Cloning and characterization of an exocellulase and synergistic effect with recombinant endocellulase and beta-glucosidase from Bacillus subtilis 1AJ3

Background: Biomass lignocellulose provide abandon cellulosic material for bioenergy. The synergy effect of the cellulase system still needs more attention to make it more efficient. Exocellulase was less of studies in former studies, especially recombinant exocellulase from bacteria. Results: An exocellulase Cbh -A from strain B. subtilis 1AJ3 was cloned and expressed in E.coli BL21. By sequence analysis, Cbh -A contains two domains: FlgJ and SH3_8. Enzymatic properties of Cbh -A were characterized, and the maximum enzyme activity was gotten under the condition of pH 6.4 and 50℃. Moreover, the enzyme reached the highest enzyme activity and kept stable under 50℃ for 4 h. Ion metal Mn 2+ had an activating effect on Cbh -A, while Cu 2+ as well as chemical EDTA had inhibition effect. Cbh -A had widely substrates specification that it could hydrolyze β-1,3-1,4/β-1,3/ β-1,4 glucan linkage, α-1,4 glucan linkage . When it took cooperation with recombinant endocellulase and β-glucosidase also from the strain B. subtilis 1AJ3, synergism effect was observed through DS (degree of synergism) value by hydrolyzing CMC-Na, Avicel, and filter paper. At the same time, the synergism effect was also examined in biomass lignocellulose by comparing the saccharification rate and DS value of each material. Conclusions: Cbh -A contained a FlgJ domain and first studied as an exocellulase cloned from B. subtilis 1AJ3. The biochemical, enzymatic properties, ion metal, and substrate specificity of Cbh -A were analyzed, and a special character of Cbh -A could keep maximum activity after pre-warm at 50℃ for 4h. Cbh-A had good synergy effect with endocellulase and β-glucosidase from the same strain in variety kind of cellulosic substrates, including simple and complex biomass cellulose. It laid a rates. All results suggest that the recombinant exocellulase is promising in industrial applications. Synergism effect of three types of cellulases from the same bacterium could cooperate well, and they have substrates specification. It provides the basis for the universal application of enzymes, and also provides new ideas for the comprehensive development and utilization of biomass energy.


Background
Lignocellulosic biomass represents the main sustainable utilize substrates of biofuels since it is a wild source and high content of cellulose of them. Cellulose in different biomass always consists of long chains of β-1,4-linked glucose units which make lignocellulose kept stable and partly dense crystalline structure [1]. The structure of cellulose is added lignin and hemicellulose crossed into it, making were from different microbial sources, including fungi, bacteria, and lactic acid bacteria cellulases [12][13][14]. Although this would be advantageous for industrial applications, the synergistic effect of cellulase produced in wild strain could not be well explained. So, if the cellulases for synergy all come from the same strain and belong to different types, do they have a stronger effect? So, we tried to use the recombinant exocellulase cloned in this study to work together with another two cellulases, endocellulase and β-glucosidase, which also cloned from the same strain, to explore their synergy effect.
In this study, a novel exocellulase Cbh-A was successfully obtained from the strain Bacillus substiles 1AJ3 by cloning and expression, and its properties were studied. Herein, an endonuclease and a βglucosidase which were also cloned and expressed from the same strain B. substiles 1AJ3 in our former work. The synergistic action between/among them were studied by enzyme activity test via a comparation of a variety of cellulose substrates such as CMC-Na, Avicel, and filter paper. Moreover, different lignocellulose biomass was used as substrates to verify the synergistic action of three types cellulases, and reducing sugar content was used as an indicator to study its synergistic effect in lignocellulose. These results showed the novel exocellulase had potential application ability. A synergy of three types of cellulases from one strain also gave us a new insight into the cellulase system. The broadness of substrates by three cellulase together shows good potential in developing cellulase cocktail due to its exquisite cooperation with various cellulase.

Results
Cloning, expression, and bioinformation analyze of Cbh-A The Cbh-A gene was successfully cloned and expressed as an active soluble recombinant protein in E.coli BL21(DE3). The recombinant exocellulase was verified by SDS-PAGE ( Fig. 1) A total of the whole ORF sequence including His-tag of Cbh-A was 897 bp, encoding 298 acids amino, whose theoretical molecular weight was 33.90 kDa. By blast in National Center for Biotechnology Information (NCBI) and analyzed domains on the website, Cbh-A contained two domains, FlgJ (21I-212L) and SH3_8 (213K-285E). It had 99.29% percent identification with exo-glucosaminidase LytG of Bacllus subtilis (WP_032726765.1). Theoretical pI of Chb-A was 7.0 by prediction, while the the instability index was 26.58, and the grand average of hydropathicity (GRAVY) was − 0.549. It showed that recombinant enzyme Cbh-A was hydrophilic with good stability protein.

