L‐Lysine α‐oxidase from Trichoderma viride i4. Purification and characterization

Abstract

A L‐lysine α‐oxidase (LOD) has been purified to homogeneity in a two‐step procedure with 300‐fold enrichment and 60% recovery from the culture extract of Trichoderma viride i4. The enzyme catalyzes the reaction between L‐lysine and molecular oxygen forming 2‐oxo‐6‐aminocaproate, ammonia and hydrogen peroxide. Numerous substrates have been tested. The Km value for L‐lysine was found to be 0.026 mM. Its apparent molecular mass is 110000 Da when determined by gel filtration on Sephadex G‐200, the estimated molecular weight of the subunits being 55000 Da in the SDS‐PAGE. The enzyme is a glycoprotein and shows absorption maxima at 276, 386 and 463 nm. It was found to contain 1 mol of FAD per subunit. The coenzyme is bound non‐covalently. Its isoelectric point is at pH 4.3. The enzyme is stable at extreme pH values, at relatively high temperatures and in diluted hydrogen peroxide.The enzyme described here differs from two other known L‐lysine oxidases previously characterized with regard to its amino acid composition and its substrate specificity.

Allosteric Enzyme-Based Biosensors—Kinetic Behaviours of Immobilised L-Lysine-α-Oxidase from Trichoderma viride: pH Influence and Allosteric Properties

“…The sigmoidal behaviour of LO kinetics of course confirmed the allosteric behaviour of this enzyme. Indeed, as pointed out in the Introduction section, LO is a dimeric enzyme consisting of two identical subunits each one containing a FAD unit [ 2 , 4 , 5 , 6 , 7 , 8 , 9 , 10 ], thus, cooperative binding could arise; nevertheless, a pH dependence of the cooperation was never reported before and accordingly a deeper study was performed. Please note that the pH dependence on the allosteric behaviour of enzymes is not surprising, since it was already described in 1904 by Christian Bohr while studying the oxygen binding affinity of haemoglobin, a striking pH effect well-known as the Bohr effect (see for example ref.…”

“…L-lysine-α-oxidase (LO) is an oxidoreductase that catalyses the oxidation of the well-known essential amino acid L-lysine (Lys) according to the following reaction: L-lysine + O 2 → α-keto-ε-aminocaproate + H 2 O 2 + NH 3 where the produced α-keto-ε-aminocaproate successively dehydrates spontaneously to Δ 1 -piperidine-2-carboxylate. The enzyme was firstly isolated from Trichoderma viride [ 1 , 2 ], but soon was found also in other Trichoderma species and strains [ 3 , 4 , 5 , 6 , 7 , 8 ] and characterised as well (see refs. [ 9 , 10 ] for a review).…”

“…LO is an L-amino acid oxidase that is almost specific to Lys, but, depending on the enzyme source, other Lys analogues (such as L-ornithine, L-tyrosine and L-arginine) are oxidised too to some extent—no matter the analogues, it is the α-amino group of the amino acid the residue that is oxidised by the enzyme, while the terminal amino group appears to be important in the binding of amino acids to the enzyme and/or to the enzyme catalysis [ 2 ]. Furthermore, LO shows a molecular weight of about 110–116 kD (depending on the source) [ 2 , 4 , 5 , 6 , 7 , 8 , 9 , 10 ] and consists of two subunits of the same molecular weight of about 55–58 kD containing one FAD per subunit [ 2 , 4 , 5 , 6 , 7 , 8 , 9 , 10 ].…”