Award Date

May 2023

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Life Sciences

First Committee Member

Brian Hedlund

Second Committee Member

Helen Wing

Third Committee Member

Kurt Regner

Fourth Committee Member

Ernesto Abel-Santos

Number of Pages

68

Abstract

The bacterium Fervidibacter sacchari is an aerobic hyperthermophile that catabolizes various polysaccharides and is the only cultivated member of the class Fervidibacteria within the phylum Armatimonadota. Among its glycoside hydrolase (GH) cache is an enzyme from GH family 50 (GH50), an understudied family with only 25 characterized representatives and two known activities from 1,518 predicted members in the Carbohydrate-Active EnZyme (CAZy) Database. Here, we expressed, purified, and functionally characterized an extracellular GH50 from F. sacchari called Fsa1572. Using colorimetric assays, we show it has novel β-1,4-glucanase activity and only weak agarose activity that is typical for GH50 enzymes. The purified enzyme has a wide temperature range of 60-95 °C (optimal 80 °C), making it the first characterized hyperthermophilic representative of GH50. The enzyme also has a broad pH range of at least 5.5-11 (optimal 6.5-10). Fsa1572 possesses KM and kcat parameters of 12.6 mM and 4.62 s-1, respectively. Finally, a phylogenetic analysis of Fsa1572 and other GH50 enzymes revealed a unique phylogenetic position for Fsa1572 that is distant from other characterized enzymes and related to yet-uncharacterized GH50s from genomes of thermophilic archaea. Fsa1572 is the first characterized GH enzyme of F. sacchari and is both functionally and phylogenetically novel.

Keywords

Gene cloning; GH50; Glycoside hydrolase; Hyperthermophilic bacterium; β-glucan; β-glucanase

Disciplines

Biochemistry | Biology | Microbiology

File Format

pdf

Degree Grantor

University of Nevada, Las Vegas

Language

English

Rights

IN COPYRIGHT. For more information about this rights statement, please visit http://rightsstatements.org/vocab/InC/1.0/

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