Expression and characterization of a thermostable sarcosine oxidase (SOX) from Bacillus sp. in Escherichia coli

Abstract

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.

Development of sarcosine quantification in urine based on enzyme-coupled colorimetric method for prostate cancer diagnosis

“…The optimal pH of the sequential reaction was 7.5. This result is in good agreement with previous studies that demonstrated the optimal pH of HRP and SOX to be 7 and 8.5, respectively (Chance and Maehly, 1955[ 11 ]; Guo et al, 2006[ 15 ]). Precision study exhibited the high reproducibility and repeatability of the assay suggesting that our method is potentially suitable for sarcosine determination in urine under given concentration range.…”

Purification and characterization of a thermostable uricase from Microbacterium sp. strain ZZJ4-1

“…At present, most enzymes applied in the clinical test are used in solution, and most proteins, including this enzyme, are relatively unstable when dissolved in aqueous solution. Therefore, for the enzymatic examination application, research was undertaken to search for a thermostable enzyme (Guo et al 2006;Huang et al 1998;Zhou et al 2005).…”

Novel integration strategy coupling codon and fermentation optimization for efficiently enhancing sarcosine oxidase (SOX) production in recombinant Escherichia coli

“…It was also reported that translational errors could be reduced via optimizing bases in the host strain (Vincentelli and Romier 2013). After gene optimization, the detectable SOX activity was improved by 33.5 % comparing to original gene expression in E. coli (Guo et al 2006). In the requisite induction process of fermentation phase, some induction toxicity for the cell growth was caused by improper induction condition.…”

“…1b. The detectable SOX activity reached 1,521 U/L, which was improved by 33.5 % comparing to original gene expression in E. coli (Guo et al 2006). …”

“…As one of the important diagnostic enzyme, sarcosine oxidase (SOX) can effectively catalyze the oxidation of methyl group in sarcosine and generate equimolar of formaldehyde, hydrogen peroxide (H 2 O 2 ) and glycine (Guo et al 2006). With this property, it has been widely used for assaying the creatinine level of human serum in the renal function examination.…”