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Investigating Enzymatic Activity Towards Alpha and Beta Glycosidic Linkages by the Unicellular Extremophilic Red Alga Galdieria sulphuraria.

Record Type:	Language materials, printed : Monograph/item
Title/Author:	Investigating Enzymatic Activity Towards Alpha and Beta Glycosidic Linkages by the Unicellular Extremophilic Red Alga Galdieria sulphuraria./
Author:	Sehar, Ujala.
Published:	Ann Arbor : ProQuest Dissertations & Theses, : 2022,
Description:	155 p.
Notes:	Source: Dissertations Abstracts International, Volume: 84-08, Section: B.
Contained By:	Dissertations Abstracts International84-08B.
Subject:	Biology. -
Online resource:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=29995989
ISBN:	9798374406191

Investigating Enzymatic Activity Towards Alpha and Beta Glycosidic Linkages by the Unicellular Extremophilic Red Alga Galdieria sulphuraria.
Sehar, Ujala.

Investigating Enzymatic Activity Towards Alpha and Beta Glycosidic Linkages by the Unicellular Extremophilic Red Alga Galdieria sulphuraria. - Ann Arbor : ProQuest Dissertations & Theses, 2022 - 155 p.

Source: Dissertations Abstracts International, Volume: 84-08, Section: B.

Thesis (Ph.D.)--New Mexico State University, 2022.

This item must not be sold to any third party vendors.

Plants convert sunlight and CO2 to biomass, and this biomass is primarily composed of lignocellulose which is the most abundant natural biopolymer and a potential feedstock for fuel and chemical production. The stringency and complexity of the cellulosic structures have resulted in the evolution of a wide array of cellulose degrading enzymes. Microorganisms with the potential to degrade cellulose produce enzymes with cellulolytic and non-cellulolytic domains that work harmoniously on the substrate to break it down, thus known as cellulases. G. sulphuraria is an extremophile with the capacity to grow photosynthetically and heterotrophically. G. sulphuraria is a unicellular, red alga that belongs to the class Cyanidiophyceae. The Cyanidiophyceae class members are known to be extremophiles in nature and are believed to be the most acidophilic photosynthetic organisms known so far. Interestingly G. sulphuraria has been previously documented to grow on a chemically diverse set of carbon substrates. This ability to use a variety of carbon substrates is linked to possessing a diverse metabolic potential. In the current study, an important paradigm of adaptation was investigated, highlighting G. sulphuraria’s natural ability to produce cellulolytic enzymes under low pH and high-temperature conditions. To carry out this study, two approaches were adopted. Firstly, the in-silico analysis which includes genome analysis for identifying genes involved in cellulose degradation homology modeling, and molecular docking was performed. Then wet lab experiments were carried out including evaluation of carbon substrate utilization, and cellulase activity screening.The in-silico analysis of the G. sulphuraria genome showed the presence of nine genes encoding β-Galactosidase. A homology model of the β-galactosidase domain of Galdieria sulphuraria was built and its molecular binding with cellopentaose yields a docking score of -8.95 kcal/mol. G. sulphuraria was grown under mixotrophic and heterotrophic conditions, we observed a 1.5-2 X increase in specific growth rates towards α and β-glycosidic linked carbohydrate polymers over autotrophic conditions. The exogenous carbohydrate substrates glucose and maltotriose were found to be best for G. sulphuraria’s growth at pH 2.5 under mixotrophic conditions. This observation is further characterized by performing different cellulolytic enzyme assays that include congo red clearing assay, DNS enzyme assay, cellulase assay, and substrate-specific fluorometric enzyme assays. The cellulase assay showed pH 4.0 to be the optimal pH for G. sulphuraria’s cellulase activity. We propose the mechanism of carbohydrate utilization by G. sulphuraria involves sensing carbon substrate in the cell’s vicinity, and induction of biosynthesis of cellulases and sugar transporters. Two types of enzymes are produced, surface-bound and secretory, that hydrolyze the alpha and beta carbohydrates into simpler sugars. The hydrolyzed product is imported into the cell through sugar transporters.

ISBN: 9798374406191Subjects--Topical Terms:

599573
Biology.
Subjects--Index Terms:

Algae

Investigating Enzymatic Activity Towards Alpha and Beta Glycosidic Linkages by the Unicellular Extremophilic Red Alga Galdieria sulphuraria.
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506 $a This item must not be sold to any third party vendors.
520 $a Plants convert sunlight and CO2 to biomass, and this biomass is primarily composed of lignocellulose which is the most abundant natural biopolymer and a potential feedstock for fuel and chemical production. The stringency and complexity of the cellulosic structures have resulted in the evolution of a wide array of cellulose degrading enzymes. Microorganisms with the potential to degrade cellulose produce enzymes with cellulolytic and non-cellulolytic domains that work harmoniously on the substrate to break it down, thus known as cellulases. G. sulphuraria is an extremophile with the capacity to grow photosynthetically and heterotrophically. G. sulphuraria is a unicellular, red alga that belongs to the class Cyanidiophyceae. The Cyanidiophyceae class members are known to be extremophiles in nature and are believed to be the most acidophilic photosynthetic organisms known so far. Interestingly G. sulphuraria has been previously documented to grow on a chemically diverse set of carbon substrates. This ability to use a variety of carbon substrates is linked to possessing a diverse metabolic potential. In the current study, an important paradigm of adaptation was investigated, highlighting G. sulphuraria’s natural ability to produce cellulolytic enzymes under low pH and high-temperature conditions. To carry out this study, two approaches were adopted. Firstly, the in-silico analysis which includes genome analysis for identifying genes involved in cellulose degradation homology modeling, and molecular docking was performed. Then wet lab experiments were carried out including evaluation of carbon substrate utilization, and cellulase activity screening.The in-silico analysis of the G. sulphuraria genome showed the presence of nine genes encoding β-Galactosidase. A homology model of the β-galactosidase domain of Galdieria sulphuraria was built and its molecular binding with cellopentaose yields a docking score of -8.95 kcal/mol. G. sulphuraria was grown under mixotrophic and heterotrophic conditions, we observed a 1.5-2 X increase in specific growth rates towards α and β-glycosidic linked carbohydrate polymers over autotrophic conditions. The exogenous carbohydrate substrates glucose and maltotriose were found to be best for G. sulphuraria’s growth at pH 2.5 under mixotrophic conditions. This observation is further characterized by performing different cellulolytic enzyme assays that include congo red clearing assay, DNS enzyme assay, cellulase assay, and substrate-specific fluorometric enzyme assays. The cellulase assay showed pH 4.0 to be the optimal pH for G. sulphuraria’s cellulase activity. We propose the mechanism of carbohydrate utilization by G. sulphuraria involves sensing carbon substrate in the cell’s vicinity, and induction of biosynthesis of cellulases and sugar transporters. Two types of enzymes are produced, surface-bound and secretory, that hydrolyze the alpha and beta carbohydrates into simpler sugars. The hydrolyzed product is imported into the cell through sugar transporters.
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