Sarcosine Oxidase

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Marilyn Schuman Jorns - One of the best experts on this subject based on the ideXlab platform.

  • Selective enhancement of ligand and flavin Raman modes in charge‐transfer complexes of Sarcosine Oxidase
    Journal of Raman Spectroscopy, 2020
    Co-Authors: Yuangang Zheng, Mary Ann Wagner, Marilyn Schuman Jorns, Paul R Carey
    Abstract:

    Monomeric Sarcosine Oxidase, from a bacterial source, is a 43.8 kDa flavoenzyme that catalyzes the oxidation of Sarcosine (N-methylglycine). The enzyme forms charge-transfer (CT) complexes with substrate analogs such as (methylthio)acetic acid (MTA), pyrrole-2-carboxylic acid (PCA) and (methylseleno)acetic acid (MSA). The structure of Sarcosine Oxidase complexed to either of the former two ligands has been solved recently and shows that the ligands are binding close to the re face of the isoalloxazine ring. More structural detail can be adduced from Raman spectroscopic studies of these complexes. These involve collecting Raman difference data using 647.1 nm excitation, a wavelength that is in the resonance or pre-resonance regime with regard to the CT bands. Selective intensity enhancements are observed and the difference spectra reveal a rich assortment of isoalloxazine modes and a few features due to the bound ligands. Within the CT complexes, intensity enhancement for the isoalloxazine ring vibrations is typically 1–2 orders of magnitude and is greatest for vibrations associated with the N5—C4a—C10a—N1 region of the ring. This identifies the region of the isoalloxazine ring that receives the charge transfer from the ligand and confirms, at the same time, that this region is a prime candidate for nucleophilic attack or electron transfer in any reaction scheme. Intensity enhancements for the ligand modes are much higher, typically a few thousand-fold. Isotopic substitutions combined with quantum mechanical and vibrational calculations were used to identify the ligand modes showing high intensity enhancement. For MTA and MSA these are the methyl symmetric bending and methylene wag. For PCA the pyrrole ring CH deformations show the highest intensity enhancement. Copyright © 2001 John Wiley & Sons, Ltd.

  • structural characterization of mutations at the oxygen activation site in monomeric Sarcosine Oxidase
    Biochemistry, 2010
    Co-Authors: Marilyn Schuman Jorns, Zhiwei Chen, Scott F Mathews
    Abstract:

    Oxygen reduction and Sarcosine oxidation in monomeric Sarcosine Oxidase (MSOX) occur at separate sites above the si- and re-faces, respectively, of the flavin ring. Mutagenesis studies implicate Lys265 as the oxygen activation site. Substitution of Lys265 with a neutral (Met, Gln, or Ala) or basic (Arg) residue results in an ∼104- or 250-fold decrease, respectively, in the reaction rate. The overall structure of MSOX and residue conformation in the Sarcosine binding cavity are unaffected by replacement of Lys265 with Met or Arg. The side chain of Met265 exhibits the same configuration in each molecule of Lys265Met crystals and is nearly congruent with Lys265 in wild-type MSOX. The side chain of Arg265 is, however, dramatically shifted (∼4−5 A) compared with Lys265, points in the opposite direction, and exhibits significant conformational variability between molecules of the same crystal. The major species in solutions of Lys265Arg is likely to contain a “flipped-out” Arg265 and exhibit negligible oxygen a...

  • identification of the oxygen activation site in monomeric Sarcosine Oxidase role of lys265 in catalysis
    Biochemistry, 2008
    Co-Authors: Guohua Zhao, Robert C Bruckner, Marilyn Schuman Jorns
    Abstract:

    Monomeric Sarcosine Oxidase (MSOX) catalyzes the oxidation of N-methylglycine and contains covalently bound FAD that is hydrogen bonded at position N(5) to Lys265 via a bridging water. Lys265 is absent in the homologous but oxygen-unreactive FAD site in heterotetrameric Sarcosine Oxidase. Isolated preparations of Lys265 mutants contain little or no flavin but can be covalently reconstituted with FAD. Mutation of Lys265 to a neutral residue (Ala, Gln, Met) causes a 6000- to 9000-fold decrease in apparent turnover rate whereas a 170-fold decrease is found with Lys265Arg. Substitution of Lys265 with Met or Arg causes only a modest decrease in the rate of Sarcosine oxidation (9.0- or 3.8-fold, respectively), as judged by reductive half-reaction studies which show that the reactions proceed via an initial enzyme·Sarcosine charge transfer complex and a novel spectral intermediate not detected with wild-type MSOX. Oxidation of reduced wild-type MSOX (k = 2.83 × 105 M−1 s−1) is more than 1000-fold faster than obs...

