Identification of Phytase Producing Bacteria from Soil with Chicken Faeces and Partial Purification of Phytase

Ng, Yee Ling (2019) Identification of Phytase Producing Bacteria from Soil with Chicken Faeces and Partial Purification of Phytase. Final Year Project (Bachelor), Tunku Abdul Rahman University College.

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Abstract

Phytate acts as an anti-nutrient as a result of the formation of insoluble complexes in the presence of protein and minerals. Monogastric animals are not able to digest phytate phosphorus either due to lack of or insufficient amount of phytate degrading enzymes. Phytate is the main natural source of phosphorus. Phytases which belong to the family of histidine acid phosphatases, hydrolyse phytic acid to inorganic phosphate and myoinositol phosphate derivatives. The objectives of this study were to determine the phytase activity, partially purify the phytase extracted from isolate S1.1, S1.8 and S1.9. Besides, the isolated phytase producing bacteria would be identified. Phosphate standard curve with equation of y = 0.0003x - 0.0179 was plotted to quantify the phosphate produced by the reaction of phytase and the phytase activity was identified. S1.1 and S1.9 showed higher phytase activity which were 9.969 x 10-3 U/mL and 12.838 x 10-3 U/mL respectively after 2 days incubation and 19.868 x 10-3 U/mL and 20.721 x 10-3 U/mL respectively after 5 days incubation. These two isolates were chosen to proceed in the study. Phytase activity after 5 days incubation increased for 99.30% and 61.40% compared with 2 days incubation for S1.1 and S1.9 respectively. BSA standard curve with equation of y = 0.0002x - 0.0078 and R2 value of 0.9993 was plotted to determine the protein concentration. The protein concentration for S1.1 and S1.9 were 454 ppm and 509 ppm respectively. The phytase was partially purified using ATPS system and dialysis after precipitation by ammonium sulphate. The crude and purified enzyme were runned for SDS-PAGE. DNA of both isolates S1.1 and S1.9 were extracted. S1.1 was successfully amplified with size of about 1.5kb using PCR. The isolate S1.1 was identified as Bacillus sp. strain M2 GeneBank (Accession No. MK615818.1) using BLAST analysis with 92% of query coverage and 93.62% identity. The phylogenetic tree of phytase producing bacteria, S1.1 with related species sequence using Neighbour-Joining method and plotted by MEGA 7 software.