Studies On Sarcosine Oxidase From Bacillus Sp. BSD-8

Sarcosine oxidase (SOX, EC 1.5.3.1) is an essential enzyme for the determination of creatinine, its property, therefore, would have an important effect on the practical application of this measurement method. Although the researches about SOX have got some progress, most of the results were based on the studies of thermal labile SOX.In this study, a strain of bacteria capable of producing thermostable SOX was isolated and identified as Bacillus sp.. The conditions for producing SOX, the method for purification and properties of SOX were studied. With the application of chromosome walking method, the SOX gene (sox) of BSD-8 strain was cloned and sequenced. The results of this research were reported as following:1. The isolation and identification of SOX-producing bacteria: Five strains of bacteria, strain HZ-6, HZ-5, HZ-13, BSD-2, BSD-8, which were capable of producing SOX were isolated from soil. Among the five strains, the strain BSD-8 showed the highest capacity of SOX productivity and its sox showed the best thermal stability. Based on the morphological observation, physiological and biochemical characteristics and the result of sequences alignment of 16S rDNA, strain BSD-8 was identified as Bacillus sp..2. The conditions for the SOX production: Based on the results of studies on the fermentation condition, the best conditions were established as follow: The fermentation medium contained: creatine, 10g; yeast extract, 8g; K2HP04, 2g; KH2PO43H20, 0.5g; MgSO47H2O, 0.5g; NaCl, 3g; H2O, 1000mL and pH 6.5. When incubated with 40mL medium in a 100mL flask on a shaker of 120 rpm, the optimal fermentation temperature was 35 and between 18h-24h the SOX reach its highest activity.3. The purification of SOX: The specific activity of SOX was increased 25-fold by a series of purification steps including ammonium sulfate precipitation, DEAE-Cellulose ion-exchange chromatography, Toyopearl HW-65C hydrophobic chromatography and Sephadex G-75 gel filtration. The molecular mass was estimated to be 51 000Da bySDS-PAGE.4. The characteristics of SOX: The optimum pH for SOX was 8.0 and the optimal temperature was 60. Between pH 7.0-10.0 the SOX was stable at 60. The Km and the Kcat value of SOX for sarcosine were 3.1mmol/L and 4.5/s, respectively. The enzyme was markedly inhibited by Ag+, Hg2+, SDS, Fe2+, but not by Cu2+, Cr3+, Zn2+, EDTA or NaN3. Tween 80 inhibited the enzyme activity about 50% at the concentration of 0.1%. The enzyme activity was inhibited considerably by p-chloromeruribenzoate. The research showed that the SOX probably noncovalently.bound with a flavin5. Cloning and sequencing of SOX gene: The complete SOX gene from BSD-8 strain was cloned by chromosome walking method. The gene was composed of 1164 bp that encoded 387 amino acids, among which adenine and thymine had high ratio in the amino acids composition. The alignment result of amino acids sequences of SOX showed that the SOX gene sequence from BSD-8 strain had distinct diversity with the SOX gene sequences from other microbes.