Characterization of a Theme C Glycoside Hydrolase Family 9 Endo-Beta-Glucanase from a Biogas Reactor Metagenome

• Published in: The Protein Journal Vol. 37; no. 5; pp. 454 - 460
• Main Authors: Schröder, Carola, Burkhardt, Christin, Busch, Philip, Schirrmacher, Georg, Claren, Jörg, Antranikian, Garabed
• Format: Journal Article
• Language: English
• Published: New York Springer US 01.10.2018; Springer; Springer Nature B.V
• Subjects: Affinity chromatography; Animal Anatomy; Barley; Biochemistry; Biofuels; Biogas; Biomass energy; Bioorganic Chemistry; Bioreactors; Calcium; Calcium ions; Cellulose; Chemistry; Chemistry and Materials Science; E coli; Enzymatic activity; Enzyme activity; Enzymes; Ethylenediaminetetraacetic acid; Ethylenediaminetetraacetic acids; Glucan; Glucans - chemistry; Glycoside hydrolase; Glycoside Hydrolases - chemistry; Glycoside Hydrolases - genetics; Histology; Homogeneity; Hydrolase; Hydrolases; Magnesium; Metagenome; Morphology; Organic Chemistry; Reactors; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Substrates; Xylans - chemistry; Xyloglucan; β-Glucan; Glycoside hydrolase family 9; Barley beta-glucan; Magnesium; Calcium; Endo-beta-glucanase
• ISSN: 1572-3887; 1573-4943; 1875-8355
• DOI: 10.1007/s10930-018-9787-5

From a biogas reactor metagenome an ORF ( bp _ cel9A) encoding a bacterial theme C glycoside hydrolase family 9 (GH9) enzyme was recombinantly produced in E. coli BL21 pQE-80L. BP_Cel9A exhibited ≤ 55% identity to annotated sequences. Subsequently, the enzyme was purified to homogeneity by affinity chromatography. The endo-beta-glucanase BP_Cel9A 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 BP_Cel9A, 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 BP_Cel9A, especially at higher temperatures. EDTA inhibited the enzyme, whereas EGTA had no effect, suggesting that Mg 2+ may adopt the function of Ca 2+ . BP_Cel9A 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.