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Functional characterization of carbohydrate-active enzymes from marine bacteria

  • In contrast to its terrestrial counterpart, the metabolic degradation of marine polysaccharides is underexplored. This work aimed to functionally characterize ulvan- and xylan-degrading enzymes from marine Bacteroidetes in order to clarify the metabolic degradation pathway. For the provision of a broad polysaccharide substrate spectrum, ulvan from several different algal sources was extracted to be used in further characterization experiments. The structural differences of these ulvans could be demonstrated by enzymatic degradation with ulvan-active enzymes. In order to clarify the synergistic catalytic effects of polysaccharide sulfatases with GHs in the degradation process of ulvan, several putative sulfatases from F. agariphila were produced recombinantly in E. coli. For that, a coexpression with an FGE encoding gene was required. It could be demonstrated that several glycoside hydrolases are inhibited, if their substrate is sulfated at the cleavage position and that a previous desulfation using one of the sulfatases enabled the further degradation. Some of the sulfatases showed an endolytic or exolytic cleavage behavior like reported for several GHs. With the combined catalytic activities, it was possible to successfully elucidate the complex ulvan degradation mechanism for the first time, which enables the use of ulvan as a biotechnological source for the production of fine chemicals and pharmaceuticals. This degradation mechanism was shown to be complemented by an alternative pathway that helps with the degradation of uronic acid-containing oligosaccharides. Here, the synergistic effects of a multimodular enzyme containing a sulfatase and rhamnosidase domain were demonstrated. Furthermore, the first dehydratase participating in the degradation of oligosaccharides was revealed. The functional characterization of putative xylan-targeting PULs from two Flavobacteriia revealed the existence of marine endolytic and exolytic xylanases. The enzymes of these PULs were produced recombinantly in E. coli and were used in biocatalysis reactions on xylan from beechwood, xylan from P. palmata or commercial xylooligosaccharide standards. Further side chain-active GHs were found to exclusively be active on either standards or xylan. The great variation of genetic equipment was demonstrated by comparing the enzyme activities of these PUL structures assuming different ecological adaptations of these organisms especially, because these PULs do not code for any putative sulfatases, which is uncommon for PULs targeting xylan. A different degradation behavior of the investigated enzymes suggested a preferred conversion of β-1,4-linked xylan, potentially present in some microalgae. The acquired insight of the metabolic ulvan and xylan utilization greatly expands the scientific knowledge about the ecologic interplays in marine environments concerning the polysaccharide utilization. It indicates the necessity of backup mechanisms for metabolic processes in order to get access to complex marine carbon sources in nature. Several small degradation cascades complement each other to break down substrate compounds to monomeric level for the use of structurally diverse polysaccharides. This expands the insights into the metabolic processes in the degradation of marine polysaccharides, which are an important part of the understanding of the ecological interactions in aquatic habitats and the ocean’s carbon cycle. The characterization of ulvan- and xylan-active enzymes and the clarification of their substrate scopes allow to use these enzymes in future production of carbohydrate-derived chemical products for many industrial applications, making it possible to use algal waste for recycling to high value materials with even beneficial effect for the environment.
  • Im Gegensatz zum terrestrischen Pendant ist der metabolische Abbau von marinen Polysacchariden wenig erforscht. Ziel dieser Arbeit war die funktionelle Charakterisierung der Ulvan- und Xylan-abbauenden Enzyme aus marinen Bacteroidetes zur Aufklärung des metabolischen Abbauprozesses. Die Charakterisierung von ulvan- und xylan-aktiven Enzymen und die Klärung ihres Substratspektrums ermöglicht die Verwendung dieser Enzyme in der Produktion von aus Kohlenhydraten gewonnenen Feinchemikalien für viele industrielle Anwendungen, wodurch es möglich wird, Algenabfälle für das Recycling zu hochwertigen Materialien, mit sogar positiven Auswirkungen auf die Umwelt, zu nutzen.

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Author: Marcus Bäumgen
Title Additional (German):Funktionelle Charakterisierung von Kohlenhydrat-aktiven Enzymen aus marinen Bakterien
Referee:Prof. Dr. Uwe T. Bornscheuer, Prof. Dr. Harry Brumer
Advisor:Prof. Dr. Uwe T. Bornscheuer
Document Type:Doctoral Thesis
Year of Completion:2020
Date of first Publication:2020/07/20
Granting Institution:Universität Greifswald, Mathematisch-Naturwissenschaftliche Fakultät
Date of final exam:2020/06/24
Release Date:2020/07/20
GND Keyword:Biochemie
Faculties:Mathematisch-Naturwissenschaftliche Fakultät / Institut für Chemie und Biochemie
DDC class:500 Naturwissenschaften und Mathematik / 500 Naturwissenschaften