Cloning and sequencing of Bacillus mojavensis TH309 phytase gene

6th International Eurasian Conference on Biological and Chemical Sciences (EurasianBioChem 2023), 11 - 13 Ekim 2023, ss.322, (Özet Bildiri)

Phytate is the main storage form of phosphorous in inositol ring in the plants. Phytase hydrolyses phytate and

releases phosphorus from the structure. Monogastric animals including poultries, pigs, and fishes lack phytatedegrading

enzymes in their gastrointestinal systems. Therefore, the animals cannot benefit from the

phosphorus found in phytate in feed. Furthermore, phytate in the gastrointestinal systems of these animals

cause chelation of metallic cations such as calcium, ferric, zincs, minerals, amino acids and proteins; therefore,

it has anti-nutritional effects in feeding the monogastric animals. To eliminate its anti-nutritional effects and

utilize phosphorus from phytate, phytase is added to feeds, and it has been used in the feeding of monogastric

animals. Phytases from fungal and bacterial sources can be used for this purpose. Phytases obtained from the

Bacillus species have several advantages such as high thermal stability, requirement of low calcium level for

activation, substrate specity, pH profile, and resistance to proteolysis. Even though phytase from several

Bacillus species have been cloned and purified, phytase from Bacillus mojavensis has not studied, yet. Here,

phytase gene from B. mojavensis TH309 strain was cloned into pet20b(+) expression vector for extracellular

synthesis of phytase in E. coli BL21. Using special primers, phytase gene from B. mojavensis TH309 was

amplified by PCR. The vector and PCR product were digested by BamHI and EcoRI restriction enzymes and

ligated by T4 DNA ligase. After obtaining recombinant plasmid called pet20b(+)-phytase, transformation and

colony PCR were carried out to select clone. After isolation of the plasmid, DNA sequencing was carried out

bidirectionally. BLAST analysis indicated that B. mojavensis TH309 phytase gene contained several mutations

compared to B. mojavensis reference sequence. Three of the mutations are missense, R290G, T301K and

N366D, which indicates the unique structure of the enzyme. Expression, purification, and characterization

studies of the enzyme are in progress.