Homology modeling, molecular docking and active sites analysis
The homology modeling of Cbh-A was performed on the raptorX website. Similarity searches for the sequence were conducted using BLASTP searches of the Protein Data Bank (PDB) database at the NCBI server. Among the results of similarity proteins, two were selected as models for homology modeling, 5T1Q_A and 5JQC_A, which covered two domains separately with 40.24% and 31.17% of identity. The homology model with a p-value of 3.36E− 8 showed that the structure of Cbh-A was composed of 298 amino acid residues, which consist of 38% α-helices and 21% β-sheet (Fig. 2a).
Based on the prediction of 3D structure, domain FlgJ was the catalytic domain contained a barrel formed by 6 α-helices and 2 β-sheets which probably were active sites. The pocket sites consist of Glu88, Phe104, Gly105, Val-106, Lys107, Tyr110, Thr119, Asp120, Glu121, Leu150, Tyr183, Ala184, Thr185. Domain SH3_8 had function of cellulose binding and with binding sites Lys232, Pro233, Trp278 (Fig. 2b). All the activity sites formed a tunnel of the loops and the antiparallel strands, which seems only allow one cellulose chain into it . The domain SH3_8 possessed cellulose binding and enhanced the connection of enzyme and cellulose substrates.
Characterization of the recombinant Cbh-A
The maximum activity of recombinant Cbh-A was observed at pH 6.4 (Fig. 3a) by measurement in different pH buffers. At pH6.0 and 7.0, the relative enzyme activity also could reach 90.3% and 85.4%. After 30 min of maintaining in different pH buffers, the highest relativity enzyme activity showed that pH 6.0 (Fig. 3b) could make enzyme stale. Followed by it was pH 6.4, 7.0 and 7.4 could also reach 89.7%, 78.1%, and 71.6%, respectively. The results suggested that the neutral pH was much more suitable for enzyme activity and keep stable. For an optimum temperature of enzyme activity, Cbh-A had maximum enzyme activity at 50℃ (Fig. 3c), and it could reach 98% relative activity at 40℃. The results suggested that action temperature between 40–50℃ could keep enzyme high enzyme activity. Cbh-A was found to be highly stable under 50℃ (Fig. 3d).
When detecting thermostability of Cbh-A, we found an interesting thing that recombinant enzyme Cbh-A presented highly enzyme activity when it was maintained under 50℃ for 30 min. It brought us a new thought out of our mind, that how long it can stay under 50℃ to keep enzyme activity. So, a time-continued experiment was done, and the change of enzyme activity with time incubated at 50 ℃ was investigated. It showed that in one hour, the enzyme activity could Within one hour, the enzyme activity increased rapidly with the increase in temperature. Within 1 to 4 h, the enzyme activity continued to increase with the increase of temperature, but the rate became slower. After 4 h, the enzyme activity remained stable (Fig. 3e) which could reach 0.225 ± 0.002 U/mL under experiment conditions.
The influence of effectors on enzyme activity was determined by using various metal salts and chemicals. Table 1 showed that relative affection on enzyme activity of different metal ions and chemicals at a concentration of 1 mM, 5 mM, and 10 mM. The presence of Na+, K+, Mg2+, Fe2+, Fe3+, [NH4]+, and chemical SDS were all had little effect on enzyme relative activity under different densities. Metal ion Mn2+ could increase relative enzyme activity greatly in 10 mM, 5 mM and 1 mM, followed by Zn2+ and Ca2+ could increase Cbh-A relative activity to some extent under 10 mM and 5 mM. Cu2+ could inhibit enzyme activity under 10 mM and 5 mM, but had no effect under 1 mM. Chemical EDTA inhibited enzyme activity under all density.
