RESUMO
In order to characterize a thermostable urate oxidase (Uox) from Microbacterium sp. strain ZZJ4-1, we cloned its gene (uox). The open reading frame of uox contained 894 base pairs and encoded a protein with 297 amino acids. Alignment of gene sequences indicated there was no obvious identity with the most reported uox and that 72% identity was found with uox from Arthrobacter globiformis. We inserted the gene into the plasmid pET-15b to construct an expression vector pET-15b-uox and got it induced expression in Escherichia coli BL21 (DE3). After the purification of the recombinant Uox by the HisBind column, we studied some properties of it. It was composed of subunits with a molecular mass of about 35 kDa. The optimal temperature and pH was 30 degrees C and pH 7.5. It was stable below 65 degrees C and from pH 8.5 to 11.0. The Km value was 0.22 mmol/L with the uric acid as the substrate. Ag+, Zn2+, CU2+ and SDS could totally inhibit its activity while Tween 20, Tween 80 and Triton X-100 had a slight promotion effect. The thermal stability of this enzyme was the most excellent among the reported recombinant Uox. Based on this property, it would be very useful in the application.
Assuntos
Actinomycetales , Genética , Sequência de Aminoácidos , Clonagem Molecular , Estabilidade Enzimática , Escherichia coli , Genética , Metabolismo , Vetores Genéticos , Genética , Dados de Sequência Molecular , Proteínas Recombinantes , Genética , Urato Oxidase , Genética , MetabolismoRESUMO
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.
Assuntos
Bacillus , Proteínas de Bactérias , Química , Metabolismo , Precipitação Química , Estabilidade Enzimática , Sarcosina Oxidase , Química , Metabolismo , Microbiologia do SoloRESUMO
A new method based on adherence of cellulolytic bacteria to insoluble cellulose for isolation and purification of thermophilic cellulolytic anaerobes was reported, in which Hungate anaerobic operating techniques were used to roll tubes with insoluble cellulose powder as substrate.