Abstract:Low-temperature-adapted alginate lyase has unique advantages for alginate oligosaccharide preparation and brown algae processing. Producing a low-temperature-adapted alginate lyase with better stability remains an urgent need for industrial applications. In this study, to screen for a low-temperature- adapted alginate lyase with good stability, we identified and isolated marine bacteria, by cloning and analyzing enzyme-encoding gene and secretory expression and purification of enzyme. The effects of different factors on enzyme activity and stability, as well as analysis of enzymatic hydrolysis products were evaluated. Results showed that the marine bacterium SJ-H-12 is capable of secreting low- temperature-adapted alginate lyase when cultivated with alginate as the sole carbon source. An evolutionary tree was constructed based on 16S rDNA sequences, and the strain was identified as Yangia sp. SJ-H-12. Furthermore, the gene encoding the enzyme ALYYA was identified as belonging to the PL5 alginate lyase family. The gene was secreted and expressed in Yarrowia lipolytica, a food-grade host. The activity of recombinant ALYYA reached 34.2 U/mL; the molecular weight of ALYYA was about 39.0 kDa, and it had a strong PolyM preference. ALYYA showed more than 80% vitality at 25℃~35℃, with peak vitality at 30℃; it demonstrated good stability in the pH range of 5.0~10.0, with more than 60% vitality remaining after incubation. NaCl could activate the activity of ALYYA at a concentration of 0~2.0 mol/L. The products of ALYYA-degraded alginate are mainly disaccharides and small amounts of monosaccharides and trisaccharides, resulting from its endoalginate lyase activity. In summary, a low-temperature-adapted alginate lyase-producing bacterium was screened, and most of the alginate lyases found were mesophilic enzymes. This study showed that ALYYA is a typical low-temperature- adapted alginate lyase with excellent enzyme activity and stability, providing reference data for the screening and industrial development and utilization of low-temperature-adapted alginate lyase.