Substrate specificity of Cbh-A also was measured to identify which glucan linkage could be acted on (Table 2). Relative activity of different substrates showed that Cbh-A had a high efficiency of maltose of 277.63% relative activity, and it presented better in barley glucan, laminarin and xylan from beechwood that their relative activity was 168.44%, 161.52%, and 185.09%, respectively. For CMC-Na and filter paper, it sighed out equally hydrolyzation ability to Avicel. Results above could identify that Cbh-A could hydrolyze β-1,3 − 1,4 glucan linkage, β-1,3 glucan linkage, β-1,4 glucan linkage, and α-1,4 glucan linkage efficiency, but had no effect on α-1,6 glucan linkage.
Synergy effect of three types of cellulase
For cellulose degradation, three types of cellulases forms a system which could hydrolyze cellulose by cooperation efficiency. Three types of recombinant cellulase cloned from B. subtills 1AJ3, endocellulase Cel-5A, exocellulase Cbh-A (this study), and β-glucosidase Bgl-16A, were combined according to a single enzyme, two together, and three mixed formed seven groups (Table 3). Then seven groups of enzymes were measured, namely enzyme activity of Avicelase, CMCase, and FPase based on the substrate of Avicel, CMC-Na, and filter paper, respectively. The DS value of two by two and three mixed groups was calculated to observed the synergism among three cellulases (Table 3).
Different enzymes have their characteristics in various substrates, and not to mention the interaction between enzymes and enzymes present diversely. It showed that all the three types of cellulase could form a synergism effect in different cellulose substrates, no matter two of them or three of them. They can improve hydrolyze efficiency and cooperate when degradation cellulose. But they had different synergy ability between themselves, and also within various cellulose substrates. For CMC-Na, standing for an amorphous region of cellulose, endocellulase Cel-5A presents optimum hydrolyzation among single cellulase. Exocellulase and β-glucosidase could synergy best which had the highest DS value of 1.736, and even higher than the three enzymes together (DS value of 1.326). But when hydrolyzing Avicel, which mainly consist of crystalline cellulose, three single cellulase present relative equally in hydrolyzation by results of enzyme activity. But when they worked to cooperate, three enzymes together presented stronger synergy effect ability (DS value of 1.501). When using filter paper as a substrate, which containing both amorphous region and crystalline cellulose, endocellulase displayed strong hydrolyzation ability. Three enzymes cooperation (DS value of 1.525) was equal to that in Avicel, but endocellulase and exocellulase cooperation seemed like more efficiency for its higher DS value of 1.703.
Application in varieties of biomass based on synergism
Three types of cellulase into lignocellulose biomass were applied to observe their synergism and also examine hydrolyze ability in more complex cellulose substrates. Adding buffer without enzymes as blank, then single enzyme and three mixture enzymes were added into 5% (m/V) various biomass. Figure 4 showed raw biomass materials and hydrolyzation samples. There were five samples in each group in the test tube and from left to right were blank, endocellulase solo, exocellulase solo, β-glucosidase solo, and three cellulases mixture. The saccharification rate of different kinds of biomass was shown in Table 4. After the hydrolyzation by enzymes, all the biomass lignocellulose showed similar characteristics. On the whole, the particles of lignocellulose after hydrolyzation changed from large to small, crashed to thin, and some even appeared sandy. Because of the lignocellulose structure becoming loosen, most of the lignocellulose color changed into lighter. However, from the results of saccharification rate of different biomass substrates by a single or mixture enzymes, cellulases and enzyme mixture had special hydrolyzation characters in different lignocellulose substrates (Table 4). For example, it displayed the highest DS value (184.0) of three enzymes in corn stalks while single enzyme nearly had no hydrolyzation effect on corn stalks. Then followed by wheat straw and rice husk, coffee ground, and sugarcane stalk, three enzymes could present cooperation with each other than single ones. But in switchgrass, corn cob, pea straw, and ginger stems and leaves, enzymes synergism was not obvious. Besides, in all these ten kinds of biomass substrates, enzymes had less effect on peanut shell, no matter single cellulase or mixture together while saccharification was low. For saccharification rate, switchgrass and corn cob would be the benefit lignocellulose for obtaining more reducing sugar content, and then followed by corn stalks and wheat straw.