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Volume 270, Number 30, Issue of July 28, pp. 17723-17729, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.

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Enhanced Transglycosylation Activity of Arthrobacter protophormiae Endo--N-acetylglucosaminidase in Media Containing Organic Solvents

(Received for publication, March 29, 1995; and in revised form, May 25, 1995)

From the  (1)Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218, the Department of Bioresource Science, Faculty of Agriculture, Kagawa University, Japan, the (2)Biotechnology Research Laboratory, Takara Shuzo Co., Ltd., Ohtsu, Shiga 520-21, Japan, and the (3)Department of Biological Chemistry, The Johns Hopkins School of Medicine, Baltimore, Maryland 21205

The transglycosylation activity of endo--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 ManGlcNAcAsn as donor and 0.5 M GlcNAc as acceptor. However, in the media containing 30% (v/v) acetone, dioxane, N,N-dimethylformamide, or dimethyl sulfoxide 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 analysis of the transglycosylation product obtained in 30% acetone with ManGlcNAcAsn as donor and methyl -2-acetamido-2-deoxy-D-glucopyranoside as acceptor showed a mass ion of m/z 1249.4, consistent with the expected molecular weight. Analysis by H NMR of the product revealed that transglycosylation occurred at the C-4 of GlcNAc and the linkage was of the -configuration. In the acetone-containing medium, Glc, Man, 2-deoxy-Glc, and methyl -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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