  • covalent flavinylation of monomeric Sarcosine Oxidase identification of a residue essential for holoenzyme biosynthesis
    Biochemistry, 2008
    Co-Authors: Alshaimaa Hassanabdallah, Guohua Zhao, Marilyn Schuman Jorns
    Abstract:

    FAD in monomeric Sarcosine Oxidase (MSOX) is covalently linked to the protein by a thioether linkage between its 8α-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 f...

  • identification of a stable flavin thiolate adduct in heterotetrameric Sarcosine Oxidase
    Journal of Molecular Biology, 2006
    Co-Authors: Robert M G Hynson, Scott F Mathews, Marilyn Schuman Jorns
    Abstract:

    Abstract Heterotetrameric Sarcosine Oxidase (TSOX) is a complex bifunctional flavoenzyme that contains two flavins. Most of the FMN in recombinant TSOX is present as a covalent adduct with an endogenous ligand. Enzyme denaturation disrupts the adduct, accompanied by release of a stoichiometric amount of sulfide. Enzyme containing ≥ 90% unmodified FMN is prepared by displacement of the endogenous ligand with sulfite, a less tightly bound competing ligand. Reaction of adduct-depleted TSOX with sodium sulfide produces a stable complex that resembles the endogenous TSOX adduct and known 4a-S-cysteinyl flavin adducts. The results provide definitive evidence for sulfide as the endogenous TSOX ligand and strongly suggest that the modified FMN is a 4a-sulfide adduct. A comparable reaction with sodium sulfide is not detected with other flavoprotein Oxidases. A model of the postulated TSOX adduct suggests that it is stabilized by nearby residues that may be important in the electron transferase/Oxidase function of the coenzyme.

Yoshiaki Nishiya - One of the best experts on this subject based on the ideXlab platform.

  • reaction mechanism of n cyclopropylglycine oxidation by monomeric Sarcosine Oxidase
    Physical Chemistry Chemical Physics, 2020
    Co-Authors: Mauro Boero, Mitsuo Shoji, Yasuteru Shigeta, Yoshiaki Nishiya
    Abstract:

    Monomeric Sarcosine Oxidase (MSOX) is a fundamental – yet one of the simplest – member of a family of flavoenzymes able to catalyze the oxidation of Sarcosine (N-methylglycine) and other secondary amines. MSOX is one of the best characterized members of the amine oxidoreductases (AOs), however, its reaction mechanism is still controversial. A single electron transfer (SET) process was suggested on the basis of studies with N-cyclopropylglycine (CPG), although a hydride transfer mechanism would be more consistent in general for AOs. To shed some light on the detailed reaction mechanisms of CPG in MSOX, we performed hybrid quantum mechanical/molecular mechanical (QM/MM) simulations. We found that the polar mechanism is energetically the most favorable. The free energy profile indicates that the first rate-limiting step is the CPG binding to the flavin ring which simultaneously proceeds with the ring-opening of the CPG cyclopropyl group. This reaction step of the CPG adduct formation corresponds to the nucleophilic attack of the cyclopropyl group (C3 atom) to the flavin ring (C4a atom), whereas the expected radical species formation in the SET mechanism was not observed. The following inactivated species, which accumulates during the CPG oxidation in MSOX, can be ascribed to an imine state, and not an enamine state, on the basis of the computed UV/Vis spectra. The conformation of CPG was found to be crucial for reactions following the CPG adduct formation.

