One‐step purification and immobilization of a β‐amino acid aminotransferase using magnetic (M‐PVA) beads

Abstract

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 (Ni2+, Co2+ and Cu2+) were tested. The best activity was obtained with Ni2+ and almost no activity was detected after loading the beads with Cu2+. 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.

Enantiomer discrimination in β-phenylalanine degradation by a newly isolated Paraburkholderia strain BS115 and type strain PsJN

“…The proposed transamination of ( S )-β-PA leads to the corresponding β-keto acid 3-oxo-3-phenylpropanoic acid. However, this molecule has never been detected in medium as it spontaneously decarboxylates to AP as has been shown in previous studies (Crismaru et al 2013 ; Dold et al 2016 ). AP on the other hand was shown to emerge in parallel to the degradation of ( S )-β-PA, peaking for both strains after about 30 h when ( S )-β-PA is depleted (Additional file 1 : Figure S1).…”

“…Several ( R )-selective ω-TAs have been described for the ability to convert and synthesize ( R )-configurated amines. However, activity towards aromatic or bulky β-AAs were only observed for ( S )-selective ω-TAs (Koszelewski et al 2010 ; Seo et al 2012 ; Dold et al 2016 ) and no ( R )-PA converting transaminase has been reported until now (Mutti et al 2011 ; Mallin et al 2014 ; Jiang et al 2015 ; Skalden et al 2015 ). Only Mano et al investigated an ( R )-selective strain, Arthrobacter sp.…”

FoldX as Protein Engineering Tool: Better Than Random Based Approaches?

“…The well-known technique to immobilize proteins to gain stabilized proteins is applied for antibody to increase the thermostability [ 34 , 35 ]. Beside the improvement of thermostability using immobilization techniques, directed evolution is an alternative approach, but the existence of a robust high-throughput screening assay for the selected protein is an important prerequisite [ 11 , [36] , [37] , [38] , [39] , [40] , [41] ]. For enzymes, activity can be used as parameter for functionality at elevated temperatures, but for non-catalyzing proteins a more sophisticated assay or even protein purification is necessary.…”

The ω‐transaminase engineering database (oTAED): A navigation tool in protein sequence and structure space

“…However, considerable enzyme engineering efforts were needed to adapt enzymes to the desired substrates. In amino acid synthesis, a special focus lies on the synthesis of optically pure β‐amino acids like β‐phenylalanine which can be produced using lipase ‐ ω‐TA cascade reactions or racemic resolution reactions using Fold type I‐( S )‐selective ω‐TAs . Non‐standard amino acids might be applied in synthetic peptides or for the synthesis of the antitumor drug paclitaxel .…”

“…In amino acid synthesis, a special focus lies on the synthesis of optically pure β‐amino acids like β‐phenylalanine which can be produced using lipase ‐ ω‐TA cascade reactions or racemic resolution reactions using Fold type I‐( S )‐selective ω‐TAs . Non‐standard amino acids might be applied in synthetic peptides or for the synthesis of the antitumor drug paclitaxel . In asymmetric synthesis, the reaction equilibrium is pushed toward the desired product by removing the co‐product, by high concentrations of the amine donor or by regenerating the co‐substrate.…”