A classification of glycoside hydrolases based on amino acid sequence similarities was proposed a few years ago, wherein β-glucosidases were mainly grouped into two superfamilies of glycoside hydrolases I (GH1), and GH3 [22
]. Although, the amino acid sequence analysis indicated that BGL belongs to GH1, it shared the highest sequence similarity of 66% with the β-glucosidses from Thermoanaerobacter mathranii
(YP_003676178.1). Moreover, it shared only the 63% with the putative β-glucosidase (YP_004471891.1) the Thermoanaerobacterium xylanolyticum
LX-11, both belonging to the genus Thermoanaerobacterium
. The Phylogenies analysis showed that the BGL was distant with the glucose-tolerant β-Glucosidases from fungi and ADD96762.1 (Figure ). The results indicated that the BGL could be a novel β-glucoside with some different properties. On the other hand, β-Glucosidases may be divided into three groups on the basis of their substrate specificity. The first group is known as aryl-β-glucosidases owing to strong affinity to aryl-β-glucose. The second group consists of cellobiases that hydrolyze oligosaccharides only. The third group is broad specific β-glucosidases that exhibit activity on a wide range of substrates, and are the most commonly observed form of β-glucosidases [23
]. The BGL, which was high affinity to p-nitrophenyl-β-D-glucopyranoside, hydrolyzed cellobiose, p-nitrophenyl-β-D-glucopyranoside, and p-nitrophenyl-β-D-galactopyranoside, but not p-nitrophenyl-α-L-arabinofuranoside, p-nitrophenyl-β-D-xylopyranoside, maltose, sucrose, and CMC. These results indicated that BGL belonged to the first group.
Enzymatic hydrolysis of cellulose is a complex process, the last step being a homogenous catalysis reaction involving the action of β-glucosidase on cellobiose. Cellobiose is a strong inhibitor of both cellobiohydrolases and endocellulases. Therefore, β-glucosidase with high tolerance for glucose has become heated in these fields. Fungi, especially Aspergillus
species, are generally considered to be a good producer with high yield of β-glucosidases [24
]. But the major β-glucosidases belonging to family 3 of the glycoside hydrolases (GH3) from Aspergillus
species were subject to competitive inhibition of glucose to produce glucose, the Ki
is generally 1–20
]. The minor β-glucosidases, which molecular weights are 40–50
kDa, exhibited a tolerance to glucose (Table ). The effect of glucose on the BGL activity revealed that the enzyme is not only resistant to end-product inhibition, but is activated by glucose at concentrations from 0 to 0.2
M. Only two β-glucosidases, activated by glucose, have been reported from Scytalidium thermophilum
and marine microbial (Table ) [13
Moreover, high specific activity for cellobiose and tolerance to substrate inhibition are other advantages for β-glucosidase in enzymatic hydrolysis of cellulose. Although, several β-glucosidases from a few fungi and bacteria show high glucose tolerant with Ki
values of more than 200
mM, the Vmax
values of these enzymes for cellobiose were much lower than for p-nitrophenyl-β-D-glucopyranoside. The Vmax
value of BGL for cellobiose was 120 U/mg, which was about 2 times higher than the Vmax
value of BGL for p-nitrophenyl-β-D-glucopyranoside. To our knowledge, in only one other study have workers described the purification and characterization (from A. oryzae
) of a β-glucosidase having such a high tolerance to glucose and high specific activity for cellobiose [19
]. But the specific activity of β-glucosidase from A. oryzae
for cellobiose was much lower than for p-nitrophenyl-β-D-glucopyranoside (Table ). The BGL was only the β-glucosidase been reported that it is not only resistant to glucose, but had higher specific activity for cellobiose than for p-nitrophenyl-β-D-glucopyranoside. In addition, the BGL had high tolerance to substrate inhibition, cellobiose. The Kcat
of BGL was 67.7
at 60°C and pH 6.4, when the concentration of cellobiose was 10% (Table ).
The chemical agents had various effects on the activity of BGL. The chelating agent EDTA displayed no influence on the β-glucosidase activity, indicating that the β-glucosidase is not a metalloprotein. However, the β-glucosidase activity was greatly stimulated by Fe2+
, which implied that Fe2+
is required for the maximal activity of BGL. These results distinguish BGL from the other bacteria β-glucosidases, on which Ca2+
show positive effects [13
]. In practical applications, the high thermostability of the enzyme is desired because the longer active life means the less consumption of the enzyme. The BGL residual activity was more than 80% after being incubated at 60°C for 2
h, and it in enzymatic hydrolysis of cellulose exhibited high activity in broad temperature, which could keep at high levels at temperatures from 45 to 70°C.
The properties of the BGL demonstrated a great potential of the gene in the genetic modification of strains for biomass degradation. Differences in codon usage preference among organisms lead to a variety of problems concerning heterologous gene expression, which can be overcome by rational gene design and gene synthesis. Protein with multiple repetitive rare codons especially within the first 20 amino acids of the amino terminus of the protein may significantly reduce the protein expression. Sometimes, it shuts down the expression completely. Since the rare codons of bgl from 1–20 amino acids were all changed into optimized codons, the activity of BGL was increased by about 70% (Figure ). More optimization of codons for the other amino acid residues in the ORF of bgl may give further improvement in the gene expression levels.