A potent peroxidase from solid cell culture of Ocimum basilicum with high sensitivity for blood glucose determination

Abstract Objectives Extensive applications of peroxidase (POX) have raised the global market demand at a considerable rate during the forecast period of 2020 - 2025. Nonetheless, the large-scale POX preparation still relies on the extraction from agricultural products, while there is an accumulative driving force toward employing biotechnological processes with agricultural hassle free identity. In pursuit of this trend; Results, A novel heme peroxidase was purified to homogeneity (MW of 40 kD) from the callus culture of Ocimum basilicum L. in darkness on Murashige-Skoog medium supplemented by 2,4-dichlorophenoxyacetic acid (10-6 M) and kinetin (10-5 M). The highest activity of the purified peroxidase (ObPOX) was observed in Tris-base buffer at pH 7.5 and 80 °C. ObPOX showed high stability over pH(s) 5 to 7.5 and temperatures of 15 to 60 °C. ObPOX specific activity was 1245.142 AU mg-1 in the presence of phenol, 4 times higher than that of HRP. ObPOX showed moderate affinity for guaiacol (Km = 21.5 mM), but obtained an exceptionally high specificity constant (kcat/Km = 66743.7 s-1M-1) for GASA (4-[(4-Hydroxy-3-methoxyphenyl) azo]-benzenesulfonic acid), the introduced substrate for determination of blood sugar. Applying ObPOX instead of HRP in glucose measurements of the real samples improved the regression constant of the correlation diagram between the tests and the lab results from 0.958 to 0.981. Conclusion Physicochemical properties of ObPOX as well as the growth rate of basil callus (5.04 g L-1 per day) and the yield of ObPOX production (35 mg per 100 g dry biomass per subculture) designates O. basilicum cell culture for large-scale production of a robust peroxidase.

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A Potent Peroxidase from Solid Cell Culture of Ocimum Basilicum with High Sensitivity for Blood Glucose Determination

10.21203/rs.3.rs-227761/v1 ◽

2021 ◽

Author(s):

Parvin Mohammadnejad ◽

Saeed Soleimani Asl ◽

Zahra Rasoulian ◽

Saeed Aminzadeh ◽

Jaleh Ghashghaie ◽

...

Keyword(s):

Cell Culture ◽

Large Scale ◽

Specific Activity ◽

High Sensitivity ◽

High Specificity ◽

Global Market ◽

Ocimum Basilicum ◽

Scale Production ◽

Market Demand ◽

Glucose Determination

Abstract Extensive applications of peroxidase (POX) have raised the global market demand at a considerable rate during the forecast period of 2020 - 2025. Nonetheless, the large-scale POX preparation still relies on the extraction from agricultural products, while there is an accumulative driving force toward employing biotechnological processes with agricultural hassle free identity. In pursuit of this trend, a novel heme peroxidase was purified to homogeneity (MW of 40 kD) from the callus culture of Ocimum basilicum L. in darkness on Murashige-Skoog medium supplemented by 2,4-dichlorophenoxyacetic acid (10-6 M) and kinetin (10-5 M). The highest activity of the purified peroxidase (ObPOX) was observed in Tris-base buffer at pH 7.5 and 80 °C. ObPOX showed high stability over pH(s) 5 to 7.5 and temperatures of 15 to 60 °C. ObPOX specific activity was 1245.142 AU mg-1 in the presence of phenol, 4 times higher than that of HRP. ObPOX showed moderate affinity for guaiacol (Km = 21.5 mM), but obtained an exceptionally high specificity constant (kcat/Km = 66743.7 s-1M-1) for GASA (4-[(4-Hydroxy-3-methoxyphenyl) azo]-benzenesulfonic acid), the introduced substrate for determination of blood sugar. Applying ObPOX instead of HRP in glucose measurements of the real samples improved the regression constant of the correlation diagram between the tests and the lab results from 0.958 to 0.981. Physicochemical properties of ObPOX as well as the growth rate of basil callus (5.04 g L-1 per day) and the yield of ObPOX production (35 mg per 100 g dry biomass per subculture) designates O. basilicum cell culture for large-scale production of a robust peroxidase.

