Isolation, Characterization, Molecular Gene Cloning, And Sequencing Of A Phytase From Bacillus Subtilis WHNB02

Bacillus subtil is WHNB02 producing phytase was isolated from 118 samples . An extracellular phytase produced from it was purified to homogeneity by the steps of alcohol precipitation, (NH4)2SO4 fractionation and Sephadex G-100 column chromatography. Purification of about 31.5 fold was achieved with an overall yield of 13. 0%. The molecular weight of the enzyme was estimated to be 42 KD on SDS-PAGE. The Km value for sodium phytate was 0.5 mmol/L. Its optimum temperature for phytsase activity was 60X2 and about 61% of its original activity remained after incubation at 80C for 10 min. The optimum pH for enzyme activity was 7.0 and stable from pH6. 0-10.0. Calcium ions were repuired for activity and stability . Its activity was moderately inhibited by Hg2+, Mn2+, Cu2+, Zn2+, Ba2+ and greatly inhibited by EDTA and Mn2+and Cd2+. The phyC gene of B . subtilis WHNB02 was amplified by the polymerase chain reaction(PCR) with primers designed according to the sequences of the phyC gene in GenBank. The amplified fragment was cloned and sequenced. The results show that: the coding region is 1152bp in size, and encodes a peptide of 383 amino acid residues, in which there is a signal peptide with 26 amino acids and a mature peptide of 357 amino acids. Comparison of this sequence with the first phyC gene of the B. subtilis strain (GenBank Accession:AF029053)reported by Kerovuo shows that the nucleotide identity is as high as 92. 4%, and the amino acid identity comes up to 93. 7%. The phyC gene and its amino acid sequence have been accessed by GenBank (Accession: AA043434.1).