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Synthesis of fucosylated oligosaccharides using crude enzyme extracts from Bifidobacterium longum subsp. longum RD 47

DC Field Value Language
dc.contributor.advisor지근억-
dc.contributor.author황정은-
dc.date.accessioned2017-07-19T11:56:21Z-
dc.date.available2017-07-19T11:56:21Z-
dc.date.issued2014-02-
dc.identifier.other000000018429-
dc.identifier.urihttps://hdl.handle.net/10371/133931-
dc.description학위논문 (석사)-- 서울대학교 대학원 : 식품영양학과, 2014. 2. 지근억.-
dc.description.abstractHuman milk oligosaccharides (HMOs) are mostly fucosylated at their non-reducing termini. It has been proposed that HMOs function as a prebiotic for bifidobacteria. Through the screening procedure, crude enzyme extracts from Bifidobacterium longum subsp. longum RD 47 were newly found to produce fucosylated oligosaccharides. The synthesized oligosaccharides were further purified from reaction mixture and identified to be as fucosylated galacto-oligosaccharides and galacto-oligosaccharides by thin layer chromatography. The analysis of fucosylated oligosaccharides by mass spectrometry showed that degree of polymerization ranged from 2 to 7. To improve the yield of the production of fucosylated oligosaccharides, the effect of the medium composition on the production of crude enzyme extracts from B.longum RD47 was evaluated and the oligosaccharides synthesis reaction conditions were optimized. The addition of raffinose in culture medium resulted in increased galactosidase activities in crude enzyme extracts from B. longum RD 47.The synthesized fucosylated oligosaccharides may provide a new constituent for the HMOs and novel prebiotic food ingredients.-
dc.description.tableofcontentsAbstract i
Contents iii
List of Figures vii
List of Tables ix
List of Tables x
1.Introduction 1
1.1.Human milk oligosaccharides 1
1.2. Synthesis ofnovel fucosylatedoligosaccharides 2
2.Materials and methods 4
2.1.Materials 4
2.2. Screening of galactosidase activity in crude cell extracts 4
2.2.1. Culture conditions 4
2.2.2. Preparation of crude cell extracts 5
2.2.3. Determination of hydrolytic activity in crude cell extracts 5
2.2.4. Determination of transglycosylationactivity in crude cell extracts 5
2.3. Characterization of crude cell extractsfrom a selected bacterium 6
2.3.1. Changes ofgalactosidase productionin crude cell extractsfrom RD 47during cultivation 6
2.3.2. Effect of enzyme extraction methods on galactosidase activityin crude cell extractsfrom RD 47 7
2.3.3. Substrates specificity of the crude cell extractsfrom RD 47 7
2.3.4. Effect of various metal ions and proteaseinhibitorson crude enzyme extracts from RD47 8
2.3.5. Time and temperature dependent of crude enzyme extracts from RD 47 8
2.4. Optimization of the synthesized oligosaccharides producedby RD 47 9
2.4.1. Effect of carbon sources in culture medium on galactosidaseactivityin crude enzyme extracts from RD 47 9
2.4.2. Concentration of crude enzyme extracts from RD 47 9
2.4.3. Effect of pH and temperature on galactosidase activityin crude enzyme extracts from RD 47 9
2.4.4. Effect of additional substances in reaction mixture on galactosidase activity in crude enzyme extracts from RD 47 10
2.4.5. Effect of Saccharomyces cerevisiaeon galactosidase activityin crude enzyme extracts from RD 47 10
2.5. Mass analysis of the synthesized oligosaccharides 11
2.5.1. Mass analysis by MALDI-TOF/MSand LC-ESI/MS 11
2.6. Separation of crude oligosaccharides mixtures 12
2.6.1. Size exclusion chromatography by Bio gel P 2 column 12
2.6.2. Preparativethin layer chromatography 12
3. Results 14
3.1. Screening of galactosidase activity in crude cell extracts 14
3.2. Determination of crude cell extracts from RD47 17
3.3.1. Changes in galactosidase production in crude enzyme extracts from RD 47 during cultivation 17
3.2.2. Comparison ofdifferent enzyme extraction methods on galactosidase activityin crude enzyme extracts from RD 47 20
3.2.3. Substrates specificitiesof crude cell extracts from RD 47 25
3.2.4. Effect ofvarious metalions and enzyme inhibitorson galactosidase activityin crude enzyme extracts from RD 47 28
3.2.5. Time and temperature dependent of galactosidase activityin crude enzyme extracts from RD 47 32
3.3. Optimization of transglycosylationactivity of galactosidasein crude enzyme extracts from RD 47 35
3.3.1. Effect of carbon sources in culture medium on galactosidase activity in crude enzyme extracts from RD 47 35
3.3.2. Effect of pH and temperature on galactosidase activityin crude enzyme extracts from RD 47 37
3.3.3. Effect of additional substances in reaction mixture on galactosidase activityin crude enzyme extracts from RD 47 40
3.3.4. Effect of Saccharomyces cerevisiaeon oligosaccharides production 43
3.4. Characterization of the synthesized oligosaccharides 45
3.4.1. Mass analysis of the synthesized oligosaccharides 45
3.4.2. Separation of the synthesized oligosaccharides47
3.4.3. Determination of the composition of the synthesized oligosaccharides 49
4.Discussion 51
References 57
Abstract in korean 64
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dc.formatapplication/pdf-
dc.format.extent1649894 bytes-
dc.format.mediumapplication/pdf-
dc.language.isoen-
dc.publisher서울대학교 대학원-
dc.subject올리고당-
dc.subject퓨코오스-
dc.subject비피도박테리움-
dc.subject.ddc641-
dc.titleSynthesis of fucosylated oligosaccharides using crude enzyme extracts from Bifidobacterium longum subsp. longum RD 47-
dc.typeThesis-
dc.description.degreeMaster-
dc.citation.pagesx,65-
dc.contributor.affiliation생활과학대학 식품영양학과-
dc.date.awarded2014-02-
Appears in Collections:
College of Human Ecology (생활과학대학)Dept. of Food and Nutrition (식품영양학과)Theses (Master's Degree_식품영양학과)
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