ABSTRACT
An efficient affinity-purification protocol for Bacillus monomeric sarcosine oxidase (SOX) expressed in Escherichia coli BL21 (DE3) was developed. 4-Aminopyrrole-2-carboxylic acid was chosen as the affinity ligand, which was coupled with Sepharose CL 4B via spacers composed of epichlorohydrin and ethylenediamine. With the affinity medium, the purification process consisted of only one affinity chromatography step to capture monomeric SOX. The purified SOX was 94 and 96% pure when analyzed on an HPLC Vydac C8 column and reducing SDS-PAGE. Meanwhile, the recoveries of typical SOX activity and protein were 90.8 and 37.5%, respectively, which were higher than other reported traditional protocols. Reducing SDS-PAGE analysis revealed that the enzyme was a single polypeptide with the mass of ~46 kDa. The desorption constant Kd and theoretical maximum absorption Qmax were 35 µg/mL and 52.7 mg/g, respectively, in absorption analysis. All results indicated that the method would be of great potential for purifying monomeric SOX on an industrial scale.
Subject(s)
Bacillus/enzymology , Escherichia coli/metabolism , Sarcosine Oxidase/biosynthesis , Sarcosine Oxidase/isolation & purification , Chromatography, Affinity , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Sarcosine Oxidase/metabolismABSTRACT
We purified a sarcosine oxidase from Bacillus sp. strain BSD-8 isolated from soil. We purified the enzyme by ammonium sulfate precipitation, DEAE-cellulose, Toyopearl hydrophobic and Sephadex G-75 molecular sieve chromatography and characterized the purified sarcosine oxidase. This sarcosine oxidase was a flavin enzyme containing a noncovalently bound flavin with the subunit molecular mass of 51 kDa. The optimal temperature for this enzyme was 60 degrees C and it showed its highest activity at pH 8.5. It was stable in the pH range of 8.0-10.0 and at the temperature of 60 degrees C. Estimated by Lineveaver-Burk plots, the K(m) of the enzyme was 3.1 mmol/L. Ag+, Hg2+, SDS and Tween 80 dramatically inhibted the enzyme activity, whereas Tween 20 and Triton X-100 had no effect on enzyme activity. The thermostability of this enzyme was better than reported sarcosine oxidases, and it could be applied in enzymatic measuring of creatinine.
Subject(s)
Bacillus/enzymology , Sarcosine Oxidase/isolation & purification , Sarcosine Oxidase/metabolism , Bacillus/isolation & purification , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Chemical Precipitation , Enzyme Stability , Sarcosine Oxidase/chemistry , Soil MicrobiologyABSTRACT
FAD in monomeric sarcosine oxidase (MSOX) is covalently linked to the protein by a thioether linkage between its 8alpha-methyl group and Cys315. Covalent flavinylation of apoMSOX has been shown to proceed via an autocatalytic reaction that requires only FAD and is blocked by a mutation of Cys315. His45 and Arg49 are located just above the si-face of the flavin ring, near the site of covalent attachment. His45Ala and His45Asn mutants contain covalently bound FAD and exhibit catalytic properties similar to wild-type MSOX. The results rule out a significant role for His45 in covalent flavinylation or sarcosine oxidation. In contrast, Arg49Ala and Arg49Gln mutants are isolated as catalytically inactive apoproteins. ApoArg49Ala forms a stable noncovalent complex with reduced 5-deazaFAD that exhibits properties similar to those observed for the corresponding complex with apoCys315Ala. The results show that elimination of a basic residue at position 49 blocks covalent flavinylation but does not prevent noncovalent flavin binding. The Arg49Lys mutant contains covalently bound FAD, but its flavin content is approximately 4-fold lower than wild-type MSOX. However, most of the apoprotein in the Arg49Lys preparation is reconstitutable with FAD in a reaction that exhibits kinetic parameters similar to those observed for flavinylation of wild-type apoMSOX. Although covalent flavinylation is scarcely affected, the specific activity of the Arg49Lys mutant is only 4% of that observed with wild-type MSOX. The results show that a basic residue at position 49 is essential for covalent flavinylation of MSOX and suggest that Arg49 also plays an important role in sarcosine oxidation.
Subject(s)
Flavins/metabolism , Sarcosine Oxidase/biosynthesis , Amino Acids/genetics , Amino Acids/metabolism , Flavins/chemistry , Holoenzymes/biosynthesis , Holoenzymes/genetics , Holoenzymes/isolation & purification , Kinetics , Models, Molecular , Molecular Structure , Mutation/genetics , Sarcosine Oxidase/genetics , Sarcosine Oxidase/isolation & purificationABSTRACT
A heat-stable sarcosine oxidase produced by Bacillus sp. BSD-8 (SOX) had been studied and its complete gene sequence, which contained 1,164 bp nucleotides and encoded a protein of 387 amino acids, was obtained by DNA Walking method. The sox gene was cloned and functionally overexpressed in E. coli and the recombinant SOX (rSOX) was purified to homogeneity, its properties was studied and compared with the wild type of SOX. The rSOX as well as SOX was stable at 60 degrees C and at pH 7.0 approximately 10.0, respectively. The optimal temperature for this enzyme was 60 degrees C and at pH 8.5, it showed its highest activity. The Km and Kcat of the enzyme was 3.1 mM and 20.3/s, respectively. The difference between the properties of the SOX and rSOX was that the SOX contained noncovalent FAD, whereas the rSOX contained covalent FAD. The study also showed that an increased number of alanine residues in the rSOX might have some contribution in the enzymatic thermostability.
