Cloning, expression, and characterization of a novel endo-type alginate lyase from Microbulbifer sp. BY17

BACKGROUND Alginate oligosaccharides (AOS), with various physiological effects, have been widely used in the food, agricultural, and pharmaceutical industries. The biological enzymatic method of preparing AOS, using alginate lyase, has more advantages compared with physical and chemical methods. Cloning and heterologously expressing alginate lyase are therefore very important. RESULTS A novel alginate lyase, BY17PV7, from Microbulbifer sp. BY17, isolated from Gracilaria, was cloned and expressed in Escherichia coli BL21(DE3). BY17PV7 was about 27 KDa. BY17PV7 showed the greatest activity (150.42 +/- 3.32 U/mg) at 43 degrees C and pH 8.9. It could be activated by Ca2+, Mn2+, Co2+, Fe3+, Na+, and inhibited by Mg2+, Zn2+, Ba2+, Cu2+, sodium dodecyl sulfate (SDS), ethylene diamine tetraacetic acid (EDTA). BY17PV7 had a wide range of substrate specificity and good degradation effects for poly beta-D-mannuronate (polyM) and poly alpha-L-guluronate (polyG), demonstrating that it is a bifunctional alginate lyase. The kinetic parameters showed that BY17PV7 had a greater affinity for polyG. BY17PV7 released AOS with a degree of polymerization (DP) of 3-4 in an endolytic manner from sodium alginate. Alginate oligosaccharides showed strong antioxidant ability of reducing Fe3+ and scavenging radicals such as hydroxyl, 2,2-azion-bia (3-ethylbenzo-thiazoline-6-sulfonic acid diammonium salt) (ABTS) and 2,2-diphenyl-1-picrylhydrazyl (DPPH). CONCLUSION A novel bifunctional alginate lyase, BY17PV7, was expressed and characterized in Escherichia coli BL21(DE3). The results were helpful for the analysis of the molecular mechanisms of degrading patterns in the polysaccharide lyase (PL) family. (c) 2022 Society of Chemical Industry.

Automated summary

A novel bifunctional alginate lyase, BY17PV7, was cloned and expressed in Escherichia coli BL21(DE3). BY17PV7 showed the greatest activity at 43 degrees C and pH 8.9, with a higher affinity for polyG. It exhibited a wide range of substrate specificity and good degradation effects. Furthermore, the released AOS by BY17PV7 showed strong antioxidant ability.