S-Space Graduate School of International Agricultural Technology (국제농업기술대학원) Dept. of International Agricultural Technology (국제농업기술학과) Theses (Master's Degree_국제농업기술학과)
Screening and Characterization of β-galactosidase producing Bifidobacterium animalis subsp. lactis HT 10-2 Isolated from Infant Feces
β-galactosidase를 생산하는 유아분변 유래 Bifidobacterium animalis subsp. lactis HT10-2의 선발과 특성 규명
- 국제농업기술대학원 국제농업기술학과
- Issue Date
- 서울대학교 국제농업기술대학원
- 학위논문 (석사)-- 서울대학교 국제농업기술대학원 국제농업기술학과, 2017. 8. 허철성.
- In this study, 637 of Bifidobacteria and Lactobacilli colonies were obtained from Korean infants, and 10 isolates were selected by colorimetric assay for β-galactosidase activities. We investigated the probiotic potential of the isolates, and Bifidobacterium animalis subsp. lactis HT 10-2 was identified as a promising probiotic strain with high activity of β-galactosidase. The complete genome sequence of HT 10-2 reveals a single circular chromosome of 1,923,647 bp, with 1,613 predicted proteins-encoding 1,553 of coding sequence (CDS). The genes (bgaA, ebgA, lacZ, and beta-galIII) coding for β-galactosidase were possessed by both Bifidobacterium animalis subsp. lactis DSM 10140 and HT 10-2. However, the enzyme activities of DSM 10140 and HT 10-2 have shown significant difference each other, HT 10-2 showed relatively higher β-galactosidase activity than DSM 10140 amounting to approximately 3 times. Forthermore, we investigated the relative mRNA expression of bgaA, ebgA, lacZ, and beta-galIII from HT 10-2 versus to thoses genes from DSM 10140 using the quantitative real time PCR (qRT-PCR). High transcriptional rate of the genes from HT 10-2 was observed compared to the genes from DSM 10140. ΔCt values indicated that mRNA expression from lacZ of the HT 10-2 were higher than that of bgaA, ebgA, lacZ, and beta-galIII. The β-galactosidase, expressed in lacZ gene, was targeted for isolation and characterization. Finally, β-galactosidase with a molecular mass of 119 kDa was purified from crude cell extracts of the HT 10-2 using ammonium sulfate fractionation followed by ion exchange chromatography with 10 fold to a specific activity of 30,473.8 unit/ml. The temperature optimum of β-galactosidase activity was found to be 37 ℃ and the purified β-galactosidase was stable at 37 ℃ and the residual β-galactosidase activity still maintained 80.43 % and 78 % after treatment for 0.5 h and 1.0 h relatively. β-galactosidase activities were enhanced by by CaCl2 (1.27 fold) and was remarkably enhanced by MgSO4 (2.43 fold), FeSO4 (3.00 fold), MnSO4 (2.75 fold) and MgCl2 (2.17 fold), but some ions, such as KCl, NaCl, Na2SO4 and CuSO4 inhibited the activities.