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 [15]. 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 + , Mg 2+ , Fe 2+ , Fe 3+ , [NH 4 ] + , and chemical SDS were all had little effect on enzyme relative activity under different densities. Metal ion Mn 2+ could increase relative enzyme activity greatly in 10 mM, 5 mM and 1 mM, followed by Zn 2+ and Ca 2+ could increase Cbh-A relative activity to some extent under 10 mM and 5 mM. Cu 2+ 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.  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.

Discussion
A novel exocellulase cloned from B. subtilis In nature, three types of cellulases play a major role in the synergetic degradation of cellulose: that resulting from the cooperation of endocellulase and exocellulase to hydrolyze amorphous region and crystalline region of cellulose, with a final of beta-glucosidase hydrolyzing the low molecular cellulose into oligosaccharides. Because of crystalline cellulose, with dense structure and it is hard to hydrolyze in lignocellulose. Exocellulase as a cellulase aims at crystalline cellulose can hydrolyze cellulose chains from one end to another by hydrolyzing cellobiose once, but it was always ignored of its low hydrolyze efficiency. It had also been found that most of the recombinant exocellulase studied were derived from fungal microorganisms, such as Thermobifida fusca [11,16], Thermomonospora fusca [17], Orpinomyces sp. [18], Irpex lacteus [19], but exocellulase from bacteria was rarely reported, such as Clostridium thermocellum [20]. In this study, we cloned an exocellulase Cbh-A from Bacillus subtilis 1AJ3 to found out its cellulose hydrolyzation ability. Cbh-A is a 34 kDa recombinant enzyme which identified conclude two domains: catalytic domain FlgJ and binding domain SH3_8. However, FlgJ domain was always regarded as glucosaminidase [21,22] and peptidoglycan hydrolase [23] in previous studies, some with an exo acting function [24]. SH3 domain as a binding protein, but no studies so far had been reported that weather it had cellulose-binding function in cellulose hydrolyzation. It's the first time that FlgJ domain was shown to be an exocellulase and characterized its enzyme activity using the substrate of Avicel.
Cbh-A could not tolerate acidic or alkaline environment which led to enzyme denaturation and inactivation, while pH 6.0-7.4 would maintain its enzyme activity over 70%. Similar pH conditions for exocellulase was observed in Cel48S [20]. Cbh-A had the highest enzyme activity under 40-50 ℃, that it can combine with other enzymes with same temperatures. Additionally, when considering Cbh-A temperature stability, we found out a special character with it. Prewarm under 50℃ for 4 h will make Cbh-A present remain at its maximum activity. It sighed that Cbh-A had a more changeable structure that longer time needed to stretch itself. A FlgJ belonging to GH73 family also has been observed that it flexible owing to could not be completely modeled into the electron density [25].
Mn 2+ could increase Cbh-A activity to a large extent, and EDTA and high density of Cu 2+ (5 mM and 10 mM) could inhibit its activity. Thus, Cbh-A has widely metal ion suitable ability that could provide suggestions in industrial application. Moreover, Cbh-A had widely suitability of substrate, that it could hydrolyze β-1,3 − 1,4 glucan linkage and α-1,4 glucan linkage in cellulosic material. These characters suggested that Cbh-A had promising potential application ability in cellulosic material hydrolyzation, no matter purify cellulose or natural complex cellulose such as biomass lignocellulose.

