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
Degree Grantor
University of Nevada, Las Vegas
Language
English
Repository Citation
Covington, Jonathan, "Biochemical Characterization of Fsa1572 from Fervidibacter Sacchari, the First Hyperthermophilic GH50 with Β-1, 4-Glucanase Activity" (2023). UNLV Theses, Dissertations, Professional Papers, and Capstones. 4664.
https://digitalscholarship.unlv.edu/thesesdissertations/4664
Rights
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