Abstract
Arylmalonate decarboxylase (AMD) is a monomeric enzyme of only 26 kDa. A recombinant AMDase from Bordetella bronchiseptica was expressed in Escherichia coli and the enzyme was immobilized using different techniques: entrapment in polyvinyl alcohol (PVA) gel (LentiKats®), covalent binding onto magnetic microparticles (MMP, PERLOZA s.r.o., Lovosice, Czech Republic) and double-immobilization (MMP-LentiKats®) using the previous two methods. The double-immobilized AMDase was stable in 8 repeated biocatalytic reactions. This combined immobilization technique has the potential to be applied to different small proteins.
Highlights
Escherichia coli BL21 (DE3) + pET28b carrying the amd gene was cultivated in Lysogeny broth (LB)
The work presented here demonstrates a novel approach to the immobilization of small enzymes and proteins
AMDase, which is a monomeric enzyme of only 26 kDa, was successfully covalently bonded to magnetic microparticles and entrapped in polyvinyl alcohol (PVA) gel
Summary
Arylmalonate decarboxylase [EC 4.1.1.76, AMDase] is a biotechnologically important enzyme, recently reviewed by Myiamoto and Kourist [1], which is able to catalyse an enantioselective decarboxylation of α-aryl-α-methylmalonates forming corresponding α-arylalkanoates and carbon dioxide, such as decarboxylation of α-phenylmalonic acid forming α-phenylacetic acid (Figure 1) [2].This enzyme has the unique ability to produce optically active α-arylpropionates that are part of an important class of nonsteroidal anti-inflammatory drugs (NSAIDs) [3], widely used against pain, fever and inflammation due to their analgesic, antipyretic and anti-inflammatory effects [4].The AMDase shows high enantioselectivity and has been used for the preparation of optically pure aryl, alkenyl, aliphatic and hydroxycarboxylic acids, such as flurbiprofen or naproxen [5,6,7]. Arylmalonate decarboxylase [EC 4.1.1.76, AMDase] is a biotechnologically important enzyme, recently reviewed by Myiamoto and Kourist [1], which is able to catalyse an enantioselective decarboxylation of α-aryl-α-methylmalonates forming corresponding α-arylalkanoates and carbon dioxide, such as decarboxylation of α-phenylmalonic acid forming α-phenylacetic acid (Figure 1) [2]. This enzyme has the unique ability to produce optically active α-arylpropionates that are part of an important class of nonsteroidal anti-inflammatory drugs (NSAIDs) [3], widely used against pain, fever and inflammation due to their analgesic, antipyretic and anti-inflammatory effects [4]. The mechanism of asymmetric decarboxylation of AMDase has been described; it was proposed that the Cys 188 in AMDase can act as a proton donor to form the asymmetric center of the product [9].
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