Acylase immobilised

Several L-amino acids are produced on a large scale by enzymatic resolution of N-acetyl-D,L-amino adds (Figure A8.4). Acylase immobilised on DEAE-Sephadex is for example employed in a continuous process while Degussa uses the free acylase retained in a membrane reactor. In the latter process the advantage of reuse of the enzyme and homogeneous catalysis are combined. 

Recommended model particle systems are enzymes immobilised on carriers ([27,44,45,47,49]), oil/water/surfactant or solvent/water/surfactant emulsions ([27, 44, 45] or [71, 72]) and a certain clay/polymer floccular system ([27, 42-52]), which have proved suitable in numerous tests. The enzyme resin described in [27,44,47] (acylase immobilised on an ion-exchanger) is used on an industrial scale for the cleavage of Penicillin G and is therefore also a biological material system. In Table 3 are given some data to model particle systems. 

En2yme techniques are primarily developed for commercial reasons, and so information about immobilisation and process conditions is usually Limited. A commercially available immobilised penicillin V acylase is made by glutaraldehyde cross-linking of a cell homogenate. It can be used ia batch stirred tank or recycled packed-bed reactors with typical operating parameters as iadicated ia Table 2 (38). Further development may lead to the creation of acylases and processes that can also be used for attaching side chains by ensymatic synthesis. 

Continuous process of amino acid resolution using immobilised acylase... 

Degussa AG uses immobilised acylase to produce a variety of L-amino adds, for example L-methionine (80,000 tonnes per annum). The prindples of the process are the same as those of the Tanabe-process, described above. Degussa uses a new type of reactor, an enzyme membrane reactor, on a pilot plant scale to produce L-methionine, L-phenylalanine and L-valine in an amount of 200 tonnes per annum. 

Since in the case of using immobilised enzymes (acylase enzyme resin [27,45, 47]), normally only a small decrease in the particle diameter leads to a distinct decrease in the enzyme activity, the more sensitive enzyme activity has to be taken as the measure of stress. 

As an example the deactivation of immobilised Pen G acylase, which catalyses the reaction of Pen G to 6-Aminopenicillanic acid and Phenylacetic acid, was studied. This enzyme was covalently bound on an ion-exchanger and cross-linked by glutaric aldehyde. To maintain a high reaction velocity, a neutral pH value (removal of Phenylacetic acid) and therefore the supply of NaOH and stirring for distribution of the base are required. 

Processes operate using immobilised penicillin V or G acylases derived from fungi such as Bovista plumbea, and such as E. coli respectively. Following the discovery ot penicillin acylases commercial processes were developed very rapidly. Productivities ot up to 2,000 kg of 6-APA/kg immobilized enzyme are obtained with operating lifetimes in excess of 10 h. 

Other biocatalytic methods of producing D-p-hydroxyphenylglycine have not proved competitive, for instance transaminase based processes require glutamate to be supplied. Others include the hydrolysis of N-acyl derivatives by acylase and amides by aminopeptidase (DSM), the use of L-specrfic hydantoinases and immobilised subtilisin for the resolution of D,L-2-acetamido-/>-hydroxyphenylacetic acid methyl ester (Bayer). 

The two largest scale industrial processes utilising immobilised enzymes are the hydrolysis of benzyl penicillin by penicillin acylase and the isomerisation of glucose to a glucose-fructose mixture by immobilised glucose isomerase. The immobilisation techniques used in general may be broadly categorised as ... 

Fig. 5.21 Simulation of sequential batch reactor operation with immobilised penicillin acylase at pH 7.8 and 40 C (K = 6.5mM Kj = 56.5mM K2 = 59.3 V = 625 pmol/min/g. a) considering full protection by catalytic modulators (all n, = 1 all n( = 1) b) not considering modulation (till n = 0 all nj = 0) c) with experimentally determined vtilues for modulation factors (Illanes et al. 1996)...

Illanes A, Altamirano C, Zuniga ME (1996) Thermal inactivation of penicillin acylase in the presence of substrate and products. Biotechnol Bioeng 50 609-616 Illanes A, Altamirano C, AUlapan A et al. (1998a) Packed-bed reactor performance with immobilised lactase under thermal inactivation. Enzyme Microb Technol 23 3-9 Illanes A, Wilson L, Altamirano C et al. (1998b) Reactor performance under thermal inactivation and temperature optimization with chitm-immobilized lactase. In Ballesteros A, Plou F, Iborra JL et al. (eds). Stability and stabilization of biocatalysts. Elsevier, Amsterdam, pp 27-34 Illanes A (1999) Stability of biocatalysts. Elect J Biotechnol 2 1-9... 

Calvo JC, Choconta KC, Diaz D et al. (2003) An alpha helix conformationally restricted peptide is recognized by cervical carcinoma patients sera. J Med Chem 46(25) 5389-5394 Cao L, van Rantwijk F, Sheldon R (2000) Cross-linked enzyme aggregates a simple and effective method for the immobilization of penidllin acylase. Otg Lett 2 1361—1364 Cao L, van Langen L, Sheldon R (2003) Immobilised enzymes carrier-bound or carrier free Curr Opin Biotech 14 387-394... 

Abraham EP, Newton GGE (1961) The structure of cephalosporin C. Biochem J 79(2) 377-393 Aguirre C, Toledo M, Medina V et al. (2002) Effect of cosolvent and pH on the kinetically controlled synthesis of cephalexin with immobilised penicillin acylase. Proc Biochem 38(3) 351-360... 

Basso A, De Martin L, Ebert C et al. (2003) Organically modified xerogels as novel tailor-made supports for covalent immobilisation of enzymes (penicillin G acylase). Tetrahed Lett 44 5889-... 

The synthetic challenge centred around the issue that a -lactam ring is more easily hydrolysed than a normal amide. For a selective hydrolysis, special reaction conditions are required, which differentiate between secondary and tertiary amides. Additionally, the carboxy group must be protected before hand. DSM developed a particular process for this, calling it Delft cleavage . Nowadays, the reaction can be carried out in an intrinsically more elegant way with the known penicillin-acylase, which however, in contrast to earlier methods, is now immobilised (Tab. 5.4). [49]... 

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