  • the reaction mechanism of Sarcosine Oxidase elucidated using fmo and qm mm methods
    Physical Chemistry Chemical Physics, 2017
    Co-Authors: M Shoji, Yoshiaki Nishiya, Hiroshi Aiba, Takahide Kishimoto, Kazuo Kitaura
    Abstract:

    Monomeric Sarcosine Oxidase (MSOX) is a flavoprotein that oxidizes Sarcosine to the corresponding imine product and is widely used in clinical diagnostics to test renal function. In the past decade, several experimental studies have been performed to elucidate the underlying mechanism of this oxidation reaction. However, the details of the molecular mechanism remain unknown. In this study, we theoretically examined three possible reaction mechanisms, namely, the single-electron transfer, hydride-transfer, and polar mechanisms, using the fragment molecular orbital (FMO) and mixed quantum mechanics/molecular mechanics (QM/MM) methods. We found that, of the three possible reaction pathways, hydride-transfer is the most energetically favorable mechanism. Significantly, hydrogen is not transferred in the hydride state (H−) but in a hydrogen atom state (H˙). Furthermore, a single electron is simultaneously transferred from Sarcosine to flavin through their overlapping orbitals. Therefore, based on a detailed theoretical analysis of the calculated reaction pathway, the reaction mechanism of MSOX can be labeled the “hydrogen-atom-coupled electron-transfer” (HACET) mechanism instead of being categorized as the classical hydride-transfer mechanism. QM/MM and FMO calculations revealed that Sarcosine is moved close to the flavin ring because of a small charge transfer (about 0.2 electrons in state 1 (MSOX–Sarcosine complex)) and that the positively charged residues (Arg49, Arg52, and Lys348) near the active site play a prominent role in stabilizing the Sarcosine–flavin complex. These results indicate that strong Coulombic interactions primarily control amine oxidation in the case of MSOX. The new reaction mechanism, HACET, will be important for all the flavoprotein-catalyzed oxidation reactions.

  • alteration of l proline Oxidase activity of Sarcosine Oxidase and a structural interpretation
    生物試料分析 = Journal of analytical bio-science, 2010
    Co-Authors: Yoshiaki Nishiya, Takahide Kishimoto
    Abstract:

    Summary Sarcosine Oxidase, an enzyme used for creatinine determination, acts slightly on L-proline, which may interfere with the assay. The Sarcosine Oxidase gene was randomly mutated for the purpose of altering L-proline Oxidase activity. Several mutants showing increased or decreased L-proline Oxidase activity were isolated, and their amino acid substitutions were identified. Two of these mutants, V94G and K322R, were suitable for practical use, because they exhibited lower L-proline Oxidase activities than the wild type without decreasing the Sarcosine Oxidase activities. Furthermore, the L-proline Oxidase activity of a multiple mutant constructed by site-directed mutagenesis, V94G+K322R+Y224T+E250Q, was considerably changed from that of the wild type. The catalytic efficiency of the mutant for L-proline was approximately 1/8th that of the wild type, whereas that for Sarcosine was at the same level. These mutational effects were discussed in terms of a three-dimensional structure model.

  • a mutant Sarcosine Oxidase in which activity depends on flavin adenine dinucleotide
    Protein Expression and Purification, 2000
    Co-Authors: Yoshiaki Nishiya
    Abstract:

    Abstract The covalent flavin attachment site in the Arthrobacter Sarcosine Oxidase (cysteine at position 318) was replaced with serine, and the mutational effect of C318S was analyzed. Wild type and C318S with a C-terminal 6-histidine tag were constructed and homogeneously purified by the single step. The covalently binding to flavin was not essential to the enzyme activity because the C318S mutant exhibited extremely weak activity. Moreover, the activity of the mutant was recovered in the presence of flavin adenine dinucleotide (FAD), and significantly increased as the concentration of FAD increased. This dependence of the mutant on FAD indicates that the noncovalent binding of free FAD to the mutant enzyme is reversible.

  • enzymatic assay for chloride ion with chloride dependent Sarcosine Oxidase created by site directed mutagenesis
    Analytical Biochemistry, 1997
    Co-Authors: Yoshiaki Nishiya, Yoshihisa Kawamura, Tadayuki Imanaka
    Abstract:

    Abstract Serum chloride ion is routinely assayed in clinical laboratories. We have developed a new enzymatic assay for determining the chloride ion concentration. The method involves the use of a mutant Sarcosine Oxidase, which was created as desired by site-directed mutagenesis and showed chloride-dependent activity. The enzyme which is reactivated by the chloride ion forms hydrogen peroxide from Sarcosine. The production of hydrogen peroxide is measured by the 4-aminoantipyrine perOxidase system. The increase of the reaction rate was proportional to the chloride ion concentration. A lag time of the time course was not observed, and the reaction rate for a blank was not detected. Therefore, a rate assay could be adopted. A standard curve of the assay reagent was linear up to 180 m m chloride ion of the sample. The specificity for the bromide ion was 43% of that of the chloride ion, although it was 0% for other ion species. When serum samples were used, within-day coefficient variations (CVs) and day-to-day CVs were below 1.5%. A good correlation with the comparison assay was observed by using 160 samples of normal and abnormal patient sera. This method can easily and reliably be used for the accurate determination of chloride ion concentration in serum or other samples.

