Table IV.
Products of Glycosynthase Reactions of BGlu1 E386G Glycosynthase and its Variants with Equal Amounts of α-glucosyl Fluoride Donor and pNPC2 Acceptor
| Glycosynthase | Percent of pNP in each oligo-glycoside producta | |||||||
|---|---|---|---|---|---|---|---|---|
| DP 2 | DP 3 | DP 4 | DP 5 | DP 6 | DP 7 | DP 8 | DP 9 | |
| E386G | 56.7 ± 0.07 | 26.8 ± 0.03 | 9.8 ± 0.04 | 4.75 ± 0.014 | 1.52 ± 0.005 | 0.37 ± 0.005 | 0.07 ± 0.001 | 0.009 ± 0.0001 |
| E386G/Y341A | 70.6 ± 0.32 | 24.1 ± 0.26 | 4.94 ± 0.06 | 0.35 ± 0.009 | 0.021 ± 0.0017 | — | — | — |
| E386G/Q187A | 49.0 ± 0.07 | 28.2 ± 0.10 | 12.8 ± 0.02 | 6.70 ± 0.016 | 2.42 ± 0.02 | 0.67 ± 0.006 | 0.14 ± 0.002 | 0.022 ± 0.0004 |
| E386G/Y341A/Q187A | 89.0 ± 0.24 | 10.3 ± 0.22 | 0.68 ± 0.025 | 0.009 ± 0.0004 | — | — | — | — |
| E386G/R178A | 98.1 ± 0.01 | 1.91 ± 0.01 | 0.009 ± 0.0001 | — | — | — | — | — |
| E386G/W337A | 83.1 ± 0.22 | 14.8 ± 0.23 | 1.51 ± 0.036 | 0.09 ± 0.004 | 0.005 ± 0.0001 | — | — | — |
| E386G/Y341A/R178A | 98.6 ± 0.01 | 1.38 ± 0.01 | — | — | — | — | — | — |
| E386G/Y341A/W337A | 96.3 ± 0.12 | 3.59 ± 0.12 | 0.066 ± 0.0007 | — | — | — | — | — |
| E386G/Y341A/R178A/W337A | 96.8 ± 0.24 | 3.21 ± 0.24 | — | — | — | — | — | — |
a
Reactions in 150 mM ammonium bicarbonate buffer, pH 7.0, 30°C for 24 h, at a 1:1 molar ratio of α-GlcF donor: pNPC2 acceptor, and 40 μM enzyme concentration. The relative percentages are in terms of peak area per total 300 nm absorbance in pNP-glycoside products and substrate peaks separated by HPLC on a ZORBAX carbohydrate column. The molecular mass [Mass + 35Cl]- of each eluted compound was confirmed by mass spectrometry: pNPC2 (498 m/z), pNPC3 (660 m/z), pNPC4 (822 m/z), pNPC5 (984 m/z), pNPC6 (1146 m/z), pNPC7 (1308 m/z), pNPC8 (1471 m/z), and pNPC9 (1633 m/z).