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

Alginate 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 K-m (0.0237 +/- 0.0004 mu M, 0.0105 +/- 0.0002 mg/mL) and k(cat)/K-m (1180.65 +/- 19.81 mu M-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 disaccharide and disaccharide respectively. The broad substrate specificity, endo-acting degradation pattern and high catalysis efficiency suggested that Aly7B_Wf could be utilizied as a potential tool for tailoring the size of alginate and preparing alginate oligosaccharides.