scholarly journals Mechanistic basis for understanding the dual activities of the bifunctional Azotobacter vinelandii mannuronan C-5 epimerase and alginate lyase AlgE7

2021 ◽  
Author(s):  
Margrethe Gaardløs ◽  
Tonje Marita Bjerkan Heggeset ◽  
Anne Tøndervik ◽  
David Tezé ◽  
Birte Svensson ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Functional Properties ◽  
Catalytic Mechanism ◽  
Azotobacter Vinelandii ◽  
Brown Seaweed ◽  
Alginate Lyase ◽  
High Sequence Identity ◽  
Monomer Composition ◽  
Lyase Activity ◽  
Mechanistic Basis

The structure and functional properties of alginates are dictated by the monomer composition and molecular weight distribution. Mannuronan C-5 epimerases determine the monomer composition by catalysing the epimerization of β- d -mannuronic acid residues (M) into α- l -guluronic acid residues (G). The molecular weight is affected by alginate lyases, which catalyse a β-elimination mechanism that cleaves alginate chains. The reaction mechanisms for the epimerization and lyase reactions are similar and some enzymes can perform both reactions. These dualistic enzymes share high sequence identity with mannuronan C-5 epimerases without lyase activity. The mechanism behind their activity and the amino acid residues responsible for it are still unknown. We investigate mechanistic determinants involved in the bifunctional epimerase and lyase activity of AlgE7 from Azotobacter vinelandii . Based on sequence analyses, a range of AlgE7 variants were constructed and subjected to activity assays and product characterization by NMR. Our results show that calcium promotes lyase activity whereas NaCl reduces the lyase activity of AlgE7. By using defined poly-M and poly-MG substrates, the preferred cleavage sites of AlgE7 were found to be M|XM and G|XM, where X can be either M or G. From the study of AlgE7 mutants, R148 was identified as an important residue for the lyase activity, and the point mutant R148G resulted in an enzyme with only epimerase activity. Based on the results obtained in the present study we suggest a unified catalytic reaction mechanism for both epimerase and lyase activity where H154 functions as the catalytic base and Y149 as the catalytic acid. Importance Post-harvest valorisation and upgrading of algal constituents is a promising strategy in the development of a sustainable bioeconomy based on algal biomass. In this respect, alginate epimerases and lyases are valuable enzymes for tailoring of the functional properties of alginate, a polysaccharide extracted from brown seaweed with numerous applications in food, medicine, and material industries. By providing a better understanding of the catalytic mechanism and of how the two enzyme actions can be altered by changes in reaction conditions, this study opens for further applications of bacterial epimerases and lyases in enzymatic tailoring of alginate polymers.

scholarly journals A mechanistic basis for understanding the dual activities of the bifunctional Azotobacter vinelandii mannuronan C-5 epimerase and alginate lyase AlgE7

2021 ◽  
Author(s):  
Margrethe Gaardløs ◽  
Tonje Marita Bjerkan Heggeset ◽  
Anne Tøndervik ◽  
David Tezé ◽  
Birte Svensson ◽  
...  
Keyword(s):  
Reaction Mechanism ◽  
Azotobacter Vinelandii ◽  
Alginate Lyase ◽  
Cleavage Sites ◽  
High Sequence Identity ◽  
Preferential Cleavage ◽  
Product Characterization ◽  
Lyase Activity ◽  
Mechanistic Basis ◽  
Alginate Lyases

Mannuronan C-5 epimerases and alginate lyases are important enzymes for tailoring of the functional properties of alginate. The reaction mechanisms for the epimerization and lyase reactions are similar, and some enzymes, like AlgE7 from Azotobacter vinelandii, can perform both reactions. These enzymes share high sequence identity with mannuronan C-5 epimerases without lyase activity, and the mechanism behind their dual activity is not understood. In this study, we investigate mechanistic determinants involved in the bifunctional alginate lyase and epimerase activity of AlgE7. Based on sequence analyses a range of AlgE7 variants were constructed and subjected to activity assays and product characterization by NMR. Calcium promotes lyase activity whereas NaCl reduces the lyase activity of AlgE7. By using well-defined alginate substrates, the preferential cleavage sites of AlgE7 are found to be M|XM and G|XM. From the study of variants, it was found that R148 is particularly important for the lyase activity of AlgE7, and we obtained pure epimerase variants. Furthermore, the results suggest a catalytic reaction mechanism, with H154 as the catalytic base and Y149 as the catalytic acid. This study opens for further applications of alginate epimerases and lyases, by providing a better understanding of the reaction mechanism and how the two enzyme reactions can be altered by changes in reaction conditions.