Tadayuki Imanaka - One of the best experts on this subject based on the ideXlab platform.

  • enzymatic assay for chloride ion with chloride dependent Sarcosine Oxidase created by site directed mutagenesis
    Analytical Biochemistry, 1997
    Co-Authors: Yoshiaki Nishiya, Yoshihisa Kawamura, Tadayuki Imanaka
    Abstract:

    Abstract Serum chloride ion is routinely assayed in clinical laboratories. We have developed a new enzymatic assay for determining the chloride ion concentration. The method involves the use of a mutant Sarcosine Oxidase, which was created as desired by site-directed mutagenesis and showed chloride-dependent activity. The enzyme which is reactivated by the chloride ion forms hydrogen peroxide from Sarcosine. The production of hydrogen peroxide is measured by the 4-aminoantipyrine perOxidase system. The increase of the reaction rate was proportional to the chloride ion concentration. A lag time of the time course was not observed, and the reaction rate for a blank was not detected. Therefore, a rate assay could be adopted. A standard curve of the assay reagent was linear up to 180 m m chloride ion of the sample. The specificity for the bromide ion was 43% of that of the chloride ion, although it was 0% for other ion species. When serum samples were used, within-day coefficient variations (CVs) and day-to-day CVs were below 1.5%. A good correlation with the comparison assay was observed by using 160 samples of normal and abnormal patient sera. This method can easily and reliably be used for the accurate determination of chloride ion concentration in serum or other samples.

  • highly conservative sequence in the carboxyl terminus of Sarcosine Oxidase is important for substrate binding
    Journal of Fermentation and Bioengineering, 1997
    Co-Authors: Yoshiaki Nishiya, Tadayuki Imanaka
    Abstract:

    Abstract The function of the highly conservative sequence —G(344)-F-S-G-H-G-F-K-F(352)— in the carboxyl terminus of Sarcosine Oxidase was investigated using site-directed mutagenesis. When H-348 was substituted with uncharged amino acids, the K m values of Sarcosine Oxidase markedly increased, although the k cat values remained the same as that of the wild-type enzyme. The kinetic parameters obtained with other mutations also suggested that the conservative sequence acts as the substrate-binding site. When K-351 was replaced by Ala, the mutant K351A could not bind the coenzyme FAD (flavin adenine dinucleotide). This result suggested that K-351 interacts with the FAD-binding site of the amino-terminal region. The enzymic activities of the mutants H348Q and H348A were lost at neutral and acidic pH as a result of the disappearance of the positive charge on H-348.

  • Analysis of a negative regulator, soxR, for the Arthrobacter Sarcosine Oxidase gene
    Journal of Fermentation and Bioengineering, 1996
    Co-Authors: Yoshiaki Nishiya, Tadayuki Imanaka
    Abstract:

    A negative regulatory gene, soxR, was found upstream of the Sarcosine Oxidase gene, soxA, from Arthrobacter sp. TE1826. The gene was sequenced and found to encode 289 amino acid residues (M.W. 32,746). A protein homology search revealed that the soxR gene product belongs to the LysR family of regulatory proteins. When soxA was cloned in Escherichia coli, the gene was constitutively expressed. In contrast, Sarcosine was required as an inducer for the expression of soxA in the parental Arthrobacter strain. When soxA and soxR were simultaneously cloned in E. coli, Sarcosine Oxidase production was drastically decreased. This strongly suggests that SoxR acts as a negative regulator for soxA.

  • active site analysis and stabilization of Sarcosine Oxidase by the substitution of cysteine residues
    Applied and Environmental Microbiology, 1995
    Co-Authors: Yoshiaki Nishiya, S Zuihara, Tadayuki Imanaka
    Abstract:

    Two cysteine residues (C-265 and C-318) in the putative hydrophilic regions of Sarcosine Oxidase were substituted by using site-directed mutagenesis. Since the mutant with the C-to-S mutation at position 318 (C318S) lost the enzyme activity, C-318 (conserved among Sarcosine Oxidases) is most likely a part of the active site. C265S, C265A, C265D, and C265R showed nearly the same enzymatic properties as those of the wild type. However, they were much more stable than the wild type in the presence of inhibitors that modified the thiol group. Moreover, they were extremely stable throughout the cultivation of the recombinant strains or even in cell extracts.

