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Item type:Publication, Access status: with files , Comparative analysis and biochemical characterization of two endo-β-1,3-glucanases from the thermophilic bacterium fervidobacterium sp.(Multidisciplinary Digital Publishing Institute (MDPI), 2019-10-01); ; ; ; Laminarinases exhibit potential in a wide range of industrial applications including the production of biofuels and pharmaceuticals. In this study, we present the genetic and biochemical characteristics of FLamA and FLamB, two laminarinases derived from a metagenomic sample from a hot spring in the Azores. Sequence comparison revealed that both genes had high similarities to genes from Fervidobacterium nodosum Rt17-B1. The two proteins showed sequence similarities of 62% to each other and belong to the glycoside hydrolase (GH) family 16. For biochemical characterization, both laminarinases were heterologously produced in Escherichia coli and purified to homogeneity. FLamA and FLamB exhibited similar properties and both showed highest activity towards laminarin at 90 °C and pH 6.5. The two enzymes were thermostable but differed in their half-life at 80 °C with 5 h and 1 h for FLamA and FLamB, respectively. In contrast to other laminarinases, both enzymes prefer β-1,3-glucans and mixed-linked glucans as substrates. However, FLamA and FLamB differ in their catalytic efficiency towards laminarin. Structure predictions were made and showed minor differences particularly in a kink adjacent to the active site cleft. The high specific activities and resistance to elevated temperatures and various additives make both enzymes suitable candidates for application in biomass conversion.Publicationtype: Journal ArticleScopus© Citations 7 250 403 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Characterization of a Theme C Glycoside Hydrolase Family 9 Endo-Beta-Glucanase from a Biogas Reactor Metagenome(2018-10-01); ; ; ; From a biogas reactor metagenome an ORF (bpcel9A) encoding a bacterial theme C glycoside hydrolase family 9 (GH9) enzyme was recombinantly produced in E. coli BL21 pQE-80L. BPCel9A exhibited ≤ 55% identity to annotated sequences. Subsequently, the enzyme was purified to homogeneity by affinity chromatography. The endo-beta-glucanase BPCel9A hydrolyzed the beta-1,3–1,4-linked barley beta-glucan with 24 U/mg at 30 °C and pH 6.0. More than 62% of activity was measured between 10 and 40 °C. Lichenan and xyloglucan were hydrolyzed with 67% and 40% of activity, respectively. The activity towards different substrates varied with different temperatures. However, the enzyme activity on CMC was extremely low (> 1%). In contrast to BPCel9A, most GH9 glucanases act preferably on crystalline or soluble cellulose with only side activities towards related substrates. The addition of calcium or magnesium enhanced the activity of BPCel9A, especially at higher temperatures. EDTA inhibited the enzyme, whereas EGTA had no effect, suggesting that Mg2+ may adopt the function of Ca2+. BPCel9A exhibited a unique substrate spectrum when compared to other GH9 enzymes with great potential for mixed-linked glucan or xyloglucan degrading processes at moderate temperatures.Publicationtype: Journal Article146 - Some of the metrics are blocked by yourconsent settings
Item type:Publication, Access status: without files , Characterization of two novel heat-active α-galactosidases from thermophilic bacteriaTwo genes (agal1 and agal2) encoding α-galactosidases were identified by sequence-based screening approaches. The gene agal1 was identified from a data set of a sequenced hot spring metagenome, and the deduced amino-acid sequence exhibited 99% identity to an α-galactosidase from the thermophilic bacterium Dictyoglomus thermophilum. The gene agal2 was identified from the whole genome sequence of the thermophile Meiothermus ruber. The amino-acid sequences exhibited structural motifs typical for glycoside hydrolase (GH) family 36 members and were also differentiated into different subgroups of this family. Recombinant production of the heat-active GH36b enzyme Agal1 (87 kDa) and GH36bt enzyme Agal2 (57 kDa) was carried out in E. coli. Agal1 exhibited a specific activity of 1502.3 U/mg at 80 °C, pH 6.5, and Agal2 225.4 U/mg at 60–70 °C, pH 6.5. Half-lives of 14 h (Agal1) and 39 h (Agal2) were obtained at 50 °C, and Agal1 showed half-lives of 4 and 2 h at 70 and 80 °C, respectively. In addition to the natural substrates melibiose, raffinose, and stachyose, 4NP α-d-galactopyranoside was hydrolyzed. Galactose was also liberated from locust bean gum. Both heat-active enzymes are attractive candidates for application in food and feed industry for high-temperature processes for the degradation of raffinose family oligosaccharides.Publicationtype: Journal Article195