Transaminases immobilization

Another alternative for co-transaminase immobilization on chitosan beads is the immobilization on catechol-chitosan-iron oxide nanoparticles. Ni et al. established a protocol for this composition carrying adhesive moieties for surface linkages [137]. After immobilization the enz3une showed higher thermal and pH stability compared to the free enz3une, and after 15 cycles of usage 50% of its activity remained. 

The aminotransferase class of enzymes (E.C. 2.6.1.x), also known as transaminases, are ubiquitous, PLP-requiring enzymes that have been used extensively to prepare natural L-amino acids and other chiral compounds.30 123 124 The L-aminotransferases catalyze the general reaction shown in Scheme 19.19 where an amino group from one L-amino acid is transferred to an a-keto acid to produce a new L-amino acid and the respective a-keto acid (see also Chapter 3). Those enzymes most commonly used as industrial biocatalysts have been cloned, overexpressed, and generally used as whole-cell or immobilized preparations. These include branched chain aminotransferase (BCAT) (E.C. 2.6.1.42), aspartate aminotransferase (AAT) (E.C. 2.6.1.1), and tyrosine aminotransferase (TAT) (E.C. 2.6.1.5). 

Production of L-Amino Acids Using Immobilized Transaminases... 

The pH-rate profile for the reaction catalyzed by the E. coli broad-range transaminase was determined using the immobilized transaminase with p-fluorophe-... 

Employment of glutamate oxidase permits the sequential assay of both transaminases without the need for co-immobilizing a second enzyme. Using a glutamate oxidase sensor, preincubation of the sample for 30 min has been found to be required [379]. Optimization of this type of sensor permitted the preincubation time to be lowered to 10 min [380]. 

Aspartic acid, alanine, phenylalanine, and lysine were manufactured by enzymatic route. Immobilized E. coli cells expressing aspartate or the immobilized enzyme has been used in the commercial production of aspartic acid from ammonia and fumaric acid. Chibata and coworkers also produced alanine by microbial Pseudomonas dacunhae) L-aspartate P-decarboxylase with aspartate as the starting material. Phenyl alanine was manufactured from fw s-cinnamic acid and ammonia by the enzymatic route by phenyl alanine ammonia lyase as catalyst or from phenyl pyruvate and aspartic acid using transaminase. 

Scheme 10.16 Schematic diagram of continuous flow dual reaction kinetics. The dual reaction system consists of two nickel-derivatized silica microreactors connected in series as described in Section 2.3.4. His6-enzymes transketolase and transaminase were immobilized inside each of the microreactors...

Tmppo, M.D., Strotman, H., and Hughes, G. (2012) Development of an immobilized transaminase capable of operating in organic solvent. ChemCatChem, 4, 1071-1074. 

Another successful example in which an immobilized enz)mie was stabilized for biocatalysis in organic media was reported by Merck researchers for a Uansaminase-catalyzed biotransformation [33]. The transaminase was immobilized on a range of commerdally available polymer-based resins. The adsorption support SEPABEAD EXE-120 for binding enzymes through hydrophobic interadions turned out to be most suitable and gave a residual activity of 45%... 

For the in vitro screening of enzyme pairs and enzymatic pathways, immobilized enzyme microreactor systems (lEMR) were established. This includes the reversible immobilization of His -tagged enzymes via nickel-nitriloacetic acid (Ni-NTA) linkage on the surface-derivatized silica. First of all the kinetic parameters for a new enzymatic pathway have to be defined via a transketolase and a w-transaminase in a continuous flow system for a two-step asymmetric synthesis of chiral amino alcohols [147]. The apparenf value is found to be flow rate dependent with different flow rates ranging from 2 to 30 j1/min. The turnover rate was also found to be lower than that in solu-... 

Synthesis of 2-amino-1,3,4-butanetriol by a transketolase/ transaminase enzyme cascade via continuous flow dual reactor. The enzymes were immobilized inside the silica microreactors separately. 

The development of rapid HTS assays is important to test the substrate scope, suitable amino donors/acceptors, and the stability under different reaction conditions, like temperature, pH, different solvents, and immobilization methods. Furthermore the rapid progress in protein engineering like directed evolution requires fast selection methods. This subject was extensively reviewed by Mathew et al. [148]. In the following a colorimetric, photometric, and kinetic assay for rapid transaminase activity screening is described and illustrated in Scheme 29.17. 

Recently immobilized transaminases in neat organic solvents have been examined for the asymmetric synthesis of amines [153]. The immobilization of the co-transaminase with Sepabeads was carried out by incubation of the transaminase with the beads in buffer for 48 h. By means of the enzyme achvity, the screening of several organic solvents was carried out among which isopropyl acetate was the most suitable solvent. The immobilized enzyme showed high activity and stability under the chosen reachon conditions, as well as good yields of various tested amines. 

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