enzyme with respect to sarcosine were used in the simulations Simple s - PDF document

1 enzyme with respect to sarcosine were us
enzyme with respect to sarcosine were used in the simulations. Simple sequential enzyme reaction SoxCa for the endpoint assay of creatinine. When equal Sox activity was used in the reaction, the SoxCa-containing reagent quickly reached an endpoint, as evidenced by the high rate of hydrogen peroxide production. The estimated minimum that of the other enzymes. To obtain the same results, approximately 35 or 7 times the amount of SoxCa would be required compared with SoxB or Thus, SoxCa is the first diagnostic enzyme selected by in silico analysis with both high predicted stability and substrate afnity. The use of sp. TE1826. 2.Nishiya Y and Imanaka

2 , T: Cloning and sequencing sp. 3.Wagne
, T: Cloning and sequencing sp. 3.Wagner MA and Jorns MS: Monomeric sarcosine oxidase: 2. Kinetic studies with sarcosine, alternate substrates, and a substrate analogue. Biochemistry, 4.Nishiya Y, Yamamoto K, Kawamura Y, and Emi S: Development of creatinine-degrading enzymes for application to clinical assays [Jpn]. Nippon 5.Nishiya Y and Imanaka T: Alteration of substrate specicity and optimum pH of sarcosine oxidase by random and site-directed mutagenesis. Appl Environ 6.Nishiya Y, Zuihara S, and Imanaka T: Active site analysis and stabilization of sarcosine oxidase by the substitution of cysteine residues. Appl Environ 7.Nishiya Y and Kishimot

3 o T: Alteration of L-proline oxidase act
o T: Alteration of L-proline oxidase activity of sarcosine oxidase and a structural 8.Nishiya Y: Altered substrate affinity of monomeric nine-103 or histidine-348. Int J Anal Bio-Sci, 1: 9.Nagata K, Sasaki H, Hua M, Oka M, Kubota K, Kamo M, Ito K, Ichikawa T, Koyama Y, Tanokura 10.Nishiya Y and Abe Y: Comparison of the substrate specificity of L-pipecolate oxidase and bacterial tation of the enzymes. Int J Anal Bio-Sci, 3: 55-62, 11.Wagner MA, Trickey P, Chen Z, Mathews FS, and 1. Flavin reactivity and active site binding determinants. 12.Kommoju P-R, Chen Z, Bruckner RC, Mathews FS, flavoprotein oxidases using chloride as an oxygen Int J Anal Bio-Sc

4 i Vol. 4, No 3 (2016) described in this
i Vol. 4, No 3 (2016) described in this paper) was demonstrated experiSoxA, respectively (Table 2). Further structural investigations using site-directed mutagenesis are now in progress in order to determine whether the high substrate affinity and catalytic efficiency of To examine the influence of the substrate simulated endpoint assays using SoxCa, SoxB, and SoxA (Fig. 6). The kinetic parameters of each Fig. 6. Simulation of endpoint assays of creatinine with SoxCa, SoxA, and SoxB. (A) It was assumed that the assay (70 L), and 10 mg/dL of the creatinine sample (10 L) were mixtured and incubated at 37°C for 5 minutes. The time courses of

5 creatinine, creatine, sarcosine, and hy
creatinine, creatine, sarcosine, and hydrogen peroxide were, then, compared to those of SoxA and SoxB. (B) The time courses of endpoint assays simulated as concentrations of hydrogen peroxide were compared to each other. It was assumed that the assay EnzymeKmfor sacosinekcatkcat/Km(mM)(s-1)(%)SoxCaSoxB0.49173.63.72410018 Table 2Comparison of kinetic parameters Int J Anal Bio-Sci Vol. 4, No 3 (2016) value of SoxCa for sarcosine was 1/7 that of SoxB and SoxA, respectively (Table 2). The high substrate affinity of SoxCa (which was predicted by comparisons of the active site structures Fig. 5. triangles). (C) Effect of pH on the activity of SoxCa. The

6 reaction mixtures contained 50 mmol/L p
reaction mixtures contained 50 mmol/L potassium StepProtein(mg)Yield(%)Activity(U)110096083461.5109.1136.81009013068Crude extracC, 10minAmmonium sulfatC, 5minAfnity chromatograph Table 1Purication of SoxCa previously yielded an RMSD for C positions of 1.08 Å. The structure modeled in the present study Close-up views of the active site structures of SoxCa and SoxB are shown in Figure 3. As a remarkable difference, the side chain of the Q255 of the A Sox activity of 0.20 U/mL was detected in BL21 (DE3) (pET-SoxCaHT). SoxCa was then purified to homogeneity as described in the Materials and Methods section. The purification of SoxCa is summar

7 ized in Table 1 and Figure 4. The purifi
ized in Table 1 and Figure 4. The purified preparation gave a single protein band on SDS-PAGE and exhibited an absorption spectrum characteristic mated at 44.0 kDa by SDS-PAGE (Fig. 4). Gel filtration analyses showed that the enzyme was of SoxCa and its His-tagged derivative were calculated from the deduced amino acid sequences as 42.1 and 43.1 kDa, respectively. The molecular weight of His-tagged SoxCa agreed with the Various enzymatic properties of SoxCa were investigated. As expected, the thermal stability of When SoxA was kept at 60°C for 10 min, it was almost completely denatured, as previously . Therefore, the thermal stability of SoxA the Sox e

8 nzyme used significantly impacts the Gen
nzyme used significantly impacts the Generally, thermostable enzymes exhibit excellent long-term stability in refrigerated storage. SoxCa might be more suitable for practical use than conventional enzymes. The pH profile of SoxCa is Fig. 3. Close-up views of active sites. Tertiary structures of SoxCa (light cyan) and SoxB (dark orange) were compared. In both structures, oxygens, nitrogens, and sulfurs are in red, blue, and yellow, respectively. Amino acid residues and flavin adenine dinucleotide (FAD) were shown Fig. 4. BL21(DE3)(pET-SoxCaHT); B: purification by an immobilized metal affinity chromatography; C: the purified protein; M: molecular weig