초록
l-Alanine dehydrogenase from Bacillus subtillis is a NADH-dependent enzyme that catalyzes the reversible reductive amination of pyruvate using ammonia as amine source. This enzyme has been intensively exploited in biocatalysis but its immobilized form is rarely used. In this work, we have immobilized this enzyme on agarose microbeads activated with glyoxyl groups (aliphatic aldehydes). 85% of the offered enzyme was immobilized on these microbeads and the enzyme recovered 45% of its initial reduction amination activity upon the immobilization. The resulting immobilized preparation was 13 times more thermostable than the soluble enzyme because the immobilization attenuated the negative conformational changes induced by the temperature. The optimally immobilized enzyme was also stabilized against acidic pH. Finally, we have applied this heterogeneous biocatalyst to the synthesis of L-[<SUP>13</SUP>N]alanine using pyruvate and [<SUP>13</SUP>N]NH<SUB>4</SUB>OH obtaining a radiochemical yield of >95% in 20min. This immobilized enzyme was re-used for up to 5 cycles keeping the maximum yield. Herein, we have fabricated a highly stable and active heterogeneous biocatalyst for reductive aminations of α-ketoacids that has an enormous potential in biocatalysis.