Purification and characterisation of uricase from Bacillus subtilis SP6. (February 2022)

• Record Type: Journal Article
• Authors: Pustake, Sneha O.; Bhagwat, Prashant; Pillai, Santhosh; Dandge, Padma B.
• Is Part Of: Process biochemistry. Volume 113(2022)
• Page Start: 55
• Page End: 61
• Publication Date: 2022-02
• Subjects: Uricase -- Bacillus subtilis -- Ion-exchange chromatography -- Biophysical characterisation -- Allantoin -- Pharma-cosmeceutical industries; Biochemical engineering -- Periodicals; Biotechnology -- Periodicals; Biochemistry -- periodicals; Biotechnology -- periodicals; Chemical Engineering -- periodicals; Génie biochimique -- Périodiques; Biotechnologie -- Périodiques; Biochemical engineering; Biotechnology; Periodicals; 660.63
• DOI: 10.1016/j.procbio.2021.12.010
• Languages: English
• ISSNs: 1359-5113

Abstract

Graphical abstract: Highlights: Bacillus subtilis SP6 was evaluated for its ability to produce uricase and allantoin. Uricase was purified with 8.65-fold purification and 26.37 % yield and had a weight of 44 kDa. Purified uricase comprises of 35.7 % α-helix, 41.8 % β-sheet and 3.8 % turns and Tm 42 °C. Presence of allantoin was confirmed using LCMS. Abstract: Uricase enzyme was purified to homogeneity from the culture filtrates of Bacillus subtilis SP6 in a two-step process involving ammonium sulphate precipitation and ion-exchange chromatography. The purified uricase was 8.65-fold pure with 26.35 % yield and had a molecular weight of 44 kDa. The purified uricase had an optimal pH of 9.0 and was stable at a pH range of 6.0–9.0. The optimum temperature for uricase was 37 °C and exhibited thermostability in the range of 10 °C–37 °C. The uricase activity was induced moderately by CuSO4 while SDS, EDTA, Triton X-100, HgI and K-ferricyanide significantly diminished the activity. The kinetic parameters ( K m and V max) for the purified uricase were 0.17 mM and 1.5 × 10 −4 mol l -1 min -1, respectively. Biophysical characterisation of uricase revealed that the enzyme has 35.7 % α-helix, 41.8 % β-sheet and 3.8 % turns and Tm 42 °C. The byproduct allantoin generated by the action of uricase on uric acid was identified by LCMS. Thus, this study highlights the potential of uricase from B. subtilis SP6 which displayed significant activity at normal physiological conditions. This may accommodate for alluring applications in pharma industries, while its efficacy to generate the byproduct 'allantoin' also opens an avenue for its use in pharma-cosmeceutical industries.