Amino acid acylase stability

From the above results it can be observed that Co ions increase the activity of amino acid acylase to a high extent. This is possible by binding of Co ions at the active site of enzyme due to which the stability of enzyme-substrate complex increases which leads to... 

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. 

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. 

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. 

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. 

Penicillin G acylase (PGA) has pivotal role in industry for the synthesis of penicillin antibiotics. PGA catalyzes the hydrolysis of peniciUm and other P-lactam antibiotics to produce 6-amino penicillinic acid [53, 54]. Stability of PGA was investigated by assaying the enzyme activity at different time points after incubation of PGA in various ILs [53]. In the absence of substrate, about 2,000-fold increase in t, was observed in a hydrophobic IL, [EMIM][Tf2N] with respect to twpropanol. Whereas in the presence of substrate, PGA showed less stability in [Tf N]" containing ILs. The reverse trend was found for PGA in a water miscible IL [BMIM][PFg], i.e., PGA showed 9 times increase in tj in [BMIM][PF ] in the presence of substrate even at an elevated temperature of 40°C [54]. These altered t, values in hydrophobic ILs in the presence of substrates were explained on the basis of cumulative outcome of specific interaction of substrates with the active site of enzyme and inhibitory effect of the hydrolytic products formed in the reaction medium. It can be speculated that in a more hydrophilic IL like [BMIM][PF ], substrates shield the enzyme active site from direct interaction with the ionic matrix and thus imparts a stabilizing effect whereas in hydrophobic IL [EMIM][TfjN], the inhibitory effect of hydrolytic products predominates and destabilizes the enzyme. 

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