Enzymatic production of lactobionic acid: From laboratory to pilot scale
Integration of life sciences & engineering
Bio-transformation in the Laboratory and in Large Scale Production (T5-3)
Keywords: lactobionic acid, carbohydrate oxidase, kinetics, enzyme deactivation, mixing
Currently, lactobionic acid and its salts are mainly used in high-price specialty products, e.g. as an ingredient in solutions used for organ stabilization. However, lactobionic acid can also be used as a biodegradable cobuilder in detergents, and it has several applications in food technology, e.g. as an ingredient in pizza-type cheese. With lower production costs it could therefore become a bulk chemical.
Novozymes A/S has recently cloned a carbohydrate oxidase from Microdochium nivale and expressed it in Fusarium venenatum [1]. The carbohydrate oxidase can oxidize several mono-, oligo-, and polysaccharides using oxygen as terminal electron acceptor. Oxidation of lactose to lactobionic acid by this enzyme has been investigated both in laboratory-scale and pilot-scale equipment.
Kinetic investigation in laboratory scale
The kinetics for the oxidation of lactose to lactobionic acid by the carbohydrate oxidase was studied in a 1 L bioreactor with control of pH, temperature, and dissolved oxygen. Due to the negative influence of the byproduct hydrogen peroxide on the oxidase, catalase was added in excess to remove the byproduct, also providing extra oxygen.
The Michaelis constant (38C, pH=6.4) of the oxidase for lactose was very low, 0.066 mM, while the Michaelis constant for oxygen was high (0.97 mM) compared to the solubility of oxygen from air at 38C and 1 bar. Surprisingly, the study showed that mixing is very important even in a 1 L bioreactor stirred by 2 Rushton turbines revolving at 1000 RPM. Thus, the oxidase deactivated due to gradients in pH when 2 M NaOH was used for neutralization even though the medium was buffered with 50 mM phosphate buffer. This was not the case when the weak base ammonia was used for neutralization.
Scale-up
On the basis of the experiments in 1 L scale, experiments were performed in a 600 L reactor equipped with a rotary jet head (http://www.iso-mix.com) for mixing and oxygen mass transfer. Deactivation could also be avoided in this system when ammonia was used for neutralization. Compared to mechanically stirred systems, high values of the volumetric mass transfer coefficient were obtained, especially at low values of the specific power input and the superficial gas velocity. This is of importance since insufficient oxygen transfer will result in a low dissolved oxygen concentration and thereby a low volumetric rate of reaction.
Further optimization
Through further optimization it is expected that lactobionic acid can be produced at a price that is attractive for e.g. the food industry. Currently, inhibition and deactivation of the oxidase by hydrogen peroxide and the effect of hydrogen peroxide on the catalase is under investigation to determine the optimal quantity of catalase used in a given reaction. By the use of rotary jet heads it will be easy to retrofit existing tanks in the food industry since the rotary jet heads can be installed without welding, without reinforcing the tanks, and without using mechanical seals.
References
[1] Xu, F., Golightly, E. J., Fuglsang, C. C., Schneider, P., Duke, K. R., Lam, L., Christensen, S., Brown, K. M., Jørgensen, C. T. & Brown, S. H. (2001). A novel carbohydrate:acceptor oxidoreductase from Microdochium nivale. Eur. J. Biochem. 268: 1136-1142.
Presented Thursday 20, 16:00 to 16:20, in session Bio-transformation in the Laboratory and in Large Scale Production (T5-3).
