Purification, characterization and synthetic application of a thermally stable laccase from Hexagonia tenuis MTCC-1119.

A thermally stable laccase was purified from the culture filtrate of Hexagonia tenuis MTCC-1119. The method involved concentration of the culture filtrate by ammonium sulphate precipitation and an anion-exchange chromatography on diethylaminoethyl (DEAE) cellulose. The sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and native polyacrylamide gel electrophoresis (native-PAGE) both gave single protein bands, indicating that the enzyme preparation was pure. The molecular mass of the enzyme determined from SDS-PAGE analysis was 100 kDa. The purification fold and percentage recovery of the enzyme activity were 12.75 and 30.12%, respectively. The pH and the temperature optima were 3.5 and 45○C, respectively. The enzyme was most stable at pH 4.0 when exposed for 1 h. Using 2,6-dimethoxyphenol (DMP), 2,2’ [azino-bis-(3-ethylbonzthiazoline-6-sulphonic acid) diammonium salt] (ABTS) and 3,5-dimethoxy-4-hydroxybenzaldehyde azine (syringaldazine) as the substrates, the Km, kcat and kcat/Km values of the laccase were 80 μM, 2.54 s-1, 3.17 × 104 M-1s-1, 36 μM, 2.54 s-1, 7.05 × 104 M-1s-1 and 87 μM, 2.54 s-1, 2.92 × 104 M-1s-1, respectively. The purified laccase was finally used for the selective biotransformation of aromatic methyl group to aldehyde group in presence of diammonium salt of ABTS as the mediator and products were characterized by HPLC, IR and 1H NMR. The percentage yields of these transformed products were >91%.

Correlation of 2 hour, 4 hour, 8 hour and 12 hour urine protein with 24 hour urinary protein in preeclampsia

To find shortest and reliable time period of urine collection for determination of proteinuria. It is a prospective study carried out on 125 pregnant women with preeclampsia after 20 weeks of gestation having urine albumin >/= 1 using dipstick test. Urine was collected in five different time intervals in colors labeled containers with the assistance of nursing staff; the total collection time was 24 hours. Total urine protein of two-hour, four-hour, eight-hour, 12-hour and 24-hour urine was measured and compared with 24-hour collection. Data was analyzed using the Pearson correlation coefficient. There was significant correlation [p value < 0.01] in two, four, eight and 12-hour urine protein with 24-urine protein, with correlation coefficient of 0.97, 0.97, 0.96 and 0.97, respectively. When a cut off value of 25 mg, 50 mg. 100 mg, and 150 mg for urine protein were used for 2-hour, 4-hours, 8-hour and 12-hour urine collection, a sensitivity of 92.45%, 95.28%, 91.51%, and 96.23% and a specificity of 68.42%, 94.74%, 84.21% and 84.21% were obtained, respectively. Two-hour urine proteins can be used for assessment of proteinuria in preeclampsia instead of gold standard 24-hour urine collection for early diagnosis and better patient compliance