Open AccessOne‐step purification and immobilization of a β‐amino acid aminotransferase using magnetic (M‐PVA) beads
Author(s) -
Dold SarahMarie,
Cai Liyin,
Rudat Jens
Publication year - 2016
Publication title -
engineering in life sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.547
H-Index - 57
eISSN - 1618-2863
pISSN - 1618-0240
DOI - 10.1002/elsc.201600042
Subject(s) - enantiopure drug , immobilized enzyme , chemistry , iminodiacetic acid , amino acid , biocatalysis , combinatorial chemistry , kinetic resolution , covalent bond , thermal stability , chromatography , enzyme , organic chemistry , catalysis , biochemistry , chelation , enantioselective synthesis , ionic liquid
Aminotransferases (ATs) received much attention as biocatalyst in the last decades and are utilized for the synthesis of chiral amines, amino alcohols and amino acids. More recently, enzymatic synthesis of enantiopure β‐amino acids is of increasing interest, for their application in peptidomimetics or other drug related compounds. For economic process management it is of major interest to acquire reusable biocatalysts. This is achievable by immobilization of the enzymes. A wide range of immobilization techniques for ATs exists. Two of the most common procedures are entrapment in sol‐gel or calcium‐alginate and alternatively covalent binding to chitosan support. For these traditional immobilization techniques, high amounts of purified enzyme solutions are required which is cost intensive. A major step toward faster and straightforward immobilization is the insertion of affinity tags, like a His‐tag, to the enzyme coding gene. Immobilization on non‐porous polyvinyl magnetic micro beads functionalized with IDA (iminodiacetic acid) leads to a fast and easy one‐step purification and immobilization of a β‐amino acid AT from Variovorax paradoxus . For loading of the functionalized beads, three divalent metal ions (Ni 2+ , Co 2+ and Cu 2+ ) were tested. The best activity was obtained with Ni 2+ and almost no activity was detected after loading the beads with Cu 2+ . The immobilized AT was successfully tested to synthesize several enantiopure β‐amino acids via kinetic resolution. Further examination and characterization of this immobilized AT showed improved stability compared to the crude cell extract. The immobilized AT revealed enhanced pH and thermal stability compared to the crude cell extract. High activity was preserved up to 38 days of storage at 4°C. Moreover, the use of magnetic beads allows an easy and mild recycling of the immobilized enzyme after the reaction, leading to an efficient reuse of the immobilized enzyme, for at least seven cycles.