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

Marine Drugs
Haifeng LiLiehuan Chen

Abstract

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 substrat...Continue Reading

References

Nov 1, 2012·Journal of Industrial Microbiology & Biotechnology·Lishuxin HuangYuguang Du
Jan 9, 2015·Preparative Biochemistry & Biotechnology·Michael M YueHongbiao Ding
Nov 7, 2017·International Journal of Biological Macromolecules·Benwei ZhuZhong Yao

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Citations

Jan 3, 2019·Critical Reviews in Biotechnology·Wan-Cang LiuPing Zhu
Mar 4, 2020·Marine Drugs·Maochen XingShuliang Song
Jun 14, 2021·Carbohydrate Polymers·Ushasree Mrudulakumari VasudevanEun Yeol Lee
Feb 21, 2019·Journal of Agricultural and Food Chemistry·Kiira S VuoristoVincent G H Eijsink

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Datasets Mentioned

BETA
AB898059
MH234061
MG792316

Methods Mentioned

BETA
PCR
glycosylation
Assay

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