Optimization of clinical uricase production by Bacillus cereus under submerged fermentation, its purification and structure characterization

Abstract

The uricase production from Bacillus cereus after statistical optimization of process variables designed by L18 orthogonal array gave enhanced enzyme activity by 13% (29.7 U/mL) as compared with un-optimized medium (26.3 U/mL). The optimized process variables used for scale up of uricase production in a bioreactor gave 39.7 U/mL of uricase activity resulting 51% increase as compared with unoptimized medium. The Luedeking-Piret model classified the uricase production from Bacillus cereus as mixed-growth associated. The culture supernatant having uricase activity (7.7 U/mg) on purification by (NH4)2SO4 precipitation followed by anion-exchange and size-exclusion chromatography resulted 87 U/mg giving 11-fold purification. The purified uricase can be used for treating gout disease. The de novo amino acid sequence analysis of purified uricase by MALDI-TOF MS showed 301 amino acids and molecular mass of 34.4 kDa, which was corroborated by 35 kDa from SDS-PAGE. The phylogenetic tree generated by homologous amino acid sequences displayed the clustering of Bacillus uricase with fungal uricase. The secondary structure analysis of uricase by circular dichroism showed 18.6% α-helices and 27.4% β-strands. These results were corroborated by secondary structure prediction analysis by PsiPred. The homology modelling of uricase displayed the conserved T-fold structure. © 2018 Elsevier Ltd