Enhanced transglycosylation activity of Arthrobacter protophormiae endo- β-N-acetylglucosaminidase in media containing organic solvents

J. Q. Fan, Kaoru Takegawa, S. Iwahara, A. Kondo, I. Kato, C. Abeygunawardana, Y. C. Lee

Research output: Contribution to journalArticle

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Abstract

The transglycosylation activity of endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (endo-A) was enhanced by inclusion of organic solvents in the reaction mixture. In aqueous solution, the transglycosylation yield relative to starting substrate was 32% using Man9GlcNAc2Asn as donor and 0.5 M GlcNAc as acceptor. However, in the media containing 30% (v/v) acetone, dioxane, N,N-dimethylformamide, or dimethyl surfoxide with 0.5 M GlcNAc as acceptor, the transglycosylation attained yields of 89, 13, 28, and 75%, respectively, as analyzed by high performance anion exchange chromatography. The enzyme was stable in media containing up to 30% acetone, 30% dimethyl sulfoxide, or 20% N,N-dimethylformamide at 37 °C for at least 30 min. The acceptor (GlcNAc) concentration must be greater than 0.2 M for efficient transglycosylation. Electrospray mass spectrometry analysts of the transglycosylation product obtained in 30% acetone with Man5GlcNAc2Asn as donor and methyl α-2-acetamido-2-deoxy-D-glucopyranoside as acceptor showed a mass ion of m/x 1249.4, consistent with the expected molecular weight. Analysis by 1H NMR of the product revealed that transglycosylation occurred at the C-4 of GlcNAc and the linkage was of the β-configuration. In the acetone-containing medium, Glc, Man, 2-deoxy-Glc, and methyl α-D-GlcNAc can serve as a good acceptor as GlcNAc. Less favorable acceptors are xylose, fructose, 6-deoxy-Glc, and 3-O-methyl-D-glucose. On the other hand, GalNAc, Gal, allose, and 3-deoxy-Glc could not serve as acceptors of the enzyme transglycosylation.

Original languageEnglish
Pages (from-to)17723-17729
Number of pages7
JournalJournal of Biological Chemistry
Volume270
Issue number30
DOIs
Publication statusPublished - Jan 1 1995

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Arthrobacter
Acetylglucosaminidase
Acetone
Organic solvents
Dimethylformamide
3-O-Methylglucose
Xylose
Enzymes
Chromatography
Dimethyl Sulfoxide
Fructose
Anions
Mass spectrometry
Mass Spectrometry
Molecular Weight
Molecular weight
Nuclear magnetic resonance
Ions
endo-alpha-sialidase
Substrates

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Enhanced transglycosylation activity of Arthrobacter protophormiae endo- β-N-acetylglucosaminidase in media containing organic solvents. / Fan, J. Q.; Takegawa, Kaoru; Iwahara, S.; Kondo, A.; Kato, I.; Abeygunawardana, C.; Lee, Y. C.

In: Journal of Biological Chemistry, Vol. 270, No. 30, 01.01.1995, p. 17723-17729.

Research output: Contribution to journalArticle

Fan, J. Q. ; Takegawa, Kaoru ; Iwahara, S. ; Kondo, A. ; Kato, I. ; Abeygunawardana, C. ; Lee, Y. C. / Enhanced transglycosylation activity of Arthrobacter protophormiae endo- β-N-acetylglucosaminidase in media containing organic solvents. In: Journal of Biological Chemistry. 1995 ; Vol. 270, No. 30. pp. 17723-17729.
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abstract = "The transglycosylation activity of endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (endo-A) was enhanced by inclusion of organic solvents in the reaction mixture. In aqueous solution, the transglycosylation yield relative to starting substrate was 32{\%} using Man9GlcNAc2Asn as donor and 0.5 M GlcNAc as acceptor. However, in the media containing 30{\%} (v/v) acetone, dioxane, N,N-dimethylformamide, or dimethyl surfoxide with 0.5 M GlcNAc as acceptor, the transglycosylation attained yields of 89, 13, 28, and 75{\%}, respectively, as analyzed by high performance anion exchange chromatography. The enzyme was stable in media containing up to 30{\%} acetone, 30{\%} dimethyl sulfoxide, or 20{\%} N,N-dimethylformamide at 37 °C for at least 30 min. The acceptor (GlcNAc) concentration must be greater than 0.2 M for efficient transglycosylation. Electrospray mass spectrometry analysts of the transglycosylation product obtained in 30{\%} acetone with Man5GlcNAc2Asn as donor and methyl α-2-acetamido-2-deoxy-D-glucopyranoside as acceptor showed a mass ion of m/x 1249.4, consistent with the expected molecular weight. Analysis by 1H NMR of the product revealed that transglycosylation occurred at the C-4 of GlcNAc and the linkage was of the β-configuration. In the acetone-containing medium, Glc, Man, 2-deoxy-Glc, and methyl α-D-GlcNAc can serve as a good acceptor as GlcNAc. Less favorable acceptors are xylose, fructose, 6-deoxy-Glc, and 3-O-methyl-D-glucose. On the other hand, GalNAc, Gal, allose, and 3-deoxy-Glc could not serve as acceptors of the enzyme transglycosylation.",
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AU - Abeygunawardana, C.

