Production, partial purification and characterization of alkaline phosphatase from a thermo-alkaliphile Geobacillus thermodenitrificans I2 isolate. (January 2021)

• Record Type: Journal Article
• Authors: Yousef, Samar M.; El-Gendi, Hamada; Ghozlan, Hanan; Sabry, Soraya A.; Soliman, Nadia A.; Abdel-Fattah, Yasser R.
• Is Part Of: Biocatalysis and agricultural biotechnology. Number 31(2021)
• Publication Date: 2021-01
• Subjects: Crude enzyme -- Experimental design -- Optimization -- Purification -- Thermophilic -- Geobacillus sp; Agricultural biotechnology -- Periodicals; Enzymes -- Biotechnology -- Periodicals; 660.6
• DOI: 10.1016/j.bcab.2020.101853
• Languages: English
• ISSNs: 1878-8181

Abstract

Abstract: Alkaline phosphatases producing microbes are wide spread in nature; ALP is a hydrolase enzyme functioning at alkaline pH, able to hydrolyze phosphates from many types of molecules; this initiating many applications. Experimental designs were applied to evaluate the culture conditions affecting ALP production by Geobacillus thermodenitrificans strain I2, where glucose, yeast-extract and agitation were the most significant variables according to Plackett-Burman design (PBD). The optimum levels of these variables were assessed through Box–Behnken design (BBD) to be (g/L): glucose 26.4, yeast-extract 29.72 and agitation 150rpm, with a predicted enzyme activity of 8.149U/ml. The measured activity upon application the optimized medium was 7.896U/ml. Enzyme was partially purified (42%; 12 fold) through precipitation by ammonium sulfate and anion column, with two protein bands (>70 & ~40kDa) in SDS-PAGE. A partial gene translated sequence proved that the natively expressed ALP by studied strain is related to the band with ~40kDa. The partially purified enzyme exhibited maximum activity at 60°C and pH9.0, with remarkable temperature stability up to 65°C. The results indicated an enhancement in enzyme activity with metals (Mg 2+, Mn 2+ ) and reducing agents (DTT & ß-mercaptoethanol) whereas, other metals (Zn 2+, Hg 2+ ), and anionic surfactant (SDS); EDTA; and PMSF inhibited the enzyme activity. The dependence on enzyme and substrate concentration was represented in Lineweaver-Burk plot where, K m and V ma x were 40mM and 25U/mg protein, respectively. In conclusion, the promising traits of studied ALP would underpin its efficient exploitation in several industries to cope with demands of worldwide enzyme markets. Graphical abstract: Image 1 Highlights: Optimization the production of ALP through applying statistical experimental design. Intensive biochemical characterization of the partial purified produced ALP. Characterization of the expressed thermo -ALP. gene.