Biochemical characterization and elucidation the degradation pattern of a new cold-adapted and Ca2+activated alginate lyase for efficient preparation of alginate oligosaccharides

Alginate lyase as the excellent tool enzyme could produce alginate oligosaccharides under mild reaction con-ditions and good specificity. Herein, the new Ca2+ activated alginate lyase gene (TAPL7B) with metal ions stability and cold-adapted was cloned and expressed heterologously from Thalassotalea algicola. TAPL7B was identified as a polysaccharide lyases family 7 (PL7) alginate lyase with endolytic activity. TAPL7B is a polyM-preferred and cold-adapted enzyme with an optimal temperature of 20 degrees C and pH 11.0. The pH stability anal-ysis indicated that TAPL7B possessed broad pH tolerant range (4.0-12.0) and preferred alkaline environments. Interestingly, the enzymatic activity of TAPL7B was greatly activated by Ca2+ and it increased 9-fold in the presence of 1.2 mM Ca2+, which is rare in other alginate lyases. The activity of TAPL7B is not only activated by Ca2+, but also by some other metal ions (such as K+, Na+, Ni+, Cu2+, Mg2+, Zn2+, Co2+, Fe3+). The results of fast protein liquid chromatography (FPLC) and electrospray ionization mass spectrometry (ESI-MS) indicate that TAPL7B adopts an endo-action mode on all three substrates, and the products are all oligosaccharides with a degree of polymerization of 2-6. Therefore, the cold adaptability, broad pH stability, excellent metal ions sta-bility, and Ca2+ activation of TAPL7B make it a potential industrial biocatalyst for the preparation of alginate oligosaccharides.