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Cloning of the β-Glucosidase Genes from Bifidobacterium Strains and Their Heterologous Expression in B. bifidum BGN4

DC Field Value Language
dc.contributor.advisor지근억-
dc.contributor.author윤소윤-
dc.date.accessioned2017-07-13T16:50:52Z-
dc.date.available2017-07-13T16:50:52Z-
dc.date.issued2013-02-
dc.identifier.other000000009846-
dc.identifier.urihttps://hdl.handle.net/10371/120321-
dc.description학위논문 (박사)-- 서울대학교 대학원 : 식품영양학과, 2013. 2. 지근억.-
dc.description.abstractβ-Glucosidase is necessary for the bioconversion of glycosidic phytochemicals in food. Two Bifidobacterium strains (Bifidobacterium animalis subsp. lactis SH5 and B. animalis subsp. lactis RD68) with relatively high β-glucosidase activities were selected among 46 lactic acid bacteria. A β-glucosidase gene (bbg572) from B. lactis was shotgun cloned, fully sequenced, and analyzed for its transcription start site, structural gene, and deduced transcriptional terminator. The structural gene of bbg572 was 1,383 bp. Based on amino sequence similarities, bbg572 was assigned to family 1 of the glycosyl hydrolases. To overexpress bbg572 in Bifidobacterium, several bifidobacteria expression vectors were constructed by combining several promoters and terminator sequence from different bifidobacteria. The maximum activity of recombinant Bbg572 was achieved when it was expressed under its own promoter and terminator. Its enzyme activity increased 31-fold compared with those of its parental strains. The optimal pH for Bbg572 was pH 6.0. Bbg572 was stable at 37-40°C. Bbg572 hydrolyzed isoflavones, quercetins and disaccharides with various β-glucoside linkages. Bbg572 converted the ginsenoside Rb1 and Rb2. These results suggest that this new β-glucosidase-positive Bifidobacterium transformant can be utilized for the production of specific aglycone products.
To characterize and overexpress the β-glucosidases of Bifidobacterium, two novel β-glucosidase encoding genes (bbg504 and bbg1176) from Bifidobacterium pseudocatenulatum Int57 and SJ32 were identified and cloned. These cloned β-glucosidase genes (bbg504 and bbg1176) were fully sequenced and analyzed for the transcription start site, structural genes, and putative transcriptional terminator. The structural genes of bbg504 and bbg1176 contained 2,247 bp open reading frame (ORF) encoding a protein containing 729 amino acids (molecular weight of 82.39 kDa) and 1,176 bp ORF encoding a protein containing 392 amino acids (molecular weight of 43.12 kDa), respectively. Based on their amino sequence similarities to the well characterized carbohydrate hydrolases, Bbg504 and Bbg572 were assigned to family 3 and family 1 of the glycosyl hydrolases, respectively. Several Bifidobacterium expression vectors were constructed by combining various promoters and a terminator sequence from different Bifidobacterium. Bp504bbg504t with its own promoter and terminator exhibited the highest β-glucosidase activity. Additionally, Bbg1176 also showed high β-glucosidase activity in B. bifidum BGN4 when cloned into p504 promoter-mediated expression system (Bp504bbg1176). These β-glucosidase activities of Bp504bbg504t and Bp5041176 were about 321- fold and 30-fold greater than those of their respective parental strains. Furthermore, Bbg504 exhibits similar pH optimum (pH 6.0) and thermostability to those of Bbg1176. Bbg504 and Bbg1176 showed substrate specific activities across a broad substrate range. Interestingly, the two β-glucosidases, Bbg504 and Bbg1176, showed different hydrolysis patterns with ginsenosides. Bbg504 hydrolyzed ginsenoside Rb1, F2 and Rg3(S), while Bbg1176 hydrolyzed Rd, Rb1, Rb2, Rg1, Rg3(S) and Rg3(R). This is the first research on cloning and expression of β-glucosidase from B. pseudocatenulatum. Based on these results, two novel β-glucosidases from Bifidobacterium transformants Bp504bbg504 and Bp504bbg1176 converted glycosides more efficiently and can be utilized for the production of specific aglycone products.		-
dc.description.tableofcontentsAbstract........................................................................i
Contents......................................................................iii
List of Tables..............................................................vii
List of Figures.............................................................viii