  • alteration of substrate specificity and optimum ph of Sarcosine Oxidase by random and site directed mutagenesis
    Applied and Environmental Microbiology, 1994
    Co-Authors: Yoshiaki Nishiya, Tadayuki Imanaka
    Abstract:

    Abstract The Sarcosine Oxidase gene was randomly mutated, and F103V, which showed altered specificity, was screened with the plate assay. Five different mutants at position 103 were also constructed. Each mutant exhibited a rather striking change in substrate specificity, except for F103W. The optimum pH was also shifted, depending on the substrate.

Chandra Shekhar Pundir - One of the best experts on this subject based on the ideXlab platform.

  • fabrication of an amperometric Sarcosine biosensor based on Sarcosine Oxidase chitosan cunps c mwcnt au electrode for detection of prostate cancer
    Enzyme and Microbial Technology, 2018
    Co-Authors: Vinay Narwal, Parveen Kumar, Pooja Joon, Chandra Shekhar Pundir
    Abstract:

    Abstract An amperometric Sarcosine biosensor was fabricated based on covalent immobilization of Sarcosine Oxidase (SarOx) onto the nanocomposite of carboxylated multi-walled carbon nanotubes (cMWCNT)/chitosan (CHIT) and copper nanoparticles (CuNPs), electrodeposited on gold (Au) electrode. The SarOx/CHIT/CuNPs/c-MWCNT/Au electrode was characterized by scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The enzyme electrode exhibited optimum current within 2 s at a potential of 0.2 V against Ag/AgCl, pH 7.0 and 35 °C. A linear relationship was obtained between Sarcosine concentration in the range, 0.1–100 μM and current (mA) under optimum conditions. The biosensor exhibited a high sensitivity of 277.5 μA/μM/cm2, a low detection limit of 0.1 pM and excellent storage stability (180 days). The analytical recoveries of added Sarcosine in sera at 0.5 μM and at 1.0 μM concentration were 95.5% and 97.30 respectively. The precision i.e. within and between-batch coefficients of variation (CVs) were 1.08% and 1.70% respectively. There was a good correlation (R2 = 0.99) between the level of Sarcosine in sera as measured by the standard immuno kit method and the present biosensor. The biosensor measured Sarcosine level in sera of prostate cancer patients, which was significantly higher than those of apparently healthy persons (p value

  • Fabrication of an amperometric Sarcosine biosensor based on Sarcosine Oxidase/chitosan/CuNPs/c-MWCNT/Au electrode for detection of prostate cancer
    Enzyme and Microbial Technology, 2018
    Co-Authors: Vinay Narwal, Parveen Kumar, Pooja Joon, Chandra Shekhar Pundir
    Abstract:

    Abstract An amperometric Sarcosine biosensor was fabricated based on covalent immobilization of Sarcosine Oxidase (SarOx) onto the nanocomposite of carboxylated multi-walled carbon nanotubes (cMWCNT)/chitosan (CHIT) and copper nanoparticles (CuNPs), electrodeposited on gold (Au) electrode. The SarOx/CHIT/CuNPs/c-MWCNT/Au electrode was characterized by scanning electron microscopy (SEM), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The enzyme electrode exhibited optimum current within 2 s at a potential of 0.2 V against Ag/AgCl, pH 7.0 and 35 °C. A linear relationship was obtained between Sarcosine concentration in the range, 0.1–100 μM and current (mA) under optimum conditions. The biosensor exhibited a high sensitivity of 277.5 μA/μM/cm2, a low detection limit of 0.1 pM and excellent storage stability (180 days). The analytical recoveries of added Sarcosine in sera at 0.5 μM and at 1.0 μM concentration were 95.5% and 97.30 respectively. The precision i.e. within and between-batch coefficients of variation (CVs) were 1.08% and 1.70% respectively. There was a good correlation (R2 = 0.99) between the level of Sarcosine in sera as measured by the standard immuno kit method and the present biosensor. The biosensor measured Sarcosine level in sera of prostate cancer patients, which was significantly higher than those of apparently healthy persons (p value

