Cloning, expression and characterization of an endo-acting bifunctional alginate lyase of marine bacteria Wenyingzhuangia fucanilytica

Publication date: Available online 21 September 2018Source: Protein Expression and PurificationAuthor(s): Xiaojie Pei, Yaoguang Chang, Jingjing ShenAbstractAlginate is the major constituent of brown algae and a commercially important polysaccharide with wide applications. Alginate lyases are desired tools for degrading alginate. Based on the genome mining of marine bacterium Wenyingzhuangia funcanilytica, an alginate lyase Aly7B_Wf was discovered, cloned and expressed in Escherichia coli. Aly7B_Wf belonged to subfamily 6 of PL7 family. Its biochemical properties, kinetic constants, substrate specificity and degradation pattern were clarified. The enzyme is an endo-acting bifunctional alginate lyase, and preferably cleaved polymannuronate (polyM). The Km (0.0237 ± 0.0004 μM, 0.0105 ± 0.0002 mg/mL) and kcat/Km (1180.65 ± 19.81 μM−1 s−1, 2654.34 ± 44.54 mg−1 ml s−1) indicated relatively high substrate-binding affinity and catalysis efficiency of Aly7B_Wf. By using mass spectrometry, final products of alginate degraded by Aly7B_Wf were identified as alginate hexasaccharide to disaccharide, and final products of polyguluronate (polyG) and polyM were confirmed as tetrasaccharide to disaccharide. The most predominant oligosaccharide in the final products of polyG and polyM was trisaccharide and disaccharide respectively. The broad substrate specificity, endo-acting degradation pattern and high catalysis efficiency suggested that Aly7B_Wf c...
Source: Protein Expression and Purification - Category: Biochemistry Source Type: research