Improvement of bilirubin oxidase productivity of Myrothecium verrucaria and studies on the enzyme overproduced by the mutant strain in the solid-state fermentation.

Bilirubin oxidase has applications in the health and environmental sectors. Hence, several attempts have been made to increase enzyme yields. However, improvements were not very high. We report here the development of a mutant strain of Myrothecium verrucaria by using UV-rays, which produced 28.8 times more enzyme compared with the parent and was higher than the yields reported in earlier submerged cultures. The mutant strain produced 35.6 times more enzyme than the parent in solid-state fermentation, which is better than that previously reported for a solid-state fermentation process. The specific activity of the enzyme produced by the mutant was higher than that of the parental enzyme. Bilirubin oxidase from both strains showed an optimum activity at pH 7 and 40°C. However, the time required to inactivate half of the initial enzyme activity at 60°C was much higher in the case of the enzyme obtained from the mutant compared with the parental enzyme. The improved thermostability of the enzyme from the mutant strain could be due to the point mutations induced during the UV irradiation, since there was no change in the mass of the enzyme compared with the parental enzyme. The bilirubin oxidase of the mutant strain degraded the bilirubin faster than the enzyme obtained from the parent under similar conditions. Faster activity of the enzyme obtained from the mutant strain could be due to its lower Km (79.4 µM) compared with that of the parental enzyme (184 µM). Hence, the mutant enzyme showed a better functionality and thermostability, which will be beneficial for industrial applications.

Development of a bioautographic method for the detection of lipase inhibitors.

An autobiographic method based on the thin layer chromatogram was developed by using the chemical system that comprises p-Nitrophenyl butyrate and bromothymol blue for detecting the lipase inhibitor. Lipase inhibitory zones were visualized as blue spots against the greenish yellow background. This method could able to detect the well known lipase inhibitor, orlistat up to the concentration of 1ng which is better than the earlier method. This method could also able to detect the lipase inhibition activities from the un-explored species of Streptomyces. The developed method can be used not only for the screening of unknown samples for the lipase inhibitors but also for the purification of the lipase inhibitors from the unknown samples.