Elucidation of non-catalytic domain alterations effects on properties and action pattern of a novel alginate lyase

The discovery and characterization of new alginate lyases with high activity and stability have been the research topic in utilization of alginate biomass. Herein, we characterized a new Thalassotalea algicola-derived polyM-preferred PL6 family alginate lyase TAPL6 with two-domains (catalytic domain NTD and non-catalytic domain CTD). TAPL6 is a bifunctional enzyme that could endolytically degrade polyM, and could exolytically degrade polyG. It could degrade alginate in a hybrid action mode. The specific activities of polyM, polyG and sodium alginate of TAPL6 were 7174 U/mg, 4140.2 U/mg and 414.3 U/mg, respectively. The optimal temperature and pH for TAPL6 are 25 degrees C and 8.0, respectively. Deletion of CTD resulted in a loss of 99.8%-98.2% of TAPL6 activity and a decrease in pH adaptability, but greatly improved the activity and temperature stability above 30 degrees C. The CTD truncation is more resistant to metal ions than TAPL6, and the promotion effect of Ca2+ on the activity is stronger. Deletion of CTD results in a change in the action mode of TAPL6 to an endo-mode. In addition, the integrity of the linker between NTD and CTD is very important for the soluble expression of re-combinant protein, and the complete linker is beneficial to the catalytic activity of NTD. This study provides a new tool enzyme for the production of alginate oligosaccharides, and explores the role of the non-catalytic domain CTD in the PL6 family alginate lyases, providing a rational design basis for the PL6 family alginate ly-ases to change the enzymatic properties and action mode.

Automated summary

This study characterized a new alginate lyase TAPL6 with high activity and stability in degrading polyM and polyG. Deletion of the non-catalytic domain CTD affected the activity and action mode of the enzyme, while improving its stability. The integrity of the linker between NTD and CTD played an important role in the expression and catalytic activity of the recombinant protein.