Assay of dehydrogenases with an O2-consuming biosensor

Haouz, Ahmed; Geloso-Meyer, A.; Burstein, Claude

doi:10.1016/0141-0229(94)90169-4

Cited by 19 publications

(5 citation statements)

References 9 publications

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“…Likewise, when oxygen-consuming enzymes applicable to the medicine and the food industries, as well as the environment, are properly linked with bacterial hemoglobins, it may be possible to develop more sensitive biosensors (Haouz et al, 1994;Sacchi et al, 1998). …”

Section: Discussionmentioning

confidence: 99%

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

Khang

Kim

Hah

et al. 2003

Biotech & Bioengineering

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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.

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Section: Discussionmentioning

confidence: 99%

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

Khang

Kim

Hah

et al. 2003

Biotech & Bioengineering

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“…These assays were performed in 2 ml of solution in the presence of an excess of the glucose substrate (100 mM of β‐ D ‐glucose=10 K m ). The reaction rate was expressed as the oxygen uptake per minute and per unit of enzyme concentration [14].…”

Section: Methodsmentioning

confidence: 99%

Involvement of protein dynamics in enzyme stability

Haouz¹,

Glandières²,

Alpert³

2001

FEBS Letters

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“…The major reason for using this test instead of the more rapid coupled spectrophotometric assay (30) is that some inhibitors absorb light at 340 nm (4 and 5), thereby rendering difficult the evaluation of NADH concentration in the coupled reaction. Also, coupled enzymatic tests might be errorprone in the determination of the true value of K i and K m of inhibitors and substrate, respectively, through the recycling of some substrate or product during the reaction coupling (31). The reaction is carried out in a 1-ml final volume of a solution of 50 mM Tris-HCl, pH 7.5, 20 mM magnesium acetate, 100 mM KCl, 1 mM EDTA, 0.5 mM dithiothreitol using different initial concentrations of ATP and TMP in the presence or absence of various inhibitors.…”

Section: Enzymatic Assaymentioning

confidence: 99%