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Impact of carbohydrate binding modules on transglycosylation efficiency in the glycoside hydrolase family 5 proteins
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Title
Impact of carbohydrate binding modules on transglycosylation efficiency in the glycoside hydrolase family 5 proteins
Name
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Kasture
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Madhura
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1994-
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Madhura Kasture
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author
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Chundawat
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Shishir
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Shishir Chundawat
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Advisory Committee
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chair
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Rutgers University
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degree grantor
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School of Graduate Studies
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school
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theses
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2020
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2020-01
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English
Abstract
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Oligosaccharides are significant in many biological processes such as modulating protein folding, cell interactions, and binding and are widely used in prebiotics and pharmaceutical industries. This rising popularity has accelerated the research for their sustainable source of generation. Enzymatic synthesis offers an alternative over standard chemical synthesis to produce these oligosaccharides with higher specificity and product purity. Transglycosylases are a class of glycoside hydrolases (GH) present in nature and are ideal biocatalysts for this process. Since these are few in number and limited in substrate specificity, over the past couple of years, extensive studies and advancements have been made to engineer glycoside hydrolases to reduce the hydrolytic activity of these enzymes to perform transglycosylation to produce oligosaccharides.
This thesis investigates the transglycosylation activity of glycoside hydrolases family five that has tethered carbohydrate-binding module (CBM) CBM3a. CBMs improve the catalytic efficiency by binding to the polysaccharide chain, making the substrate more accessible to the enzyme. The catalytic nucleophile of GH5 enzymes was substituted for alanine, glycine, and serine amino acids. Activity assay in the presence of an activated soluble sugar was performed to obtain a product profile for the enzymes. With the data from the activity assays, a kinetic model was developed. A structure-functional analysis was attempted to explain the results. It was observed that few enzymes with a mutated catalytic nucleophile show transglycosylation like activity in the presence of an appended CBM3a, suggesting that the presence of CBM3a affected the mechanism of the enzyme activity other than just increasing the local concentration of the substrate at the catalytic pocket. In summary, this study provides results that led further confidence in the active role of the CBM3a in the transglycosylation activity of the GH5 enzymes and understanding the mechanism.
This thesis investigates the transglycosylation activity of glycoside hydrolases family five that has tethered carbohydrate-binding module (CBM) CBM3a. CBMs improve the catalytic efficiency by binding to the polysaccharide chain, making the substrate more accessible to the enzyme. The catalytic nucleophile of GH5 enzymes was substituted for alanine, glycine, and serine amino acids. Activity assay in the presence of an activated soluble sugar was performed to obtain a product profile for the enzymes. With the data from the activity assays, a kinetic model was developed. A structure-functional analysis was attempted to explain the results. It was observed that few enzymes with a mutated catalytic nucleophile show transglycosylation like activity in the presence of an appended CBM3a, suggesting that the presence of CBM3a affected the mechanism of the enzyme activity other than just increasing the local concentration of the substrate at the catalytic pocket. In summary, this study provides results that led further confidence in the active role of the CBM3a in the transglycosylation activity of the GH5 enzymes and understanding the mechanism.
Subject
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Chemical and Biochemical Engineering
Subject
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Topic
Glycosidases
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Rutgers University Electronic Theses and Dissertations
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ETD_10542
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1 online resource (xiii, 80 pages) : illustrations
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M.S.
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Includes bibliographical references
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School of Graduate Studies Electronic Theses and Dissertations
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rucore10001600001
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doi:10.7282/t3-c78h-4e12
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ETD graduate
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Rights
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The author owns the copyright to this work.
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Name
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Kasture
GivenName
Madhura
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Permission or license
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2020-01-13 00:46:56
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Madhura Kasture
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Rutgers University. School of Graduate Studies
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Author Agreement License
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I hereby grant to the Rutgers University Libraries and to my school the non-exclusive right to archive, reproduce and distribute my thesis or dissertation, in whole or in part, and/or my abstract, in whole or in part, in and from an electronic format, subject to the release date subsequently stipulated in this submittal form and approved by my school. I represent and stipulate that the thesis or dissertation and its abstract are my original work, that they do not infringe or violate any rights of others, and that I make these grants as the sole owner of the rights to my thesis or dissertation and its abstract. I represent that I have obtained written permissions, when necessary, from the owner(s) of each third party copyrighted matter to be included in my thesis or dissertation and will supply copies of such upon request by my school. I acknowledge that RU ETD and my school will not distribute my thesis or dissertation or its abstract if, in their reasonable judgment, they believe all such rights have not been secured. I acknowledge that I retain ownership rights to the copyright of my work. I also retain the right to use all or part of this thesis or dissertation in future works, such as articles or books.
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2020-01-31
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2022-01-30
Detail
Access to this PDF has been restricted at the author's request. It will be publicly available after January 30th, 2022.
Copyright
Status
Copyright protected
Availability
Status
Open
Reason
Permission or license
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