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

Sarcosine oxidase (SOX) was an important diagnostic enzyme in the renal function examination. An integrated strategy coupling codon and fermentation optimization was firstly proposed for improving SOX production from recombinant E. coli in 3-L fermentor. The expression suppression (gene phase) and poor balance between SOX expression and cell growth (fermentation phase) in the traditional SOX production were respectively improved by the multiple strategies. Based on the codon bias, the expression suppression was weakened via codon optimization and SOX activity reached 1,521 U/L. The induction toxicity was reduced with the optimal induction condition and SOX production increased to 4,015 U/L. Based on the kinetic analysis of µ x and µ p , a better balance between cell growth and expression was achieved by the two-stage pH-stat control strategy. The SOX activity was further improved to 8,490 U/L and fermentation cycle was also significantly shortened from 44 to 32 h. The substrate inhibition was weakened with a constant feeding fed-batch. With the assistance of integrated strategy, the activity and productivity reached 12,466 U/L and 389.6 U/(L h), respectively, or 3.1-fold and 4.3-fold of the uncontrolled fermentation. The strategy would be also useful in the industrial application of other similar enzymes.

[Preparation and identification of recombinant sarcosine oxidase].

An important index determination for clinical diagnosis of renal function is to assay the creatinine concentration in serum. In the analytical process applied with coupled-enzyme, the quality control of sarcosine oxidase (SOX) as a key enzyme is the first problem to be solved. In order to establish an efficient and laboratory-scale production of SOX, the recombinant sarcosine oxidase (r-SOX) gene was a high-level expression in E. coli induced with lactose on a large-scale fermentation in 300 L fermenter. The results suggested that the biomass concentration reached OD600 of 22 and the expression of recombinant sarcosine oxidase in E. coli accounted for about 25% of total soluble protein in culture after fermentation. The cell-free extract obtained from high pressure homogenizer was processed by selective thermal denaturation and then purified with Ni-Sepharose FF chromatography. The sarcosine oxidase with 97% purity, 25 U/mg specific activity and 92.4% activity recovery was obtained. The molecular weight with single peptide chain of 53 kD and 55 kD of recombinant sarcosine oxidase was assessed by SDS-PAGE in presence or absence of 2-mercaptoehanol and Sephacryl S-200 chromatography. This sarcosine oxidase was found to be a conjugated protein, yellow enzyme, which combined with FAD as prosthetic group by covalent linkage. The contaminant of catalase was not detected in the sample pool of this enzyme. In addition, a further test to the thermal stability of sarcosine oxidase was done. According to the above results, the development and utilization of this enzyme has been set up on a reliable foundation.

Novel affinity purification of monomeric sarcosine oxidase expressed in Escherichia coli.

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.

Promote the expression and corrected folding of an extremely stable N-demethylase by promoter reconstruction, native environment simulation and surface design.

Thermomicrobium roseum sarcosine oxidase (TrSOX) was a N-demethylase with specific substrate chiral selectivity, outstanding thermostability and environmental resistance. To promote the expression of TrSOX in Bacillus subtilis W600, the HpaII promoter of pMA5 plasmid was replaced by constitutive or inducible promoters. Through orthogonal experiment, the expression process was optimized, B. subtilis W600 cells containing pMA5-Pxyl-trSOX plasmid were cultivated until OD600nm reached 2.0 and were then induced with 1.6% xylose at 37 °C for 2 h, and the native environment of T. roseum was simulated by heating at 80 °C, with the productivity of TrSOX increased from ~8.3 to ~66.7 µg/g wet cells; and the simulated high temperature was the key switch for the final folding. To reduce the surface hydrophobicity, a S320R mutant was built to form a hydrophilic lid around the entrance of the substrate pocket, and the yield of TrSOX (S320R) was ~163.0 µg/g wet cells, approximately 20 folds as that in the initial expression system. This mutant revealed the similar secondary structure, stability, resistance, chiral substrate selectivity and optimal reaction environment with wild type TrSOX; however, the N-demethylation activities for amino acid derivative substrates were dramatically increased, while those for hydrophobic non-amino acid compounds were repressed.

Enhancement of soluble expression of codon-optimized Thermomicrobium roseum sarcosine oxidase in Escherichia coli via chaperone co-expression.

The codon-optimized sarcosine oxidase from Thermomicrobium roseum (TrSOX) was successfully expressed in Escherichia coli and its soluble expression was significantly enhanced via the co-expression of chaperones. With the assistance of whole-genome analysis of T. roseum DSM 5159, the sox gene was predicated and its sequence was optimized based on the codon bias of E. coli. The TrSOX gene was successfully constructed in the pET28a plasmid. After induction with IPTG for 8h, SDS-PAGE analysis of crude enzyme solutions showed a significant 43 kDa protein band, indicating SOX was successfully expressed in E. coli. However, the dark band corresponding to the intracellular insoluble fraction indicated that most of TrSOX enzyme existed in the inactive form in "inclusion bodies" owing to the "hot spots" of TrSOX. Furthermore, the co-expression of five different combinations of chaperones indicated that the soluble expression of TrSOX was greatly improved by the co-expression of molecular chaperones GroES-GroEL and DnaK-DnaJ-GrpE-GroES-GroEL. Additionally, the analysis of intramolecular forces indicated that the hydrophobic amino acids, hydrogen bonds, and ionic bonds were favorable for enhancing the interaction and stability of TrSOX secondary structure. This study provides a novel strategy for enhancing the soluble expression of TrSOX in E. coli.

Efficient improvement on stability of sarcosine oxidase via poly-lysine modification on enzyme surface.

A novel modification method was proposed for improving the stability of sarcosine oxidase. In this process, sarcosine oxidase surface was efficiently linked with poly-lysine (poly-Lys) covalently after activation with N-ethyl-N'-3-dimethylaminopropyl carbodiimide (EDC); the optimal conditions for this reaction were also investigated. The molar ratios of enzyme-COOH to EDC and enzyme-COOH to poly-Lys-NH2 were 1:2 and 1:50, respectively, while the optimal reaction pH was 7.0. The covalently binding of poly-Lys onto enzyme surface was confirmed by mass spectrum (MS) and Fourier transform infrared spectroscopy (FTIR). The catalytic kinetic parameters (Km and Vmax) of modified enzyme were determined as 47.94mM and 0.157µmol/min, respectively. Moreover, compared to the native enzyme, the pH, thermal and storage stabilities of modified sarcosine oxidase were significantly improved. More than 90% of initial activity of modified enzyme was maintained at a broad pH range from 5.0 to 10.0. Most activity of modified enzyme could be detected after being incubated at 60°C for 10min. The storage stability was enhanced ∼12-fold after being stored at 37°C for 7 days. The novel modification was highly efficient for improving the stability of sarcosine oxidase and might be a good reference for other similar enzymes.

Covalent flavinylation of monomeric sarcosine oxidase: identification of a residue essential for holoenzyme biosynthesis.

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.