywords: NAD(P)H Oxidase, Bubble Column Reactor, Gas-Liquid Interface, Enzyme Deactivation, Mass Transfer, Biocatalysis, Hydrodynamics, Biotechnology
• language: English
• id: 9203853
• date added to LUP: 2025-06-24 11:45:42
• date last changed: 2025-06-24 11:45:42