  • an improved amperometric creatinine biosensor based on nanoparticles of creatininase creatinase and Sarcosine Oxidase
    Analytical Biochemistry, 2017
    Co-Authors: Parveen Kumar, Ranjana Jaiwal, Chandra Shekhar Pundir
    Abstract:

    Abstract An improved amperometric biosensor for detection of creatinine was developed based on immobilization of nanoparticles (NPs) of creatininase (CA), creatinase (CI), and Sarcosine Oxidase (SOx) onto glassy carbon (GC) electrode. Transmission electron microscopy (TEM) and fourier transform infrared spectroscopy (FTIR) were employed for characterization of enzyme nanoparticles (ENPs). The GC electrode was characterized by scanning electron microscopy (SEM), cyclic voltammetry (CV) and electrochemical impedance spectra (EIS) at different stages of its amendment. The biosensor showed optimum response within 2s at pH 6.0 in 0.1 M sodium phosphate buffer and 25 °C, when operated at 1.0 V against Ag/AgCl. Biosensor exhibited wider linear range from 0.01 μM to 12 μM with a limit of detection (LOD) of 0.01 μM. The analytical recoveries of added creatinine in sera were 97.97 ± 0.1% for 0.1 mM and 98.76 ± 0.2% for 0.15 mM, within and between batch coefficients of variation (CV) were 2.06% and 3.09% respectively. A good correlation (R2 = 0.99) was observed between sera creatinine values obtained by standard enzymic colorimetric method and the present biosensor. This biosensor measured creatinine level in sera of apparently healthy subjects and persons suffering from renal and muscular dysfunction. The ENPs electrode lost 10% of its initial activity within 240 days of its regular uses, when stored at 4 °C.

  • immobilization of creatininase creatinase and Sarcosine Oxidase on iron oxide nanoparticles chitosan g polyaniline modified pt electrode for detection of creatinine
    Enzyme and Microbial Technology, 2012
    Co-Authors: Sandeep Yadav, Rooma Devi, Pratibha Bhar, Sapna Singhla, Chandra Shekhar Pundir
    Abstract:

    Abstract Commercial enzymes, creatininase (CA) from Pseudomonas sp, creatinase (CI) from Pseudomonas sp, Sarcosine Oxidase (SO) from Bacillus sp were co-immobilized onto iron oxide nanoparticles/chitosan-graft-polyaniline (Fe 3 O 4 -NPs/CHIT-g-PANI) composite film electrodeposited on surface of Pt electrode through glutaraldehyde coupling. Transmission electron microscopy (TEM) was used for characterization of Fe 3 O 4 -NPs. A creatinine biosensor was fabricated using Enzymes/Fe 3 O 4 -NPs/CHIT-g-PANI/Pt electrode as working electrode, Ag/AgCl as reference electrode and Pt wire as auxiliary electrode. The enzyme electrode was characterized by cyclic voltammetry (CV), scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopic and electrochemical impedance spectroscopy (EIS). The biosensor exhibited an optimum response within 2 s at pH 7.5 and 30 °C, when polarized at 0.4 V vs Ag/AgCl. The electrocatalytic response showed a linear dependence on creatinine concentration ranging from 1 to 800 μM. The sensitivity of the biosensor was 3.9 μA μM −1  cm −2 , with a detection limit of 1 μM (S/N = 3). Apparent Michaelis–Menton ( K m ) value for creatinine was 0.17 mM. The biosensor showed only 10% loss in its initial response after 120 uses over 200 days, when stored at 4 °C. The biosensor measured creatinine in the serum of apparently healthy persons which correlated well with a standard colorimetric method ( r  = 0.99).

  • Immobilization of Arthrobacter Sarcosine Oxidase onto alkylamine and arylamine glass and its application in serum Sarcosine determination
    2011
    Co-Authors: Chandra Shekhar Pundir, Nidhi Chauhan, Garima Kumari, Vandana
    Abstract:

    Commercial Sarcosine Oxidase (Sarcosine: O2 oxidoreductase, EC 1.5.3.1) from Arthrobacter sp. has been immobilized onto alkyl and arylamine glass-beads through glutaraldehyde cross linking and diazotization with a conjugation yield of 0.86 mg/g & 0.91 mg/g support and 75.8% & 84.6% retention of initial activity of free enzyme, respectively. The enzyme showed decrease in optimum pH and Km for Sarcosine but increase in temperature for maximum activity and time for linearity after immobilization. The glass beads were employed for colorimetric determination of Sarcosine in serum. The minimum detection limit of the method was 0.05 mM. The glass beads were reused 100-times without considerable loss of activity, when stored in distilled water at 4°C for a period of two months.

