Fusion protein of Vitreoscilla hemoglobin with D‐amino acid oxidase enhances activity and stability of biocatalyst in the bioconversion process of cephalosporin C

Abstract

In this study we constructed an artificial flavohemoprotein by fusing Vitreoscilla hemoglobin (VHb) with D-amino acid oxidase (DAO) of Rhodotorula gracilis to determine whether bacterial hemoglobin can be used as an oxygen donor to immobilized flavoenzyme. This chimeric enzyme significantly enhanced DAO activity and stability in the bioconversion process of cephalosporin C. In a 200-mL bioreactor, the catalytic efficiency of immobilized VHb-DAO against cephalosporin C was 12.5-fold higher than that of immobilized DAO, and the operational stability of the immobilized VHb-DAO was approximately threefold better than that of the immobilized DAO. In the scaled-up bioprocess with a 5-L bioreactor, immobilized VHb-DAO (2500 U/L) resulted in 99% bioconversion of 120 mM cephalosporin C within 60 min at an oxygen flow rate of 0.2 (v/v) x min. Ninety percent of the initial activity of immobilized VHb-DAO could be maintained at up to 50 cycles of the enzymatic reaction without exogenous addition of H(2)O(2) and flavin adenine dinucleotide (FAD). The purity of the final product, glutaryl-7-aminocephalosporanic acid, was confirmed to be 99.77% by high-performance liquid chromatography (HPLC) analysis. Relative specificity of immobilized VHb-DAO on D-alpha-aminoadipic acid, a precursor in cephalosporin C biosynthesis, increased twofold, compared with that of immobilized DAO, suggesting that conformational modification of the VHb-DAO fusion protein may be altered in favor of cephalosporin C.

Thermal inactivation of D‐amino acid oxidase from Trigonopsis variabilis occurs via three parallel paths of irreversible denaturation

“…Therefore, several authors have addressed the issues of stability and stabilization of TvDAO (Fernández-Lafuente et al, 1999;Ju et al, 2000;Khang et al, 2003). However, a clear-cut inactivation mechanism has not been established for TvDAO so far, and so enhancement of the total turnover number of the enzyme through rational stabilization of the activity remains a problem.…”

Docking of Polyaniline with D-Amino Acid Oxidase of Rhodosporidium toruloides and Pig Kidney-An Insight into the Mechanism of Binding for Immobilization in Polyaniline Supports

“…The D-aao assay (pyruvate method) of the immobilized beads was done according to the method of Khang et al [24] with some modifications. Briefly, 10 mM of D-alanine dissolved in 0.1 M sodium phosphate buffer, pH 8.0 containing 400 U catalase (HiMedia, India) was reacted with 0.3 g of the PAni-sodium alginate-D-aao beads and the pyruvic acid produced was determined by reacting 100 µl of the sample solution with 40 µl of 0.2% 2,4-dinitrophenylhydrazine (in 2 N HCl) at 37°C for 3 min.…”

Kinetic Resolution of 3-Fluoroalanine Using a Fusion Protein ofD-Amino Acid Oxidase withVitroscillaHemoglobin

“…It scavenges oxygen molecules from water to provide oxygen to increase cellular activities in heterologous organisms. 10) Artificial flavohemoprotein (VHb-DAAO), developed by fusing VHb with DAAO from R. gracilis, significantly enhances DAAO activity and stability in the bioconversion process of cephalosporin C. 11) In this study, a chimeric enzyme (VHb-DAAO) was used in the kinetic resolution of rac-3-fluoroalanine into (R)-3-fluoroalanine (Fig. 1A).…”

“…The chimeric enzyme (VHb-DAAO) gene was amplified from the pET-VHbDAO 11) by PCR using P2 and P3 (5 0 -CGCCATATGTTAGACC-AGCAAACC-3 0 ) primers. The PCR product was ligated and inserted into IPTG-inducible expression vector pET24ma.…”