Immobilization of phenylalanine dehydrogenase onto Eupergit CM for the synthesis of (S)-2-amino-4-phenylbutyric acid (original) (raw)
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Reports on chemical immobilization of proteins and enzymes first appeared in the 1960s. Since then, immobilized proteins and enzymes have been widely used in the processing of variety of products and increasingly used in the field of medicine. Here, we present a review of recent developments in immobilized enzyme use in medicine. Immobilized enzymes are widely used for variety of applications. Based on the type of application, the method of immobilization and support material can be selected. The immobilized enzymes can be separated from the reaction mixture and reused and also immobilized in order to prevent the enzyme from being exposed to harsh conditions, high temperature, surfactants, and oxidizing agents etc. the immobilized enzymes are also widely used in food industry, pharmaceutical industry, bioremediation, detergent industry, textile industry, etc. Enzyme immobilization improves the operational stability and is also due to the increased enzyme loading which causes the controlled diffusion. Several hundreds of enzymes are immobilized and used for various large scale industries. Immobilization technique reduces the effluent treatment costs and this paper reviews the methods and applications of immobilized enzymes.
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Enzyme and Microbial Technology, 2001
The immobilization parameters were optimized for the hydantoinase and the L-N-carbamoylase from Arthrobacter aurescens DSM 3747 or 3745, respectively. To optimize activity yields and specific activities for the immobilization to Eupergit C, Eupergit C 250 L, and EAH-Sepharose wild-type, recombinant and genetically modified ('tagged') enzymes were investigated concerning the influence of the protein concentration, the kind of support and the immobilization method. For both enzymes, the use of the recombinant proteins resulted in enhanced specific activities especially when using a hydrophilic support for immobilization such as Sepharose. In the case of a genetically modified hydantoinase carrying a His 6-tag, affinity coupling led to a loss of activity of higher than 80%. Both enzymes were significantly stabilized by immobilization: In packed bed reactors, Eupergit C 250 L (NH 2)-immobilized hydantoinase and EAH-Sepharose-immobilized L-N-carbamoylase showed half-life times of approx. 14000 and 900 hours, respectively. Together with specific activities of the immobilized enzymes of 2.5 U/g wet carrier (hydantoinase) and 10 U/g wet carrier (L-N-carbamoylase) the newly developed biocatalysts are sufficient to fulfill industrial requirements. In comparison to the free enzymes, temperature and pH-optima were increased by 10°C and one pH unit, respectively, after immobilization. The pH and temperature optima of the hydantoinase (L-N-carbamoylase) were determined to be pH 8.5-10 (pH 9.5) and 45-60°C (60°C). In order to provide sufficient amounts of biocatalyst for the process development in mini plant scale, a 50 fold scale-up of the optimized immobilization procedure was carried out for both enzymes. Because of the overlapping optima, both immobilized enzymes can be operated together in one reactor.
Immobilization of synthetically useful enzymes by condensation polymerization
Journal of the American Chemical Society, 1978
Immobilization of Synthetically Useful Enzymes by Condensation Polymerization Sir.' We wish to describe a new procedure for the immobilization of enzymes in cross-linked organic polymer gels. This procedure rivals or surpasses in its operational simplicity and generality methods presently widely used (BrCN-agarose, glutaraldehydc, functionalized glass, preformed activated organic polymer gels),r and has proved especially valuable in immobilization of reliatively delicatc enzymcs of interest for enzyme-cata ly zed organic synthesis.2
Enzyme and Microbial Technology, 2007
A study of an engineered phenylalanine dehydrogenase (PheDH) mutant, L307V, from Bacillus sphaericus, catalysing the oxidation of lphenylalanine (Phe) and five non-natural p-substituted derivatives (4-F/Cl/CH 3 /OCH 3 /NO 2-l-Phe) in water-miscible organic solvents, methanol, ethanol and acetonitrile, is reported. Results showed that the enzyme still had high activity with 0.2 mM 4-CH 3 /OCH 3 /NO 2-l-Phe in 10% methanol. The kinetic parameters of the enzyme were determined with three substrates, 4-F/Cl/CH 3-l-Phe, in the absence/presence of 10% methanol and with two less water-soluble substrates, 4-OCH 3 /NO 2-l-Phe, only in the presence of 10% methanol. Data indicated that catalytic efficiency of the enzyme with 4-Cl/CH 3-Phe as substrates was little changed in 10% methanol as compared to purely aqueous solvent. The stability of L307V, WT and N145A PheDH was assessed over 1 week at two pH values and in the presence/absence of organic solvents. In pH 8.0 buffer with either 10% methanol or 10% ethanol, L307V PheDH retained almost 85% original activity after 7 days, and WT PheDH was similarly stable. In contrast, N145A PheDH, also a versatile biocatalyst, was much less stable, the best performance being 68% retention of activity after 7 days at pH 8.0 with 10% methanol. Practical application of L307V PheDH, with baker's yeast alcohol dehydrogenase for co-enzyme recycling, to synthesise a non-natural amino acid, p-OCH 3-l-Phe is also reported, illustrating the utility of this versatile biocatalyst for potential industrial uses.