Immobilization of l-aspartate oxidase from Sulfolobus tokodaii as a biocatalyst for resolution of aspartate solutions

Abstract

L-Aspartate oxidase from the thermophilic archaebacterium Sulfolobus tokodaii (StLASPO) catalyzes the stereoselective oxidative deamination of L-aspartate to yield oxaloacetate, ammonia and hydrogen peroxide. The recombinant flavoenzyme shows distinctive features that make it attractive for biotechnological applications (it is highly thermostable, it is stable in a broad pH range, it tightly binds the FAD cofactor and it shows a low K m for dioxygen). In order to set up an efficient and economically feasible bioconversion process, in this work, we investigated the immobilization of this novel biocatalyst. The best results in terms of immobilization yield have been obtained when StLASPO was immobilized on the amino support Relizyme™ HA403/S R after activation with glutaraldehyde and on the epoxy support SEPABEADS® EC-EP/S (in both cases, through the free amino groups of the enzyme), as well as prepared as cross-linked enzyme aggregates (CLEA). By using the Relizyme™ HA403/S R support, as well as by the CLEA preparation procedure, full immobilization in terms of enzymatic activity was obtained. Immobilized StLASPO was used for the resolution of racemic solutions of 50 mM D,L-aspartate achieving full resolution in <1 hour. Indeed, the Relizyme-StLASPO immobilized enzyme (the one showing the highest immobilization yield) was efficiently reused in 5 cycles keeping full oxidation of L-aspartate in ≤2 hours. In a semi-preparative scale reaction, 66 mg of enantiomerically pure D-aspartate were produced in 1 day using 20 units of enzyme.

Immobilization of an Industrial β-Glucosidase from Aspergillus fumigatus and Its Use for Cellobiose Hydrolysis

“…The hydrolysis reaction of pNPG occurs at a high rate, so that the total rate of the process would be determined by the arrival of more substrate to the active center of the enzyme, which in this case would be the slow and, therefore, limiting stage of catalysis. It should be noted that the ReliZyme™ HA403 support has been used in the immobilization of other enzymes of biotechnological interest, appearing in several literature citations to date [15,[30][31][32][33][34].…”

l-aspartate oxidase magnetic nanoparticles: synthesis, characterization and l-aspartate bioconversion

“…5): aer 420 min of reaction, #70% of the substrate was converted in the h and sixth cycle. Notably, under optimized conditions, the Relizyme-StLASPO fully converted 25 mM L-aspartate for three sequential cycles of 4 hours onlyd, 7 pointing to partial inactivation of the immobilized a-voenzyme. Taking into consideration the four cycles that resulted in full L-aspartate oxidation, 50 mmoles of substrate were converted per unit of NP-LASPO.…”

“…7 The separation allows to easily distinguish and quantify L-aspartate from glycine (used as internal standard) and thus to calculate the bioconversion yield; R t (glycine): 10.5 min, R t (L-aspartate) 4.5 min.…”