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Title:Alginate and laminarin degrading enzymes from Vibrio splendidus and Vibrio breoganii
Author(s):Badur, Ahmet H
Director of Research:Rao, Christopher V
Doctoral Committee Chair(s):Rao, Christopher V
Doctoral Committee Member(s):Jin, Yong-Su; Schroeder, Charles M; Zhao, Huimin
Department / Program:Chemical & Biomolecular Engineering
Discipline:Chemical Engineering
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):alginate
alginate lyase
laminarin
laminarinases
brown seaweed
macroalgae
glycoside hydrolase
polysaccharide lyase
vibrio
vibrio splendidus 12B01
vibrio breoganii 1C10
vibrio splendidus 13B01
Abstract:The marine bacteria V. splendidus 12B01, V. splendidus 13B01, and V. breoganii 1C10 metabolize brown seaweeds. Brown seaweeds have many advantages over terrestrial feedstocks, including fast growth and non-recalcitrant carbon feedstocks, so brown seaweeds are an attractive target for industrial fermentation. Alginate and laminarin are two major components of brown seaweeds, comprising up to 60% of the dry weight. Alginate is a polysaccharide consisting of the 1,4-linked epimers α- L - guluronate and β- D -mannuronate. Laminarin is a storage glucan consisting of β-1,3- and β-1,6-linked glucose monomers. In order to utilize these carbon sources, many organisms express enzymes that cleave the bonds linking the constituent monomers within alginate and laminarin. These enzymes are called alginate lyases and laminarinases. V. splendidus 12B01, V. splendidus 13B01, and V. breoganii 1C10 each contain between four and twelve putative alginate lyases. We have over-expressed and purified 21 alginate lyases from these organisms and determined under what conditions these enzymes are most active. We found these enzymes are optimally active between pH 6.5 and 10 and between 20 to 30 °C. Additionally, these enzymes were broadly salt tolerant between 50 mM and 1 M NaCl. We also determined the enzyme kinetics for these enzymes and found K m parameters towards alginate between 22 and 300 µM alginate. The computed turnover numbers range from 0.6 to 18 s -1 . Alginate lyases have preferential specificity toward specific dyads within alginate. We found alginate lyases with all potential dyad specificities: G-G, G-M, M-G, and M-M specific alginate lyases. Having characterized the alginate lyases in 12B01, 13B01, and 1C10, we can begin to understand the metabolism of alginate by these organisms. 12B01 was found to poorly degraded and metabolize alginate, and we found 12B01 to express and secrete its enzymes at low levels. In addition we found the 12B01 alginate lyases have low enzymatic activity and narrow dyad specificity. 13B01 was found to degrade and metabolize alginate at high levels. We identified the presence of a unique enzyme to 13B01, which upon knockout, resulted in eight-fold less secreted alginate lyase activity. We found that this high activity enzyme allows 13B01 to degrade alginate efficiently and iii then metabolize the liberated monomers of alginate. 1C10 contains eleven alginate lyases within its genome. While this organism has 70% of the 13B01 secreted alginate lyase activity, we found that the 1C10 lyases do not have large enzymatic activity. Rather, the concerted action of enzymes with broad dyad specificity allow 1C10 to efficiently degrade alginate. Overall, we identified several attractive alginate lyases for future metabolic engineering to produce biofuels from alginate. While this would require expression of additional metabolic pathways, we present the first step to the industrial utilization of alginate. V. breoganii 1C10 contains four laminarinases which we over-expressed and purified. These enzymes had optimal enzymatic activity between pH 6.5 and 8.0 and between 25 and 40 °C. These enzymes were shown to have especially broad tolerance to salt between 50 mM and 1 M NaCl. The 1C10 laminarinases had K m parameters towards laminarin between 3.4 and 6 mM laminarin. These enzymes also had computed turnover numbers ranging from 0.69 to 6.1 s -1 . As the degraded monomer of laminarin is glucose, these enzymes can be expressed in fermentative hosts with no additional metabolic pathways, so laminarin utilization is an attractive target for biofuel production.
Issue Date:2015-11-04
Type:Thesis
URI:http://hdl.handle.net/2142/89188
Rights Information:Copyright 2015 Ahmet Hakan Badur
Date Available in IDEALS:2016-03-02
Date Deposited:2015-12


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