1. Literature review - Bifidobacterium and carboxyl
hydrolase..................................................................1
1.1 Bifidobacterium.....................................................2
1.2 Carbohydrate-modifying enzymes in Bifidobacterium
sp.......................................................................3
1.3 Classification and structure of β-glucosidase............5
1.4 Catalytic mechanisms ..........................................9
1.5 Mechanism of substrate binding and specificity.......11
1.6 Sources and characteristics of recombinant β-
glucosidase........................................................12
1.7 Functional roles and applications based on hydrolytic
activity of β-glucosidase.......................................15
References...............................................................19
2. Identification of the β-Glucosidase Gene from
Bifidobacterium animalis subsp. lactis and its
Expression in B. bifidum BGN4 ............................... 29
2.1 Introduction.........................................................30
2.2 Materials and methods......................................32
2.2.1 Bacterial strains and culture conditions.............32
2.2.2 Screening of lactic acid bacteria for their β-
glucosidase activities.....................................34
2.2.3 Construction of cosmid library of Bifidobacterium
...................................................................37
2.2.4 Cloning of the Bifidobacterium β-glucosidase
genes and sequence analysis........................38
2.2.5 Analysis of transcription start sites...................39
2.2.6 Construction of a vector encoding β-glucosidase
and transformation of Bifidobacterium...............40
2.2.7 Analysis of β-glucosidase activity in recombinant
B. bifidum BGN4............................................43
2.2.8 Optimal pH, temperature, and time of the β-
glucosidase........44
2.2.9 Substrate specificity of β-glucosidase..............45
2.3 Results and discussion........................................47
2.3.1 Sequence analysis of Bifidobacterium β-
glucosidase genes.........................................47
2.3.2 Analysis of the transcription start point..............52
2.3.3 Expression of β-glucosidase in B. bifidum BGN4
...................................................................54
2.3.4 Biochemical characterizations of the β-
glucosidase activity of cell extracts from
transformants harboring p572bbg572t................58
2.3.5 Substrate specificity of the recombinant β-
glucosidase from B. bifidum BGN4......................60
References...............................................................70

3. Molecular cloning, characterization and expression in comparison of β-glucosidases from Bifidobacterium pseudocatenulatum in B. bifidum BGN4..........................76
3.1 Introduction.........................................................77
3.2 Materials and method...........................................79
3.2.1 Bacterial strains, plasmids, media, and culture
conditions.....................................................79
3.2.2 Assessment of β-glucosidase for the various
experimental bacteria and recombinant B.
bifidum BGN4................................................81
3.2.3 Construction of cosmid library of Bifidobacterium
pseudocatenulatum.......................................82
3.2.4 Cloning of the Bifidobacterium β-glucosidase
genes and sequence analysis........................83
3.2.5 Analysis of transcription start sites...................86
3.2.6 Construction of a vector encoding β-glucosidase
and transformation of Bifidobacterium......... .....87
3.2.7 Optimal temperature and pH of the β-glucosidases
...................................................................89
3.2.8 substrate specificity of β-glucosidase..............90
3.3Results and discussion.........................................91
3.3.1 Identification and sequence analysis of
Bifidobacterium β-glucosidase genes................91
3.3.2 Analysis of the transcription start point.............100
3.3.3 Expression of β-glucosidase in B. bifidum BGN4
.................................................................102
3.3.4 Biochemical characterizations of the β-
glucosidase activity of cell extracts from
Bp504bbg504t and Bp504bbg1176....................106
3.3.5 Substrate specificity of the recombinant β-
glucosidases from B. bifidum BGN4...............109
References.............................................................121		-
dc.formatapplication/pdf-
dc.format.extent2369781 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subject.ddc641-
dc.titleCloning of the β-Glucosidase Genes from Bifidobacterium Strains and Their Heterologous Expression in B. bifidum BGN4-
dc.typeThesis-
dc.description.degreeDoctor-
dc.citation.pages140-
dc.contributor.affiliation생활과학대학 식품영양학과-
dc.date.awarded2013-02-
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