High-Level Expression of a Thermally Stable Alginate Lyase Using Pichia pastoris, Characterization and Application in Producing Brown Alginate Oligosaccharide

Abstract

An alginate lyase encoding gene <i>sagl</i> from <i>Flavobacterium</i> sp. H63 was codon optimized and recombinantly expressed at high level in <i>P.pastoris</i> through high cell-density fermentation. The highest yield of recombinant enzyme of <i>sagl</i> (rSAGL) in yeast culture supernatant reached 226.4 μg/mL (915.5 U/mL). This was the highest yield record of recombinant expression of alginate lyase so far. The rSAGL was confirmed as a partially glycosylated protein through EndoH digestion. The optimal reaction temperature and pH of this enzyme were 45 °C and 7.5; 80 mM K⁺ ions could improve the catalytic activity of the enzyme by 244% at most. rSAGL was a thermal stable enzyme with T50¹⁵ of 57⁻58 °C and T50³⁰ of 53⁻54 °C. Its thermal stability was better than any known alginate lyase. In 100 mM phosphate buffer of pH 6.0, rSAGL could retain 98.8% of the initial activity after incubation at 50 °C for 2 h. Furthermore, it could retain 61.6% of the initial activity after 48 h. The specific activity of the purified rSAGL produced by <i>P. pastoris</i> attained 4044 U/mg protein, which was the second highest record of alginate lyase so far. When the crude enzyme of the rSAGL was directly used in transformation of sodium alginate with 40 g/L, 97.2% of the substrate was transformed to di, tri, tetra brown alginate oligosaccharide after 32 h of incubation at 50 °C, and the final concentration of reducing sugar in mixture reached 9.51 g/L. This is the first report of high-level expression of thermally stable alginate lyase using <i>P. pastoris</i> system.