Acylase operational stability

Bommarius, A.S., Drauz, K., Klenk, H. and Wandrey, C. (1992) Operational stability of enzymes - acylase catalyzed resolution of /V-acetyl amino acids to enantiomerically pure L-amino acids. Annals of the New York Academy of Sciences, 672, 126-136. 

As in all catalytic processes, catalyst stability is an essential feature. We have investigated the stability of acylase in conditions that are pertinent for large-scale processes. Instead of just determining thermal stability, which can be done by measuring storage stability of the enzyme in particular conditions of temperature and pH, we have also determined operational stability. The relevant parameter for operational stability studies of enzymes is the product of active enzyme concentration [E] and residence time t, [E] t. For a CSTR, the quantities [E] and t are linked by Eq. (19.37), where [S0] = initial substrate concentration, % = degree of conversion, and r(x) = reaction rate (Wandrey, 1977 Bommarius, 1992b). 

The reuse of the expensive biocatalysts is a prerequisite for the economy of the biocatalytic process. On a lab-scale the carrier-fixed enzymes can be used for more than 100 cycles (DAO) and 180 cycles (GA), before reaching half of the starting activity [15]. Prolonging the reaction time can compensate for the decreasing activity. As claimed by reference [15] for the lab-scale preparation of 1 kg 7-ACA about 1.2 kU D-amino acid oxidase and 1.5 kU glutaryl-7-ACA acylase are consumed, but operational stability is dependent on scale. In production vessels gradients, e.g., pH value and shear stress, are different and could influence the operational stability of the biocatalysts, therefore a higher biocatalyst consumption is usually realistic. 

Since the early 1980s this process has been scaled-up to a production level of hundreds of tons per year. Fig. 6 shows the Degussa process for manufacturing l-methionine, l-6 [9 a]. The biocatalyst is produced in bulk quantities and its operational stability is high hence this continuous EMR-acylase process demonstrates high efficiency, especially on a large-scale [9]. 

Results on operational stability of both acylases in a recycle reactor at constant conversion1641 with reaction conditions close to intended large-scale conditions demonstrated much better stability of the Aspergillus enzyme, while renal enzyme is not stable enough for long-term operation164,651. Moreover, on the process scale achieved today the supply of renal acylase is insufficient, so that fungal acylase is used almost exclusively nowadays, especially since the price per unit is comparable. 

The N-acetyl-D,L-amino acid precursors are conveniently accessible through either acetylation of D,L-amino acids with acetyl chloride or acetic anhydride in a Schotten-Baumann reaction or via amidocarbonylation I801. For the acylase reaction, Co2+ as metal effector is added to yield an increased operational stability of the enzyme. The unconverted acetyl-D-methionine is racemized by acetic anhydride in alkali, and the racemic acetyl-D,L-methionine is reused. The racemization can also be carried out in a molten bath or by an acetyl amino acid racemase. Product recovery of L-methionine is achieved by crystallization, because L-methionine is much less soluble than the acetyl substrate. The production is carried out in a continuously operated stirred tank reactor. A polyamide ultrafiltration membrane with a cutoff of 10 kDa retains the enzyme, thus decoupling the residence times of catalyst and reactants. L-methionine is produced with an ee > 99.5 % and a yield of 80% with a capacity of > 3001 a-1. At Degussa, several proteinogenic and non-proteinogenic amino acids are produced in the same way e.g. L-alanine, L-phenylalanine, a-amino butyric acid, L-valine, l-norvaline and L-homophenylalanine. 

Regarding the economical viability of the plant, the retention and stability of acylase are essential features for the process. An ultrafiltration unit retains acylase as the mobile catalyst in the reactor. Alternatively, acylase can be immobilized in a fixed or fluidized bed. A mobile catalyst system is preferred compared to the immobilized form, as the mobile catalyst system avoids mass-transfer limitations. Additionally, regeneration of the catalyst and scale-up of the reactor are much easier as compared to the process with the immobilized acylase. With respect to the deactivation of the catalyst, the thermal as well as the operational stability of acylase has been evaluated extensively [128, 129]. At a pH of 7, acylase appears to be sufficiently stable for L-amino acid manufacture. 

The acylase-catalyzed resolution of N-acetyl-D,L-amino acids to obtain enantiomerically pure i-amino acids (see Chapter 7, Section 7.2.1) has been scaled up to the multi-hundred ton level. For the immobilized-enzyme reactor (Takeda, 1969) as well as the enzyme membrane reactor technology (Degussa, 1980) the acylase process was the first to be scaled up to industrial levels. Commercially acylase has broad substrate specificity and sufficient stability during both storage and operation. The process is fully developed and allowed major market penetration for its products, mainly pharmaceutical-grade L-methionine and L-valine. 

In switching from buffered to pH-stat operation one should be aware of changes in kinetics as discussed above. The pH of the solution is important not only for enzyme activity but also for enzyme stability. Unfortunately, the optimal pH values for enzyme activity and stability are not necessarily identical, as is well documented in the literature for the hydrolysis of penicillin G by penicillin acylase. In such cases, the method for controlling pH and mixing behavior of the reactor may become crucial. 

Enzymes are labile catalysts that require to be stabilized under operating conditions. Penicillin acylase is a moderately expensive enzyme so that its efficient... 

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