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

1
College of Medicine, Hangzhou Normal University, Hangzhou 311121, China
2
Department of Microbiology, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou 310051, China
3
College of Animal Sciences and Technology, Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China
*
Authors to whom correspondence should be addressed.
Mar. Drugs 2018, 16(5), 158; https://doi.org/10.3390/md16050158
Received: 25 March 2018 / Revised: 30 April 2018 / Accepted: 7 May 2018 / Published: 11 May 2018
An alginate lyase encoding gene sagl from Flavobacterium sp. H63 was codon optimized and recombinantly expressed at high level in P.pastoris through high cell-density fermentation. The highest yield of recombinant enzyme of sagl (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 P. pastoris 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 P. pastoris system. View Full-Text
Keywords: Pichia pastoris; alginate lyase; expression; oligosaccharide Pichia pastoris; alginate lyase; expression; oligosaccharide
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MDPI and ACS Style

Li, H.; Wang, S.; Zhang, Y.; Chen, L. High-Level Expression of a Thermally Stable Alginate Lyase Using Pichia pastoris, Characterization and Application in Producing Brown Alginate Oligosaccharide. Mar. Drugs 2018, 16, 158. https://doi.org/10.3390/md16050158

AMA Style

Li H, Wang S, Zhang Y, Chen L. High-Level Expression of a Thermally Stable Alginate Lyase Using Pichia pastoris, Characterization and Application in Producing Brown Alginate Oligosaccharide. Marine Drugs. 2018; 16(5):158. https://doi.org/10.3390/md16050158

Chicago/Turabian Style

Li, Haifeng, Shuling Wang, Yunyi Zhang, and Liehuan Chen. 2018. "High-Level Expression of a Thermally Stable Alginate Lyase Using Pichia pastoris, Characterization and Application in Producing Brown Alginate Oligosaccharide" Marine Drugs 16, no. 5: 158. https://doi.org/10.3390/md16050158

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