Immobilization of a multi‐enzyme system for L‐amino acids production

Abstract

BACKGROUND Four enzymes were immobilized for the production of optically pure natural and non‐natural L‐amino acids via the ‘Double‐Racemase Hydantoinase Process’. Immobilization constitutes an empirical process, and for each enzyme we tested 11 carriers with different functional groups; epoxide, EC‐EP, EC‐HFA, IB‐150 and IB‐350, carboxylic acid IB‐C435, quaternary ammonium IB‐A161, IB‐A171 and IB‐A369, aromatic group IB‐S861, and hydroxyl group IB‐S60S and IB‐S60P. RESULTS Each protein showed preference for binding on one or several supports: D,L‐hydantoinase/IB‐350, hydantoin racemase/EC‐EP, L‐carbamoylase/EC‐EP and carbamoyl racemase/IB‐A161. The process was optimized for each enzyme by modifying temperature, pH and ionic strength. For the enzymatic cascade, it was demonstrated that it was essential to use supports having homogeneous characteristics. The product of the first reaction is the substrate of the next one, and so on. Free mass diffusion from one enzyme to the other is crucial to avoid retention on the support and to maintain the protein–substrate interaction constant. CONCLUSION It was proved that support size, weight and hydrophobicity must be homogeneous to avoid the formation of separate layers of the matrix selected. For this reason, the support IB‐350 was used for the four enzymes, even though it may not be the most efficient one for some of them. © 2015 Society of Chemical Industry

Concomitant cell‐free biosynthesis of optically pure D‐(−)‐acetoin and xylitol via a novel NAD+ regeneration in two‐enzyme cascade

“…Over the last few decades, enzymatic biosynthesis in cell‐free biosystems has revealed numerous advantages, including a high product TPY (titer, volumetric productivity and yield), easy product separation, high product quality and easy process control and optimization . To address the major problems of AC production methods that employ living microorganisms, enzymatic biosynthesis of AC is increasingly being favored.…”

l-Amino Acid Production by a Immobilized Double-Racemase Hydantoinase Process: Improvement and Comparison with a Free Protein System

“…Optimum pH and temperature conditions, which allow maximum activity of the mixture of proteins, must first be established. Subsequently, the quantity of each protein must be varied according to their specific activities and velocity in order to avoid reaction intermediates [15]. However, it should be noted that, although the fine tuning of the system requires a great deal of effort, once it is achieved, the cascade of activity has been stable for more than one year, opening future lines of work.…”

“…When the four immobilized enzymes were mixed, the different density and nature of the supports impeded the matter transfer and thus led to the accumulation of intermediate reaction products. Finally, the four proteins were immobilized in Sepabeads IB350, containing an epoxy group for covalent binding [15]. The rate of production and number of cycles over which the system could be reused increased at greater quantities of protein per mg of beads.…”

“…Several supports from two companies have been tested to immobilize the proteins of the double-racemase hydantoinase process [15], and in the present study we have optimized the enzymatic cascade of four enzymes to produce a broad spectrum of optically pure L-amino acids. Protein saturation of all binding sites in the support does not always result in greater effectiveness due to crowding and substrate diffusion limitations [16].…”

Evaluation of Designed Immobilized Catalytic Systems: Activity Enhancement of Lipase B from Candida antarctica

“…On the other hand, no activity was observed when IB-A161, IB-A171, IB-A369, IB-EC1, IB-S861, and IB-S500 were applied. Rodríguez-Alonso et al [55] immobilized four enzymes necessary for L-amino acid formation on, among others, IB-150, IB-C435, IB-161, IB-A171, IB-A369, IB-S861, IB-S60S, and IB-S60P. Each enzyme demonstrated a preference for binding on one or several carriers.…”