Next Article in Journal
Diterpenes from the Marine Algae of the Genus Dictyota
Previous Article in Journal
The Maxi-K (BK) Channel Antagonist Penitrem A as a Novel Breast Cancer-Targeted Therapeutic
Article Menu
Issue 5 (May) cover image

Export Article

Open AccessArticle
Mar. Drugs 2018, 16(5), 158; https://doi.org/10.3390/md16050158

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.
Received: 25 March 2018 / Revised: 30 April 2018 / Accepted: 7 May 2018 / Published: 11 May 2018
Full-Text   |   PDF [2726 KB, uploaded 11 May 2018]   |  

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 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
Figures

Figure 1

This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).
SciFeed

Share & Cite This Article

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.

Show more citation formats Show less citations formats

Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Related Articles

Article Metrics

Article Access Statistics

1

Comments

[Return to top]
Mar. Drugs EISSN 1660-3397 Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top