Synergy effect of Cbh-A with Cel-5A and Bgl-16A
In previous studies, the synergy effect among cellulases was from different microbes, even different species, in which fungal cellulases were the majority or some consist of commercial cellulases [26], some were mixed with bacterial cellulase [27]. And in this study, for the first time, the synergy effect of three different types of cellulases derived from the same bacterium was studied. Three cellulose substrates were chosen to evaluate the hydrolyzation synergic ability of cellulase, in which CMC-Na stands for amorphous cellulose, Avicel present crystalline cellulose, while filter paper was complex cellulose consist of amorphous and crystalline cellulose. When Cel-5A, Cbh-A, and Bgl-16A hydrolyzed the cellulose by itself, the results of the enzyme activity (Table 3) indicated that Cel-5A had high enzyme activity in CMC-Na and less in Avicel. Oppositely, Bgl-16A had higher enzyme activity in Avicel than that in CMC-Na. Cbh-A showed its equal hydrolyzation ability in different substrates.
Another interesting thing is that three enzymes had similarity enzyme activity when hydrolyzing Avicel. It mostly probably because of the structure of CMC-Na was more loosen that suitable for endocellulase hydrolyze beta-1,4 glucan linkage randomly. But Avicel with compact structure prevented endocellulase attachment and action. β-glucosidase was aimed at cellobiose and cellotriose further degraded into glucose [18], so when it singly hydrolyzes cellulose substrate, no substrates were provided for it. However, β-glucosidase had higher enzyme activity than that in CMC-Na, it should be attributed to Avicel was insoluble, easy to gathered than CMC-Na for β-glucosidase to connected, while enzyme proximity is the most important factor for activity enhancement [28], and βglucosidase don't have cellulose binding domain. While cellulase mixed with another, things got much more complex. Endocellulase and exocellulase cooperated well, especially in filter paper, DS value reached 1.703. Similarly, endocellulase have been observed predominates in the initial stage of hydrolysis when hydrolyze filter paper with exocellulase [29]. And it was observed that when considered a mixture ratio and adjust the combination rations of endocellulase and exocellulase into 5, the maximum DS reached 1.8 [30] also indicated that Cel-5A and Cbh-A in this study had efficiency synergism. It also suggests the exocellulase could help to released crystalline cellulose chains and make it structure getting loosen, which benefit for endocellulase to have a chance acting on glucan linkage. Furthermore, endocellulase could produce more shorter cellulose chains for exocellulase.
Exocellulase and β-glucosidase had stabilized cooperation effect in hydrolyzing, while exocellulase can produce more available substrate for β-glucosidase and β-glucosidase can remove substrate inhibition for exocellulase [31]. Exocellulase was the speed limit factor for β-glucosidase. When three enzymes mixed, they all could get better synergism and the DS of more difficult hydrolyze cellulose substrate, filter paper and Avicel, over 1.5. Additionally, we have to consider of the synergy effect among different types of cellulase is complicated, depending on such factors as their ratio, enzyme concentration, reaction time, and also the substrate characteristics [32,33], so the maximum of three types in this study still need further study. All the results suggesting the cellulases from B. subtilis 1AJ3 has the ability of good synergism for biotechnological applications.
Due to these three recombinant cellulases belong to different types, and they have absolutely synergy effect with each other and all together, we tried to enlarge the substrates ranges to natural biomass lignocellulose, evaluate their synergism in various cellulosic materials. Therefore, solo cellulase and three cellulases mixture were applied to the ten different common biomass materials, with the same ratio of that in the CMC-Na, Avicel, and filter paper above. The statement of the raw materials and the state after hydrolysis were shown in Fig. 4. Not all the biomass substrates can be well hydrolyzed. The degree of hydrolysis is not only related to the single or mixed action of the enzyme, but also the raw material [34]. Switchgrass was much easier to hydrolyze [35], also with the maximum saccharification rate of 9.62% of total solid. Studies had also proved that it is considered the most promising cellulosic material as an energy crop due to its high yield, high nutrient use efficiency and its wide growth region [36,37]. Then followed corn cob, which had an 8.72% saccharification ratio hydrolyzed by three enzymes mixture. All of the biomass substrates, peanut shell is the most difficult to hydrolyze, no matter by single enzymes or mixture enzymes, it kept stubborn. It mainly owing to peanut shell is suborned, dense, and hard structure, no pretreatment made it much harder to hydrolyze. Crude enzymes from Aspergillus aculeatus Iizuka FR60 has been reported that could hydrolyze peanut shell effectively, but the peanut shell still had been pretreatment by removing lignin [38]. Secondly, size of cellulose fiber also could impact cellulase synergy [39]. Besides, cellulase has substrate specificity during the hydrolysis process, even if it is synergistic [40]. Corn stalks hydrolyzation perfectly exhibits the synergism of three cellulases, with the DS value of it reached 184, and the meantime with a saccharification rate of 5.74%. The same phenomenon was observed in wheat straw, rice husk, sugarcane stalk, and coffee ground, they all present the cooperation effect of three enzymes than a single one. A difference of synergism effect ability suggesting that enzymes in this study have substrate specificity. However, considering maximizing of the effects of each enzyme to achieve the larger degradation and utilization of biomass, the method of synergistic of three enzymes should be optimized. In this study, three enzymes were mixed in 1 × PBS buffer and added into a biomass substrate for synergistic degradation test. Different adding order or batch hydrolyzation should be considered in further studies because of enzyme reaction was under different suitable pH. Besides, other enzymes should be attempt to add into the enzymatic hydrolysis reaction system, such as xylanase, lignin-degrading enzymes while the presence of hemicellulose and lignin could prevent cellulose hydrolyzation.

