This review describes the most recent developments in the biotechnological applications of penicillin acylases. This group of enzymes is involved mainly in the industrial production of 6-aminopenicillanic acid and the synthesis of semisynthetic β-lactam antibiotics. In addition, penicillin acylases can also be employed in other useful biotransformations, such as peptide synthesis and the resolution of racemic mixtures of chiral compounds. Particular emphasis is placed on advances in detection of new enzyme specificities towards other natural penicillins, enzyme immobilization, and optimization of enzyme-catalyzed hydrolysis and synthesis in the presence of organic solvents.

Biotechnological applications of penicillin acylases: state-of-the-art.

Kinetics of β-lactam antibiotics synthesis by penicillin G acylase (PGA) from the viewpoint of the industrial enzymatic reactor optimization

Biotransformations catalyzed by multimeric enzymes: stabilization of tetrameric ampicillin acylase permits the optimization of ampicillin synthesis under dissociation conditions.

Ammonolysis of d , l -phenylglycine methyl ester catalysed by Novozym 435 at 40°C in tert -butyl alcohol gave d -phenylglycine amide in 78% ee at 46% conversion, corresponding to an enantiomeric ratio ( E ) of 16. Lowering the temperature improved the enantioselectivity ( E =52 at −20°C). Combination of ammonolysis with pyridoxal-catalysed in situ racemisation of the unconverted ester (dynamic kinetic resolution), at −20°C, gave d -phenylglycine amide with 88% ee at 85% conversion. The amide racemised much slower than the ester at this low temperature.

Dynamic kinetic resolution of phenylglycine esters via lipase-catalysed ammonolysis

Although the extractive biotransformation in two-phase partitioning systems have been studied extensively, such as the water–organic solvent two-phase system, the aqueous two-phase system, the reverse micelle system, and the room temperature ionic liquid, etc., this has not yet resulted in a widespread industrial application. Based on the discussion of the main obstacles, an exploitation of a cloud point system, which has already been applied in a separation field known as a cloud point extraction, as a novel two-phase partitioning system for biotransformation, is reviewed by analysis of some topical examples. At the end of the review, the process control and downstream processing in the application of the novel two-phase partitioning system for biotransformation are also briefly discussed.

The potential of cloud point system as a novel two-phase partitioning system for biotransformation

The enzymatic, thermodynamically controlled synthesis of amoxicillin in aqueous solution was measured in order to study the feasibility of a `solid-to-solid' conversion. In aqueous solution, however, the synthetic yield of amoxicillin remains lower than the amoxicillin solubility. Therefore, a `solid-to-solid' synthesis of amoxicillin in aqueous solution is not feasible. Synthetic yields in enzymatic condensation reactions can often be improved by adding organic solvents in monophasic systems. Addition of cosolvents is calculated to improve the apparent equilibrium constant of amoxicillin synthesis considerably, but probably not the synthetic yield, due to solubility restrictions of the reactants.

Feasibility of the thermodynamically controlled synthesis of amoxicillin

Course of pH during the formation of amoxicillin by a suspension-to-suspension reaction.

In the present downstream processing of penicillin G, penicillin G is extracted from the fermentation broth with an organic solvent and purified as a potassium salt via a number of back-extraction and crystallization steps. After purification, penicillin G is hydrolyzed to 6-aminopenicillanic acid, a precursor for many semisynthetic beta-lactam antibiotics. We are studying a reduction in the number of pH shifts involved and hence a large reduction in the waste salt production. To this end, the organic penicillin G extract is directly to be added to an aqueous immobilized enzyme suspension reactor and hydrolyzed by extractive catalysis. We found that this conversion can exceed 90% because crystallization of 6-aminopenicillanic acid shifts the equilibrium to the product side. A model was developed for predicting the equilibrium conversion in batch systems containing both a water and a butyl acetate phase, with either potassium or D-p-hydroxyphenylglycine methyl ester as counter-ion of penicillin G. The model incorporates the partitioning equilibrium of the reactants, the enzymatic reaction equilibrium, and the crystallization equilibrium of 6-aminopenicillanic acid. The model predicted the equilibrium conversion of Pen G quite reasonably for different values of pH, initial penicillin G concentration and phase volume ratio. The model can be used as a tool for optimizing the enzymatic hydrolysis.

Equilibrium modeling of extractive enzymatic hydrolysis of penicillin G with concomitant 6-aminopenicillanic acid crystallization.

Synthetic yields in enzymatic condensation reactions can often be improved by using organic solvents in monophasic systems. However, the addition of these cosolvents may result in loss of stability of the enzyme. Therefore, knowing that stabilization of the enzyme is necessary and often laborious, it would be useful to predict the potential improvement of the reaction equilibrium in the presence of the solvents before modifying the enzyme. This paper describes a model for predicting reaction equilibria in cosolvent-water mixtures by simply measuring the equilibrium in water, and the apparent pKa of all reactants in those mixtures. In the model a reference reaction must be chosen. This choice of reference reaction is crucial for the outcome of the model. The reference reaction should be chosen such that the same functional groups are present in the same ionic state both in the reactants as in the products. The model works quite well for the model system studied, i.e. synthesis of penicillin G from 6-aminop...

Predicting enzyme catalyzed reaction equilibria in cosolvent-water mixtures as a function of pH and solvent composition

To facilitate experimental studies on countercurrent reactors, a discrete contacting mode was worked out experimentally and theoretically. Enzymatic hydrolysis of penicillin G to phenylacetic acid and 6-aminopenicillanic acid was carried out in biphasic aqueous organic systems without pH control. The two phases were countercurrently contacted in a discrete manner, so that equilibrium was reached in each stage. Sets of three and five shake flasks served to mimic equilibrium stages in the countercurrent setup. It was shown that discrete countercurrent contact leads to the same extent of improvement of the equilibrium conversion as continuous countercurrent contact does, when compared to the batch situation. Therefore, discrete experiments may be used to simplify the development of continuous countercurrent reactors.

M. B. Diender

Papers

Feasibility of the thermodynamically controlled synthesis of amoxicillin

Course of pH during the formation of amoxicillin by a suspension-to-suspension reaction.

Equilibrium modeling of extractive enzymatic hydrolysis of penicillin G with concomitant 6-aminopenicillanic acid crystallization.

Predicting enzyme catalyzed reaction equilibria in cosolvent-water mixtures as a function of pH and solvent composition

Discrete countercurrent contacting : An experimental method for developing continuous countercurrent reactors