Immobilization of grain-sorghum (Sorghum bicolor, var. CSH-14) leaf oxalate oxidase on to a modified mica chip and its application in the determination of urinary oxalate

• Published in: Biotechnology and applied biochemistry 2007-06, Vol.47 (2), p.125-129
• Main Authors: Bhambi, Manu, Pundir, Chandra Shekhar
• Format: Article
• Language: English
• Subjects: Adsorption; Aluminum Silicates - chemistry; Biological and medical sciences; Biosensing Techniques - methods; Biotechnology; Coated Materials, Biocompatible - chemistry; Colorimetry - methods; Enzymes, Immobilized - chemistry; Fundamental and applied biological sciences. Psychology; grain sorghum (Sorghum bicolor) leaf; Hordeum vulgare; immobilization; mica chip; oxalate; oxalate oxidase; Oxalic Acid - chemistry; Oxalic Acid - urine; Oxidoreductases - chemistry; Plant Leaves - enzymology; Reproducibility of Results; Sensitivity and Specificity; Sorghum; Sorghum - enzymology; Sorghum bicolor; Urinalysis - methods; urinary oxalate
• ISSN: 0885-4513; 1470-8744
• DOI: 10.1042/BA20060181

A new method is described for immobilization of oxalate oxidase purified from grain sorghum (Sorghum bicolor, var. CSH‐14) leaves on to mica chip through MnCl2 · 4H2O coupling with a conjugation yield of 0.36 mg of protein/cm2 and 65.3% retention of the initial activity of the free enzyme. The immobilized enzyme showed maximum activity at pH 6.5 when incubated at 40°C for 15 min. Km for oxalate was decreased from 0.7 to 0.53 mM after immobilization. The immobilized enzyme lost 12% of its initial activity after storage at 4°C in reaction buffer. The oxalate in urine was analysed using this immobilized enzyme. The minimum detection limit was 2.5 mg/l of urine. Analytical recovery of added oxalate (10, 20 and 30 mg/l) was 89.2, 91 and 92.1% respectively. Within‐ and between‐batch coefficients of variation were