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Whole Blood Glucose Determination Using Glucose Oxidase Immoblized on Cotton Cheese Cloth

Biosensors '94 ◽

10.1016/b978-1-85617-242-4.50135-x ◽

1994 ◽

pp. 168

Author(s):

Achyut V. Kulkarni ◽

Sangita D. Kumar

Keyword(s):

Blood Glucose ◽

Glucose Oxidase ◽

Whole Blood ◽

Glucose Determination ◽

Blood Glucose Determination ◽

Cheese Cloth

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Progress of MIR Non-Invasive Blood Glucose Determination and Effect of Stratum Corneum

Acta Optica Sinica ◽

10.3788/aos201131.0900105 ◽

2011 ◽

Vol 31 (9) ◽

pp. 0900105

Author(s):

陈星旦 Chen Xingdan ◽

王动民 Wang Dongmin ◽

卢启鹏 Lu Qipeng ◽

丁海泉 Ding Haiquan

Keyword(s):

Blood Glucose ◽

Stratum Corneum ◽

Glucose Determination ◽

Non Invasive ◽

Blood Glucose Determination

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Effect of Addition of WZB117 as an Inhibitor of Glucose Transporter 1 for Venous Blood Glucose Determination

Laboratory Medicine ◽

10.1093/labmed/lmaa051 ◽

2020 ◽

Author(s):

Lei Zhang ◽

Yaqiong Ran ◽

Yan Zhu ◽

Qianna Zhen

Keyword(s):

Blood Glucose ◽

Glucose Level ◽

Glucose Transporter ◽

Venous Blood ◽

Sugar Transport ◽

Glucose Transporter 1 ◽

Glucose Determination ◽

Glucose Levels ◽

Significant Difference ◽

Blood Glucose Determination

Abstract Objective Sodium fluoride (NaF) has been applied to inhibit glycolysis in venous specimens for decades. However, it has had little effect on the rate of glycolysis in the first 1 to 2 hours, resulting in a decrease of glucose, so a more efficient method is needed. Recently, we discovered that WZB117, a specific Glut1 inhibitor, restricts glycolysis by inhibiting the passive sugar transport of human red blood cells and cancer cells. The purpose of this study was to evaluate the results of intravenous blood glucose determination after the addition of WZB117. Methods Venous specimens from 40 pairs of healthy volunteers were collected for several days and placed in tubes containing NaF plus EDTA-disodium (Na2) without WZB117 (the A group); citric acid, trisodium citrate, and EDTA-Na2 without WZB117 (B group); and NaF plus EDTA-Na2 with WZB117 (C group). The glucose concentration was measured after venipuncture and compared with test tubes treated for 1 hour, 2 hours, and 3 hours before centrifugation. Glucose level was determined by the hexokinase method. The paired t-test was used to examine differences in glucose values at baseline and at different time points. The number of misdiagnoses and the misdiagnosis rate were calculated at 2 diagnostic stages: high risk of diabetes (glucose level of 6.1 mmol/L) and diagnosis of diabetes (glucose level of 7.0 mmol/L). Results Glucose levels decreased by 1.0% at 1 hour and by 2.1% at 3 hours in the C group tubes and simultaneously decreased by 1.7% at 1 hour and by 2.5% at 3 hours in the B group tubes. In contrast, glucose levels decreased by 4.1% at 1 hour and by 6.3% at 3 hours in the A group tubes. There was a statistically significant difference in glucose levels measured in the A group tubes and B group tubes at 1 hour, 2 hours, and 3 hours. The misdiagnosis rate of clinical diagnosis in diabetes was highest in the A group tubes (7.0‰ at 1 hour, 0.1‰ at 3 hours at 7.0 mmol/L point; 14.6‰ at 1 hour, 0.4‰ at 3 hours at 6.1 mmol/L point) and lowest in the C group tubes (2.95‰ at 1 hour, 0‰ at 3 hours at 7.0 mmol/L point; 4.8‰ at 1 hour, 0.1‰ at 3 hours at 6.1 mmol/L point). Conclusion The tube addition of WZB117 is more suitable for minimizing glycolysis and has no effect on glucose levels even if specimens are left uncentrifuged for up to 3 hours.

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Blood Glucose Determination without Deproteinization, with Use of o-Toluidine in Dilute Acetic Acid

Clinical Chemistry ◽

10.1093/clinchem/17.5.440 ◽

1971 ◽

Vol 17 (5) ◽

pp. 440-441 ◽

Cited By ~ 10

Author(s):

Giovanni Ceriotti

Keyword(s):

Acetic Acid ◽

Blood Glucose ◽

Acid Solution ◽

Acetic Acid Solution ◽

Glucose Determination ◽

Blood Glucose Determination

Abstract A method is described for determining glucose in 25 µl of plasma. o-Toluidine is used in weak acetic acid solution. Addition of the emulsifier "Cremophor" obviates the need for deproteinization. The reaction is complete after 15 min at 100°C, and the color is stable for longer than 24 h. The blank is almost colorless. The reagent is stable, colorless, not viscous, and convenient to handle.

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