PHYTASE GENE ISOLATION FROM BACTERIA OBTAINED FROM HOT SPRINGS OF AJK

Abstract

Phytases are enzymes capable of hydrolyzing phytic acid to less phosphorylated
myo-inositol derivates. Phytases are becoming essential supplement to animal feeds. Bacillu ssp.
produces a thermostable phytase which renders it suitable for commercial production. A locally
isolated strain of Bacillus subtilis was used for the amplification of phytase gene. Amplicons of 1300
and 1120bps were obtained by PCR. For the study twenty samples of water were obtained from hot
springs found at different places in district Kotli village TattaPani (AJK). Plate streaking method was
used for the isolation of pure bacterial colonies. Single bacterial colony was purified after five
passages on nutrient agar media. Screening of phytase producing bacteria was done by modified
Phytase screening medium. Isolated colonies were spread on this medium which had Calcium phytate
as substrate for phytase enzyme. Local isolate of Bacillus sp. was employed for amplification and
isolation of phytase gene. Dubous salt medium was prepared in order to obtain the growth of Bacillus
subtilis. Genomic DNA of Bacillus subtilis was extracted. Oligonucleotide primers were designed
using primer-3 software program. Primers were blast in order to check the relevance and suitability of
primers for amplification of desired gene. Consequently, a 1300bp gene, by using phy lit primer
(primer set 3) was obtained after optimization of PCR conditions. Band size of desired gene for primer
set 3 was 1300bp and 1059bp. For primer set 1 and 2 expected band sizes were 1120bp and 1236bp,
respectively and these two primer set were also optimized. Thus it is concluded from the study that
desired gene band of 1120bp as well as other non-specific bands of 100bp, 400bp and 800bp were
obtained which is a clear indication of presence of phytase gene in hot springs.