Study on Structure and Tanning Properties of Copolymer of Resorcinol and 3,5-Dihydroxyl Benzoic Acid

2012 ◽  
Vol 487 ◽  
pp. 48-52
Author(s):  
Sheng Hua Lv ◽  
Gong Rui ◽  
Di Li
Keyword(s):  
Molecular Weight ◽  
Nuclear Magnetic Resonance ◽  
Fourier Transform ◽  
Infrared Spectroscopy ◽  
Gel Permeation Chromatography ◽  
Radical Copolymerization ◽  
Mass Ratio ◽  
Monomer Composition ◽  
Chrome Tanning ◽  
Gel Permeation

The radical copolymerization of resorcinol (RSC) and 3,5-dihydroxyl benzoic (DHBA) was carried out in water by the initiator of horseradish (HRP)/H2O2. It was discussed that the effects of monomer composition on the properties of the copolymer. The best monomer mass ratio of RSC:DHBA was 60:40 and the shrink temperature can reached to 88.5oC. The tanning result indicated that the copolymer of RSC and DHPA has particularly excellent tanning properties and can be served as leather tannage substitute for chrome tanning materials. And also the mechanism of the HRP initiated copolymerization was proposed. The structure and molecular weight of the copolymer was characterized by Fourier Transform Infrared spectroscopy (FTIR), Nuclear Magnetic Resonance (NMR) and Gel Permeation Chromatography (GPC). The copolymer can be as tannage and retannage in making leather process. The results showed that it has excellent tanning properties and retanning effects.

Crystallographic and Enzymic Investigations on the Role of Ser558, His610, and Asn614 in the Catalytic Mechanism of Azotobacter vinelandii Dihydrolipoamide Acetyltransferase (E2p)

Biochemistry ◽  
1995 ◽  
Vol 34 (13) ◽  
pp. 4287-4298 ◽  
Author(s):  
Joerg Hendle ◽  
Andrea Mattevi ◽  
Adrie H. Westphal ◽  
Johan Spee ◽  
Arie de Kok ◽  
...  
Keyword(s):  
Catalytic Mechanism ◽  
Azotobacter Vinelandii

scholarly journals Structure Characteristics, Biochemical Properties, and Pharmaceutical Applications of Alginate Lyases

Marine Drugs ◽  
2021 ◽  
Vol 19 (11) ◽  
pp. 628
Author(s):  
Shu-Kun Gao ◽  
Rui Yin ◽  
Xiao-Chen Wang ◽  
Hui-Ning Jiang ◽  
Xiao-Xiao Liu ◽  
...  
Keyword(s):  
Brown Algae ◽  
Catalytic Mechanism ◽  
Degradation Products ◽  
Structural Characteristics ◽  
Catalytic Efficiency ◽  
Alginate Lyase ◽  
Biochemical Properties ◽  
Structure And Property ◽  
Alginate Oligosaccharides ◽  
Alginate Lyases

Alginate, the most abundant polysaccharides of brown algae, consists of various proportions of uronic acid epimers α-L-guluronic acid (G) and β-D-mannuronic acid (M). Alginate oligosaccharides (AOs), the degradation products of alginates, exhibit excellent bioactivities and a great potential for broad applications in pharmaceutical fields. Alginate lyases can degrade alginate to functional AOs with unsaturated bonds or monosaccharides, which can facilitate the biorefinery of brown algae. On account of the increasing applications of AOs and biorefinery of brown algae, there is a scientific need to explore the important aspects of alginate lyase, such as catalytic mechanism, structure, and property. This review covers fundamental aspects and recent developments in basic information, structural characteristics, the structure–substrate specificity or catalytic efficiency relationship, property, molecular modification, and applications. To meet the needs of biorefinery systems of a broad array of biochemical products, alginate lyases with special properties, such as salt-activated, wide pH adaptation range, and cold adaptation are outlined. Withal, various challenges in alginate lyase research are traced out, and future directions, specifically on the molecular biology part of alginate lyases, are delineated to further widen the horizon of these exceptional alginate lyases.

scholarly journals Macrophage-stimulating activities of a novel low molecular weight saccharide fragment prepared from ascophyllan with alginate lyase

2020 ◽  
Vol 67 ◽  
pp. 103839
Author(s):  
Zedong Jiang ◽  
Gang Yu ◽  
Qingyun Bao ◽  
Xu Xu ◽  
Yanbing Zhu ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Alginate Lyase ◽  
Low Molecular Weight

scholarly journals Characterization of a New Intracellular Alginate Lyase with Metal Ions-Tolerant and pH-Stable Properties

Marine Drugs ◽  
2020 ◽  
Vol 18 (8) ◽  
pp. 416
Author(s):  
Yan Ma ◽  
Jie Li ◽  
Xin-Yue Zhang ◽  
Hao-Dong Ni ◽  
Feng-Biao Wang ◽  
...  
Keyword(s):  
Metal Ion ◽  
Brown Seaweed ◽  
Alginate Lyase ◽  
Industrial Applications ◽  
Alginate Oligosaccharides ◽  
Potential Applications ◽  
Pharmaceutical Industries ◽  
Ph Range ◽  
Alginate Lyases

Alginate lyases play an important role in alginate oligosaccharides (AOS) preparation and brown seaweed processing. Many extracellular alginate lyases have been characterized to develop efficient degradation tools needed for industrial applications. However, few studies focusing on intracellular alginate lyases have been conducted. In this work, a novel intracellular alkaline alginate lyase Alyw202 from Vibrio sp. W2 was cloned, expressed and characterized. Secretory expression was performed in a food-grade host, Yarrowia lipolytica. Recombinant Alyw202 with a molecular weight of approximately 38.3 kDa exhibited the highest activity at 45 °C and more than 60% of the activity in a broad pH range of 3.0 to 10.0. Furthermore, Alyw202 showed remarkable metal ion-tolerance, NaCl independence and the capacity of degrading alginate into oligosaccharides of DP2-DP4. Due to the unique pH-stable and high salt-tolerant properties, Alyw202 has potential applications in the food and pharmaceutical industries.

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