275 JOURNAL OF BIOSCIENCE AND BIOENGINEERING © 2007, The Society for Biotechnology, Japan Vol. 104, No. 4, 275–280. 2007 DOI: 10.1263/jbb.104.275 Mathematical Modelling of NADH Oxidation Catalyzed by New NADH Oxidase from Lactobacillus brevis in Continuously Operated Enzyme Membrane Reactor Zvjezdana Findrik, 1 Ana Vrsalovic Presecki, 1 and Ðurda Vasic-Racki 1 * Faculty of Chemical Engineering and Technology, University of Zagreb, Savska c. 16, HR-10 000 Zagreb, Croatia 1 Received 22 January 2007/Accepted 10 July 2007 NADH oxidase from Lactobacillus brevis was kinetically characterized in two different buffers: Tris–HCl and glycine–sodium pyrophosphate (pH 9.0). Reaction kinetics was described using the Michaelis–Menten model with product (NAD + ) inhibition. It was found that this type of inhibition is uncompetitive. Experiments in the continuously operated enzyme membrane reactor revealed a strong enzyme deactivation at two different residence times: 12 and 60 min. A stronger deacti- vation was observed at the lower residence time in the glycine–sodium pyrophosphate buffer. Enzyme deactivation was assumed to be of the first order. The developed mathematical model for the continuously operated enzyme membrane reactor described these experiments very well. The mathematical model simulations revealed that a high enzyme concentration (up to 30 g cm –3 ) is necessary to obtain and maintain the stationary NADH conversion near 100% for a longer period of time. [Key words: NADH oxidase, coenzyme regeneration, enzyme kinetics, enzyme membrane reactor, inhibition] It is known that certain types of enzyme require coen- zymes for their action. Dehydrogenases are one of them. They are dependent on the presence of NAD(P) + or NAD(P)H in the reaction system. Since coenzymes are expensive, par- ticularly NAD(P)H, they are usually not added in stoichio- metric amounts. That is why many in-situ methods for their regeneration have been studied and developed (1–4). These methods can be divided into several different categories (1): chemical, biological, electrochemical (5), photochemical (6) and enzymatic (7–10). Enzymatic methods include the addi- tion of a second enzyme to the reaction system to catalyze the regeneration reaction, or adding a cosubstrate, which re- acts in the second reaction catalyzed by the catalytic en- zyme. Two problems can be differentiated: NAD + and NADH regeneration. Namely, formate dehydrogenase is an indus- trial enzyme for NADH regeneration, which catalyzes for- mate oxidation to CO 2 . NAD + is consumed in this reaction and forms NADH, which is required for the main reaction (2, 11). This kind of regeneration is necessary in ketone or keto acid reduction. In the oxidation catalyzed by dehydrogenases, it is neces- sary to regenerate NAD + . There are various known coen- zyme regeneration methods (12, 13), but none of them is as advantageous as that using NADH oxidase. This enzyme is found mostly in anaerobic bacteria (14–19). It catalyzes NADH oxidation with oxygen. Depending on the enzyme source, the reaction product, besides coenzyme NAD + , can be water or hydrogen peroxide. The aim of this study is to carry out the kinetic character- ization of NADH oxidase from Lactobacillus brevis. This enzyme is a tetramer that consists of four identical subunits and it is a member of the NADH oxidase group of enzymes that produce water (14, 15, 17, 18, 20). To realize an effi- cient enzymatic regeneration, it is necessary to estimate the kinetic parameters of the catalyst under operating condi- tions. Since it is known that dehydrogenase activity in the oxidation reaction is higher under alkaline conditions, the kinetic characterization of NADH oxidase was carried out at pH 9.0 in two different buffers: Tris–HCl and glycine–so- dium pyrophosphate buffers. Unfortunately, there are only a few reports wherein NADH oxidases were used as regener- ating enzymes in coupled enzyme systems in practice (14, 15, 21). MATERIALS AND METHODS Materials NADH oxidase from L. brevis was a gift from Prof. Hummel (IMET, Research Center Jülich, Germany) and was prepared as described elsewhere (15). NAD + and NADH were from Jülich Fine Chemicals (Jülich, Germany). Glycine, Trishy- droxymethylaminomethane and mercaptoethanol were from Fluka (Schnelldorf, Germany). Sodium-pyrophosphate, sodium carbonate, sodium hydrogen carbonate, citric acid, sodium hydrogen phos- phate and potassium dihydrogen phosphate were from Kemika (Zagreb, Croatia). The buffer concentration made from these chemi- cals was 75 mmol dm –3 . NADH oxidase activity assay NADH oxidase activity was assayed using a spectrophotometer at 340 nm at 30°C. Measure- * Corresponding author. e-mail: dvracki@marie.fkit.hr phone: +385-1-4597-104 fax: +385-1-4597-133