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Biotechnology Letters

, Volume 36, Issue 10, pp 2023–2028 | Cite as

Characterization and expression of glucosamine-6-phosphate synthase from Saccharomyces cerevisiae in Pichia pastoris

  • Sheng Wang
  • Piwu LiEmail author
  • Jing Su
  • Xiangkun Wu
  • Rongrong Liang
Original Research Paper

Abstract

Glucosamine-6-phosphate (GlcN-6-P) synthase from Saccharomyces cerevisiae was expressed in Pichia pastoris SMD1168 GIVING maximum activity of 96 U ml−1 for the enzyme in the culture medium. By SDS-PAGE, the enzyme, a glycosylated protein, had an apparent molecular mass of 90 kDa. The enzyme was purified by gel exclusion chromatography to near homogeneity, with a 90 % yield and its properties were characterized. Optimal activities were at pH 5.5 and 55 °C, respectively, at which the highest specific activity was 6.8 U mg protein −1. The enzyme was stable from pH 4.5 to 5.5 and from 45 to 60 °C. The Km and Vmax of the GlcN-6-P synthase towards d-fructose 6-phosphate were 2.8 mM and 6.9 μmol min−1 mg−1, respectively.

Keywords

Glucosamine-6-phosphate synthase Pichia pastoris Saccharomyces cerevisiae 

Notes

Acknowledgments

This work was financially supported by the National High-tech R&D Program of China (863 Program, No. 2012AA021504) and the Taishan Scholar Program of Shandong.

Supplementary material

10529_2014_1561_MOESM1_ESM.docx (12 kb)
Supplementary material 1 (DOCX 11 kb)

References

  1. Badet B, Vermoote P, Haumont PY et al (1987) Glucosamine synthease from Escherichia coli: purification, properties and glutamine-utilizing site location. Biochemistry 26:1940–1948PubMedCrossRefGoogle Scholar
  2. Borowski E (2000) Novel approaches in the rational design of antifungal agents of low toxicity. Farmaco 55:206–208PubMedCrossRefGoogle Scholar
  3. Chen XZ, Xu SQ, Zhu MS et al (2010) Site-directed mutagenesis of an Aspergillus niger xylanase B and its expression, purification and enzymatic characterization in Pichia pastoris. Process Biochem 45:75–80CrossRefGoogle Scholar
  4. Czarnecka J, Kwiatkowska K, Gabriel I et al (2012) Engineering Candida albicans glucosamine-6-phosphate synthase for efficient enzyme purification. J Mol Recognit 25:564–570PubMedCrossRefGoogle Scholar
  5. Deng MD, Severson DK, Grund AD et al (2005) Metabolic engineering of Escherichia coli for industrial production of glucosamine and N-acetylglucosamine. Metab Eng 7:201–214PubMedCrossRefGoogle Scholar
  6. Durand P, Golinelli-Pimpaneau B, Mouilleron S et al (2008) Highlights of glucosamine-6 P synthase catalysis. Arch Biochem Biophys 474:302–317PubMedCrossRefGoogle Scholar
  7. Gonzalez-Ibarra J, Milewski S, Villagómez-Castro JC et al (2010) Sporothrix schenckii: purification and partial biochemical characterization of glucosamine-6-phosphate synthase, a potential antifungal target. Med Mycol 48:110–121PubMedCrossRefGoogle Scholar
  8. Hebert LF, Daniels MC, Zhou J et al (1996) Overexpression of glutamine: fructose 6-phosphate amidotransferase in transgenic mice leads to insulin resistance. J Clin Invest 98:930–936PubMedCentralPubMedCrossRefGoogle Scholar
  9. Olchowy J, Kur K, Sachadyn P et al (2006) Construction, purification, and functional characterization of His-tagged Candida albicans glucosamine-6-phosphate synthase expressed in Escherichia coli. Protein Exp Purif 46:309–315CrossRefGoogle Scholar
  10. Rodriguez-Diaz J, Rubio-Del-Campo A, Yebra MJ (2012) Metabolic engineering of Lactobacillus casei for production of UDP-N-acetylglucosamine. Biotechnol Bioeng 109:1704–1712PubMedCrossRefGoogle Scholar
  11. Sachadyn P, Jedrzejczak R, Milewski S et al (2000) Purification to homogeneity of Candida albicans glucosamine-6-phosphate synthase overexpressed in Escherichia coli. Protein Exp Purif 19:343–349PubMedCrossRefGoogle Scholar
  12. Teplyakov A, Obmolova G, Badet B et al (2001) Channeling of ammonia in glucosamine-6-phosphate synthase. J Mol Biol 313:1093–1102PubMedCrossRefGoogle Scholar
  13. Teplyakov A, Leriche C, Obmolova G et al (2002) From Lobry de Bruyn to enzyme-catalyzed ammonia channelling: molecular studies of D-glucosamine-6P synthase. Nat Prod Rep 19:60–69PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Sheng Wang
    • 1
  • Piwu Li
    • 1
    Email author
  • Jing Su
    • 1
  • Xiangkun Wu
    • 1
  • Rongrong Liang
    • 1
  1. 1.Shandong Provincial Key Laboratory of Microbial Engineering, School of Food and BioengineeringQilu University of TechnologyJinanChina

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