Conclusion
For a better understanding of exocellulase from bacteria, a novel exocellulase Cbh-A was successfully cloned and expressed from B. subtilis 1AJ3. It consisted of two domains and had high enzyme activity when pre-warm under 50℃ for 4 h. Maximum enzyme activity was observed at pH 6.4, 50℃. Mn 2+ could increase enzyme activities, while Cu 2+ and EDTA had inhibited affection. It could hydrolyze β-1,3 − 1,4/β-1,3/β-1,4 glucan linkage, α-1,4 glucan linkage efficiently, but had no affection on α-1,6 glucan linkage. Cbh-A had synergism effect with endocellulase and β-glucosidase cloned from the same strain in different types of cellulose. Not only endocellulase produces more shorter cellulose chains for exocellulase, but also exocellulase hydrolyzes crystalline cellulose loosen for endocellulase. β-glucosidase enzyme activity was limited by exocellulase, and take a stable degree of synergy effect with exocellulase. Three cellulase synergism efficient in simple cellulose as well as complex biomass lignocellulose without pretreatment by DS value: corn stalks, wheat straw, rice husk, sugarcane stalk, and coffee ground. Besides, the mixture cellulase performed well in switchgrass, corn cob, and corn stalks, which presented good saccharification rates. All results suggest that the recombinant exocellulase is promising in industrial applications. Synergism effect of three types of cellulases from the same bacterium could cooperate well, and they have substrates specification. It provides the basis for the universal application of enzymes, and also provides new ideas for the comprehensive development and utilization of biomass energy.

Strains, medium, and chemicals
Mountains (China) in our previous work was growing in LB medium at 37 ℃, and its' genomic DNA was used as cellulase cloning templet. The plasmid of pET-28a(+) was used for recombinant vector construction. The E.coli BL21(DE3) was used for recombinant enzyme expression grow in LB medium with 100 µg/mL Kanamycin (Kan).
Whatman No.1 filter paper was purchased from GE Healthcare (Stockholm, Sweden), and 6 mm in diameter holes were obtained with a puncher in the same size. Avicel (PH-101), CMC-Na, barley glucan, laminarin, pullulan, and xylan from beechwood purchased from Sigma-Aldrich (St. Louis, USA).
Other chemicals in this study were all of the analytical grade.
Lignocellulose biomass, conclude switchgrass, wheat straw, corn stalks, corn cob, rice husk, sugarcane stalk, pea straw, ginger stems and leaves, peanut shell, and coffee ground were crashed into 10 mesh and dried out under 60 ℃.

Enzyme activity and reducing sugar content assay
Reducing sugar content was determined by DNS (3,5- Exocellulase activity (Avicelase), endocellulase activity (CMCase), and filter paper activity (FPase) were carried out by using 1% Avicel, CMC-Na and filter paper as a substrate separately, and reacted with the enzyme at 50 ℃ for 30 to 60 min, and then reducing sugar content of reaction solution was measured by DNS method.

