ENHANCED TRANSGLYCOSYLATION ACTIVITY OF ARTHROBACTER-PROTOPHORMIAE ENDO-BETA-N-ACETYLGLUCOSAMINIDASE IN MEDIA CONTAINING ORGANIC-SOLVENTS

The transglycosylation activity of endo beta-N-acetylglucosaminidase hom 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 Man(9)GlcNAc(2)Asn 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 degrees 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 Man(5)GlcNAc(2)Asn as donor and methyl alpha-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-1 NMR of the product revealed that transglycosylation occurred at the C-4 of GlcNAc and the linkage was of the beta-configuration. In the acetone-containing me dium, Glc, Man, a deoxy-Glc, and methyl alpha-D-GlcNAc can serve as a good acceptor as GlcNAc. Less favorable accepters 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 accepters of the enzyme transglycosylation.