Expression and Purification of BoGal36A Wild-type and W297F Mutant
(2024) KEMP20 20241Department of Chemistry
- Abstract
- This study investigated the enzyme BoGal36A, produced by the gut bacterium Bacteroides ovatus. BoGal36A is an alpha-galactosidase, an enzyme that breaks down complex sugars. Unlike most alpha-galactosidases from the GH36 family, BoGal36A appears to cleave internal glycosidic bonds. In this study, the wild-type BoGal36A and mutant W297F was expressed in E. coli BL21 (DE3) cells and purified subsequently using His-tag affinity chromatography. The proteins were analysed qualitatively using SDS-PAGE and quantitatively using nanodrop and para-nitrophenol alpha-galactosidase assay. Bioinformatics analysis for the protein predicted BoGal36A to be a stable, hydrophilic protein. The wild-type BoGal36A exhibited significantly higher specific... (More)
- This study investigated the enzyme BoGal36A, produced by the gut bacterium Bacteroides ovatus. BoGal36A is an alpha-galactosidase, an enzyme that breaks down complex sugars. Unlike most alpha-galactosidases from the GH36 family, BoGal36A appears to cleave internal glycosidic bonds. In this study, the wild-type BoGal36A and mutant W297F was expressed in E. coli BL21 (DE3) cells and purified subsequently using His-tag affinity chromatography. The proteins were analysed qualitatively using SDS-PAGE and quantitatively using nanodrop and para-nitrophenol alpha-galactosidase assay. Bioinformatics analysis for the protein predicted BoGal36A to be a stable, hydrophilic protein. The wild-type BoGal36A exhibited significantly higher specific activity (229.5 U/mg) whereas the mutant showed a specific activity of 2.86 U/mg. The W297F mutation likely affects the enzyme's structure and function due to the change in amino acid properties. X-ray crystallography or computational modeling could provide detailed structural information on how the mutation affects BoGal36A and give insights into the substrate specificity. Further research is needed to understand the unique catalytic activity of BoGal36A in the gut environment. Understanding BoGal36A's function could also contribute to improved methods for promoting the growth of Bacteroides ovatus in the gut, potentially leading to better human health. (Less)
- Popular Abstract
- This study investigated an enzyme called BoGal36A and its variant called W297F which is produced by the human gut bacterium Bacteroides ovatus. This enzyme helps people digest complex sugars that we consume in our diet. How? To address some of it, this study was conducted using a combination of scientific techniques. Unlike most other members of the family that BoGal36A belongs to, BoGal36A seems to be a special case because it has shown its ability to cleave the glycosidic bonds inside the sugar molecules instead of just cleaving the bonds at the terminals. In this study, the wild-type BoGal36A and mutant W297F were obtained from growing E. coli BL21 (DE3) cells and using the His-tag which is a repeat of Histidine amino acids that the... (More)
- This study investigated an enzyme called BoGal36A and its variant called W297F which is produced by the human gut bacterium Bacteroides ovatus. This enzyme helps people digest complex sugars that we consume in our diet. How? To address some of it, this study was conducted using a combination of scientific techniques. Unlike most other members of the family that BoGal36A belongs to, BoGal36A seems to be a special case because it has shown its ability to cleave the glycosidic bonds inside the sugar molecules instead of just cleaving the bonds at the terminals. In this study, the wild-type BoGal36A and mutant W297F were obtained from growing E. coli BL21 (DE3) cells and using the His-tag which is a repeat of Histidine amino acids that the Ni+2 prefers. This is why nickel ion beads were used in a complex system to obtain the relatively pure enzyme. The results of the study show that the enzyme in its wild-type is much more active than the mutant protein which indicates that the difference in the structure and sequence of both proteins is important in its activity and role for sugar preference. More information about the structure and function of this enzyme can be helpful in promoting people’s gut health. (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9166746
- author
- Inam, Hadiqa LU
- supervisor
- organization
- course
- KEMP20 20241
- year
- 2024
- type
- L3 - Miscellaneous, Projetcs etc.
- subject
- keywords
- biochemistry, alpha galactosidase, bacteroides ovatus, GH36A, glycoside hydrolase, BoGal36A, expression, purification
- language
- English
- id
- 9166746
- date added to LUP
- 2024-06-24 11:37:33
- date last changed
- 2024-06-24 11:37:33
@misc{9166746,
abstract = {{This study investigated the enzyme BoGal36A, produced by the gut bacterium Bacteroides ovatus. BoGal36A is an alpha-galactosidase, an enzyme that breaks down complex sugars. Unlike most alpha-galactosidases from the GH36 family, BoGal36A appears to cleave internal glycosidic bonds. In this study, the wild-type BoGal36A and mutant W297F was expressed in E. coli BL21 (DE3) cells and purified subsequently using His-tag affinity chromatography. The proteins were analysed qualitatively using SDS-PAGE and quantitatively using nanodrop and para-nitrophenol alpha-galactosidase assay. Bioinformatics analysis for the protein predicted BoGal36A to be a stable, hydrophilic protein. The wild-type BoGal36A exhibited significantly higher specific activity (229.5 U/mg) whereas the mutant showed a specific activity of 2.86 U/mg. The W297F mutation likely affects the enzyme's structure and function due to the change in amino acid properties. X-ray crystallography or computational modeling could provide detailed structural information on how the mutation affects BoGal36A and give insights into the substrate specificity. Further research is needed to understand the unique catalytic activity of BoGal36A in the gut environment. Understanding BoGal36A's function could also contribute to improved methods for promoting the growth of Bacteroides ovatus in the gut, potentially leading to better human health.}},
author = {{Inam, Hadiqa}},
language = {{eng}},
note = {{Student Paper}},
title = {{Expression and Purification of BoGal36A Wild-type and W297F Mutant}},
year = {{2024}},
}