scholarly journals The Bifunctional Flavokinase/Flavin Adenine Dinucleotide Synthetase from Streptomyces davawensis Produces Inactive Flavin Cofactors and Is Not Involved in Resistance to the Antibiotic Roseoflavin

2007 ◽  
Vol 190 (5) ◽  
pp. 1546-1553 ◽  
Author(s):  
Simon Grill ◽  
Simone Busenbender ◽  
Matthias Pfeiffer ◽  
Uwe Köhler ◽  
Matthias Mack
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Amino Acid ◽  
Sus Scrofa ◽  
Flavin Adenine Dinucleotide ◽  
Functional Expression ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Sensitive Bacterium ◽  
Antibiotic Effect

ABSTRACT Streptomyces davawensis synthesizes the antibiotic roseoflavin, one of the few known natural riboflavin analogs, and is roseoflavin resistant. It is thought that the endogenous flavokinase (EC 2.7.1.26)/flavin adenine dinucleotide (FAD) synthetase (EC 2.7.7.2) activities of roseoflavin-sensitive organisms are responsible for the antibiotic effect of roseoflavin, producing the inactive cofactors roseoflavin-5′-monophosphate (RoFMN) and roseoflavin adenine dinucleotide (RoFAD) from roseoflavin. To confirm this, the FAD-dependent Sus scrofa d-amino acid oxidase (EC 1.4.3.3) was tested with RoFAD as a cofactor and found to be inactive. It was hypothesized that a flavokinase/FAD synthetase (RibC) highly specific for riboflavin may be present in S. davawensis, which would not allow the formation of toxic RoFMN/RoFAD. The gene ribC from S. davawensis was cloned. RibC from S. davawensis was overproduced in Escherichia coli and purified. Analysis of the flavokinase activity of RibC revealed that the S. davawensis enzyme is not riboflavin specific (roseoflavin, k cat/Km = 1.7 10−2 μM−1 s−1; riboflavin, k cat/Km = 7.5 10−3 μM−1 s−1). Similar results were obtained for RibC from the roseoflavin-sensitive bacterium Bacillus subtilis (roseoflavin, k cat/Km = 1.3 10−2 μM−1 s−1; riboflavin, k cat/Km = 1.3 10−2 μM−1 s−1). Both RibC enzymes synthesized RoFAD and RoFMN. The functional expression of S. davawensis ribC did not confer roseoflavin resistance to a ribC-defective B. subtilis strain.

scholarly journals Enhancement of L-amino Acid Oxidase Production by Bacillus Subtilis H-8 With Oxygen-vector and Asymmetric Degradation of DL Arginine to D-arginine

2020 ◽  
Author(s):  
peng xu ◽  
Changpei Pan ◽  
Gongcheng Cui ◽  
ChunYan Wei ◽  
Lijuan Wang ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Oleic Acid ◽  
Amino Acid ◽  
Fermentation Broth ◽  
Cation Exchange Resin ◽  
Optical Purity ◽  
Phenyl Isothiocyanate ◽  
Acid Oxidase ◽  
Amino Acid Oxidase ◽  
Oxygen Vector

Abstract The Bacillus subtilis H-8 independently screened by our laboratory can produce L-amino acid oxidase (L-AAO), and DL-arginine can be degraded asymmetrically by suspending the wet bacteria in the degradation liquid. By adding oxygen-vectors to the fermentation medium, the collected amount of wet bacteria can be increased. Taking n-dodecane, n-hexadecane, oleic acid, paraffin, and n-hexane as oxygen-vectors, the optimal oxygen-vector oleic acid was 1.2% (v/v). The weight of wet cells increased by 66.83% compared with before, and the activity of L-AAO in fermentation broth increased by 38.88% compared with before. The standard sample DL-arginine was derivatized by phenyl isothiocyanate, and then subjected to high performance liquid chromatography(HPLC), and the obtained peak area and arginine content were used as standard curves to measure the DL-arginine. The content of D-arginine and L-arginine in the initial degradation solution was 50% each, and the bacterial cells are added to the initial degradation solution of DL-arginine. After 21 hours of reaction, L-arginine was completely Degraded, remaining 47% of D-arginine.D-alanine was easily extracted from the reaction solution using cation-exchange resin,after centrifugation, decolorization, concentration and vacuum drying, and the chemical and optical purity of the extracted d-alanine was 92.68 and 97.46%, respectively.

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