Wu Wang - One of the best experts on this subject based on the ideXlab platform.

  • structural and catalytic alteration of Sarcosine Oxidase through reconstruction with coenzyme like ligands
    Journal of Molecular Catalysis B-enzymatic, 2016
    Co-Authors: Mengling Zheng, Yanjun Tong, Ling Zhang, Qing Wang, Liushen Lu, Wu Wang
    Abstract:

    Abstract A Sarcosine Oxidase (SOX) gene from Bacillus sp. (AY626822.2) was expressed in Escherichia coli BL21 (DE3) in the form of inclusion bodies. A 3D model of SOX was then built and refined, and molecular docking was used to investigate the interactions between SOX and natural or coenzyme-like ligands, including flavin adenine dinucleotide (FAD); flavin mononucleotide (FMN); riboflavin; isoalloxazine; 7-methyl-8-chloro-10-(1′- d -ribityl) isoalloxazine (7-M-8-C); 7-bromo-8-methyl-10-(1′- d -ribityl) isoalloxazine (7-B-8-M); 7-methyl-8-bromo-10-(1′- d -ribityl) isoalloxazine (7-M-8-B); 7-chloro-8-ethyl-10-(1′- d -ribityl) isoalloxazine (7-C-8-E); 7,8-diethyl-10-(1′- d -ribityl) isoalloxazine (7,8-D); and 3-methyl-7,8-dimethyl-10-(1′- d -ribityl) isoalloxazine (3-M-7,8-D). Unfolded SOX was extracted from inclusion bodies, and reconstructed with these ligands via a refolding process. The reconstructed enzymes were then subjected to structural and catalytic analysis. After structural simulation, refinement, and molecular docking, all ligands were able to recognize the coenzyme site of SOX. In addition, when the position 7- or 8-site of the compounds was modified, new pi-cation/sigma interactions were formed in the SOX-ligand complex. Fluorescent detection revealed that all the ligands could be successfully reconstructed with unfolded SOX. Circular dichroism (CD) spectra and nano differential scanning calorimetry (DSC) analysis indicated that the loss of phosphoric acid and adeninein natural coenzymes could significantly reduce the α-helix content, transition temperature (Tm), and calorimetric enthalpy (ΔH). In addition, although reconstruction with the position 7- or 8-site modified compounds led to variations in secondary structure, no significant shifts in Tm and ΔH were observed. Furthermore, in the evaluation of catalytic kinetic parameters, when SOX was reconstructed with ligands containing halogen atoms at the 7- or 8-sites, much higher relative specificities in the presence of organic solvents were noted.

  • enhancement of soluble expression of codon optimized thermomicrobium roseum Sarcosine Oxidase in escherichia coli via chaperone co expression
    Journal of Biotechnology, 2016
    Co-Authors: Yanjun Tong, Hailin Yang, Ling Zhang, Wu Wang, Shoushuai Feng, Wei Chen
    Abstract:

    Abstract 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 8 h, 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.

  • novel integration strategy coupling codon and fermentation optimization for efficiently enhancing Sarcosine Oxidase sox production in recombinant escherichia coli
    World Journal of Microbiology & Biotechnology, 2015
    Co-Authors: Yanjun Tong, Hailin Yang, Ling Zhang, Wu Wang
    Abstract:

    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.

  • efficient improvement on stability of Sarcosine Oxidase via poly lysine modification on enzyme surface
    International Journal of Biological Macromolecules, 2014
    Co-Authors: Yanjun Tong, Hailin Yang, Ling Zhang, Wu Wang
    Abstract:

    Abstract 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-NH 2 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 ( K m and V max ) of modified enzyme were determined as 47.94 mM and 0.157 μmol/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 10 min. 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.

  • novel affinity purification of monomeric Sarcosine Oxidase expressed in escherichia coli
    IEEE Journal of Solid-state Circuits, 2013
    Co-Authors: Yanjun Tong, Hailin Yang, Ling Zhang, Hui Xu, Wu Wang
    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.

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