Abstract
Alginate is one of the most abundant polysaccharides in algae. Alginate lyase degrades alginate through a β-elimination mechanism to produce alginate oligosaccharides with special bioactivities. Improving enzyme activity and thermal stability can promote the application of alginate lyase in the industrial preparation of alginate oligosaccharides. In this study, the recombinant alginate lyase cAlyM and its thermostable mutant 102C300C were expressed and characterized in Pichia pastoris. The specific activities of cAlyM and 102C300C were 277.1 U/mg and 249.6 U/mg, respectively. Both enzymes showed maximal activity at 50 °C and pH 8.0 and polyG preference. The half-life values of 102C300C at 45 °C and 50 °C were 2.6 times and 11.7 times the values of cAlyM, respectively. The degradation products of 102C300C with a lower degree of polymerization contained more guluronate. The oligosaccharides with a polymerization degree of 2–4 were the final hydrolytic products. Therefore, 102C300C is potentially valuable in the production of alginate oligosaccharides with specific M/G ratio and molecular weights.
Highlights
Alginate is a natural anionic polysaccharide and one of the main structural components of brown algae, accounting for approximately 40% of the algal dry weight [1]
To obtain a safe alginate lyase with high enzymatic activity and good thermal stability, the alginate lyase cAlyM and its thermostable mutant 102C300C were expressed in P. pastoris, and the enzymatic properties of these two enzymes were characterized and compared in detail
The degradation products were analyzed by high performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS)
Summary
Alginate is a natural anionic polysaccharide and one of the main structural components of brown algae, accounting for approximately 40% of the algal dry weight [1]. Alginate is an unbranched binary copolymer comprised of β-D-mannuronate (M) and α-L-guluronate (G) units These units appear in three types of blocks: poly β-D-mannuronate (polyM), poly α-L-guluronate (polyG), and a heteropolymer (polyMG). We previously reported that an alginate lyase cAlyM with high enzymatic activity from Microbulbifer sp. P. pastoris expression system has significant advantages in the production of many recombinant proteins [19], but there are few reports of alginate lyases expressed in P. pastoris [20]. Due to the low activity and poor thermal stability of alginate lyases, their applications in the production of AOS have been limited [21]. To obtain a safe alginate lyase with high enzymatic activity and good thermal stability, the alginate lyase cAlyM and its thermostable mutant 102C300C were expressed in P. pastoris, and the enzymatic properties of these two enzymes were characterized and compared in detail. The degradation products were analyzed by high performance liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS)
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