Applied Microbiology and Biotechnology

, Volume 43, Issue 2, pp 228–234

Enzymatic preparation of [1, 3-13C]dihydroxyacetone phosphate from [13C]methanol and hydroxypyruvate using the methanol-assimilating system of methylotrophic yeasts

  • H. Yanase
  • M. Okuda
  • K. Kita
  • Y. Sato
  • K. Shibata
  • Y. Sakai
  • N. Kato
Biotechnology Original Paper

DOI: 10.1007/BF00172817

Cite this article as:
Yanase, H., Okuda, M., Kita, K. et al. Appl Microbiol Biotechnol (1995) 43: 228. doi:10.1007/BF00172817

Abstract

Dihydroxyacetoone synthase (EC 2.2.1.3), which is a key enzyme of the C1-compound-assimilating pathway in yeasts, catalyzes transketolation between formaldehyde and hydroxypyruvate, leading to the formation of dihydroxyacetone and CO2. When [13C]formaldehyde was used as a substrate with dihydroxyacytone synthase from Candida boidinii 2201, 13C was confirmed to be incorporated to the C-1 and C-3 positions of dihydroxyacetone, and the 13C content of each carbon (atoms/100 atoms) was estimated to be 50%. [13C]Methanol was also useful for the enrichment of dihydroxyacetone with 13C, when alcohol oxidase from a methylotrophic yeast was added for the conversion of methanol to formaldehyde. A fed-batch reaction with periodic addition of the substrates was required for the accumalation of 13C-labelled dihydroxyacetone at a higher concentration, because the enzyme system was relatively susceptible to the C donor, formaldehyde or methanol. The optimum conditions for the production gave 160mM (14.4 mg/ml) dihydroxyacetone for 180 min; the molar yield relative to methanol added was 80%. Diyhdroxyacetone kinase (EC 2.7.1.29) from methanol-grown Hansenula polymorpha CBS 4732 was a suitable enzyme for the phosphorylation of dihydroxyacytone. The phosphorylation system, comprising of dihydroxyacetone kinase, adenylate kinase, and ATP, could be coupled with the system for dihydroxyacetone production. A fed-batch reaction afforded 185 mM [1, 3-13C]dihydroxyacetone phosphate from [13C]methanol; the molar yield of the ester relative to methanol added was 92.5%

Copyright information

© Springer-Verlag 1995

Authors and Affiliations

  • H. Yanase
    • 1
  • M. Okuda
    • 1
  • K. Kita
    • 1
  • Y. Sato
    • 2
  • K. Shibata
    • 2
  • Y. Sakai
    • 3
  • N. Kato
    • 3
  1. 1.Department of BiotechnologyTottori UniversityTottoriJapan
  2. 2.Frontier Technology Research InstituteTokyo Gas Co.YokohamaJapan
  3. 3.Department of Agricultural ChemistryKyoto UniversityKyotoJapan

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