Figure 1.
Multiple alignment of the deduced amino acid sequence of mature NfBGL1 (XP_001261562) with other fungal counterparts from T. reesei (TrBgl1, 1713235A), and A. aculeatus (AaBGL1, 4IIB).
Identical and similar amino acids are indicated by solid black and gray boxes, respectively. The putative catalytic residues, D235 and E447, are indicated with asterisks. The residues probably related to subsite −1 (W236) and +1 (W36, N261, and Y449) are indicated with white and black triangles, respectively. The residues probably forming hydrogen bonds with substrate, D60, R124, K147 and H148, are indicated with black dots.
Figure 2.
The homology-modeled NfBGL1 with AaBGL1 from A. aculeatus (4IIB, 44.1% identity) as the template.
(A) Putative structure of NfBGL1. The catalytic residues D235 and E447 are indicated. (B) Putative interactions between the key residues of NfBGL1 and substrate cellobiose.
Figure 3.
Purification and high-cell-density fermentation of recombinant NfBGL1.
(A) SDS-PAGE analysis of purified recombinant NfBGL1. Lanes: 1, the molecular mass standards; 2, the purified recombinant of NfBGL1. (B) Time course of NfBGL1 production in a 3.7-l fermenter. Each value in the panel represents the means ± S.D. (n = 3).
Table 1.
Property comparison of NfBGL1 from N. fischeri P1 and its fungal counterparts. a
Figure 4.
Characterization of purified recombinant NfBGL1.
(A) Effect of pH on β-glucosidase activity. The enzyme assay was performed at 80°C for 10 min. (B) pH stability. The enzyme was pre-incubated without substrate at 37°C for 60 min, and then subjected to residual activity assay under standard conditions (pH 5.0, 80°C, 10 min). (C) Effect of temperature on β-glucosidase activity determined at pH 5.0 for 10 min. (D) Thermostability. The residual enzyme activities were measured under standard conditions after pre-incubation of the enzyme without substrate in McIlvaine buffer (pH 5.0) for various periods. Each value in the panel represents the means ± S.D. (n = 3).
Table 2.
Substrate specificities of NfBGL1.
Table 3.
Conversion of soybean isoflavone glycosides into free isoflavones by NfBGL1 and Novozyme 188. a