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N2 - The transglycosylation activity of endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (endo-A) was enhanced by inclusion of organic solvents in the reaction mixture. In aqueous solution, the transglycosylation yield relative to starting substrate was 32% using Man9GlcNAc2Asn as donor and 0.5 M GlcNAc as acceptor. However, in the media containing 30% (v/v) acetone, dioxane, N,N-dimethylformamide, or dimethyl surfoxide with 0.5 M GlcNAc as acceptor, the transglycosylation attained yields of 89, 13, 28, and 75%, respectively, as analyzed by high performance anion exchange chromatography. The enzyme was stable in media containing up to 30% acetone, 30% dimethyl sulfoxide, or 20% N,N-dimethylformamide at 37 °C for at least 30 min. The acceptor (GlcNAc) concentration must be greater than 0.2 M for efficient transglycosylation. Electrospray mass spectrometry analysts of the transglycosylation product obtained in 30% acetone with Man5GlcNAc2Asn as donor and methyl α-2-acetamido-2-deoxy-D-glucopyranoside as acceptor showed a mass ion of m/x 1249.4, consistent with the expected molecular weight. Analysis by 1H NMR of the product revealed that transglycosylation occurred at the C-4 of GlcNAc and the linkage was of the β-configuration. In the acetone-containing medium, Glc, Man, 2-deoxy-Glc, and methyl α-D-GlcNAc can serve as a good acceptor as GlcNAc. Less favorable acceptors are xylose, fructose, 6-deoxy-Glc, and 3-O-methyl-D-glucose. On the other hand, GalNAc, Gal, allose, and 3-deoxy-Glc could not serve as acceptors of the enzyme transglycosylation.

AB - The transglycosylation activity of endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae (endo-A) was enhanced by inclusion of organic solvents in the reaction mixture. In aqueous solution, the transglycosylation yield relative to starting substrate was 32% using Man9GlcNAc2Asn as donor and 0.5 M GlcNAc as acceptor. However, in the media containing 30% (v/v) acetone, dioxane, N,N-dimethylformamide, or dimethyl surfoxide with 0.5 M GlcNAc as acceptor, the transglycosylation attained yields of 89, 13, 28, and 75%, respectively, as analyzed by high performance anion exchange chromatography. The enzyme was stable in media containing up to 30% acetone, 30% dimethyl sulfoxide, or 20% N,N-dimethylformamide at 37 °C for at least 30 min. The acceptor (GlcNAc) concentration must be greater than 0.2 M for efficient transglycosylation. Electrospray mass spectrometry analysts of the transglycosylation product obtained in 30% acetone with Man5GlcNAc2Asn as donor and methyl α-2-acetamido-2-deoxy-D-glucopyranoside as acceptor showed a mass ion of m/x 1249.4, consistent with the expected molecular weight. Analysis by 1H NMR of the product revealed that transglycosylation occurred at the C-4 of GlcNAc and the linkage was of the β-configuration. In the acetone-containing medium, Glc, Man, 2-deoxy-Glc, and methyl α-D-GlcNAc can serve as a good acceptor as GlcNAc. Less favorable acceptors are xylose, fructose, 6-deoxy-Glc, and 3-O-methyl-D-glucose. On the other hand, GalNAc, Gal, allose, and 3-deoxy-Glc could not serve as acceptors of the enzyme transglycosylation.

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