Characterization of Cbh-A
The optimum pH condition of Cbh-A was determined at 50 ℃ for 60 min using the various buffers ranging from pH 2.4 to 11.0. For the pH stability assay, the enzyme was incubated in ice-water for 30 min without substrate and then measured the enzyme activity at 50 ℃ for 60 min.
The optimum temperature of Cbh-A was measured at optimum pH under different temperatures from 30 to 80 ℃ (10℃ intervals) for 60 min. For the thermal stability of Cbh-A, the enzyme was incubated at different temperatures for 30 min and measured the enzyme activity under optimum temperature and pH for 60 min.
The effect of metal ions and chemicals on enzyme activity was assayed in buffer with various metal salts, including 1, 5, and 10 mM of metal ions and chemicals.
Substrate specificity was measured by using different substrates at 1% concentration: Avicel, pullulan, maltose, CMC-Na, filter paper, barley glucan, laminarin, xylan from beech wood. The enzyme reaction was performed at 50 ℃ for 60 min in a buffer of pH 6.0. Measuring the enzyme activity and calculating relative activity in different substrate based on the Avicel activity was set to 100%.
Special characteristic of Cbh-A-pre-incubation at 50℃ can increase enzyme activity Surprisingly, Cbh-A had higher enzyme activity when it was incubated under 50 ℃ for 30 min than in ice-water before measurement. To observe whether pre-warm up at 50 ℃ can help to increase the enzyme activities, we measured the enzyme activity of pre-incubation at 50 ℃ over time. Enzyme activity of Cbh-A was measured every ten minutes within one hour, and every hour after one hour. Set the enzyme activity at 1 h as 100% and calculate the relative enzyme activity at other times.

Assessment of synergy effect
Cel-5A (GenBank: MN795058) is an endocellulase containing a GH5 catalytic domain (CD) and a CBM3 family Cellulose binding domain (CBD). Its optimum pH is 4.5 and has a wide region of pH from 4.5 to 9.0, the relative enzyme activity all over 80%. It has an optimum temperature of 50 ℃. Bgl-16A (GenBank: MN795059) is a β-glucosidase belongs to GH16 family. Its maximum enzyme activity condition is at pH 8.6 and 50 ℃. From pH 7.0 to 9.0, it can retain over 80% relative enzyme activity.
Cbh-A (GenBank: MN795060) in this study, has an optimum enzyme reaction condition of pH 6.0 and 50℃. It had a wide pH work region from 6.0 to 7.0. Based on the three enzymes suitable enzyme reaction conditions, we chose 1 × PBS (pH 7.2-7.4) buffer as an enzyme reaction system buffer, and all enzyme reaction was measured at 50 ℃ in further study (Fig. 5).
To measure the synergy between cellulases in the reaction process, we defined the "Degree of synergism" (DS) as the evaluation index.

DS = Un/(U1 + U2+…+Un)
In which, Un stands for enzyme activity of mixture enzymes. U1 to Un stands for enzyme activity by a single enzyme. When the DS value is greater than 1, it indicates that there is a synergy effect between/among the cellulases, and the larger the value, the stronger the synergy. When the DS value is less than 1, it indicates that the enzymes do not have a synergistic effect, and may even inhibit each other or produce an antagonistic effect. Three cellulases of crude enzyme solution were obtained by expression in advance. For better understanding and evaluating the synergy effect between the three enzymes and their synergistic ability in different types of cellulose substrates, three cellulose substrates were chosen for this part, CMC-Na, Avicel, and filter paper. Total enzyme reaction system contained 100µL substrate (within 1 × PBS buffer) and 100µL enzyme (mixtures) and enzyme action process take under 50℃. Exact enzyme adding volume for single or mixture was according to Table 3.

Synergy effect of cellulase in biomass
A same volume ratio of each enzyme (three enzyme mixtures) was added into different lignocellulose biomass to make the final reaction system of 5% (m/V) of straw. Blank was adding 1 × PBS buffer without any enzyme. Total reducing sugar content was measured via a DNS method of each reaction system and observed the phenomenon of each lignocellulose biomass. The saccharification rate (= Total reducing sugar content (mg) / total biomass quality (mg) × 100% ) was calculated to compare each process. Besides, for a better evaluation of enzyme's degradation ability and cellulose substrates adaption, ten biomass materials were selected in this part (switchgrass, wheat straw, corn stalks, corn cob, rice husk, sugarcane stalk, pea straw, ginger stems and leaves, peanut shell, and coffee ground).

Statistical analysis
All experiments in this study were performed three times. Data were analyzed using Origin 9.2 statistical analysis software, and results were presented as mean ± standard deviation (SD) value.  Tables Table 1 Effect of metal ions and chemical on the cellulase activity of Cbh-A Activity without any metal ion or chemicals set as 100%.

Table 2
Substrate specificity analysis of recombinant enzyme and the original enzyme Depending on the substrate, activity was determined under optimal conditions. ND, no detectable activity. Table 3 Synergistic effect reaction system and their DS value in different substrates.
No. Component of cellulase enzyme system Enzyme activity in separate system and DS value of enzymes in different substrates e CMC-Na 1% CMC-Na, 2 piece of 6mm-diameter circle filter paper, and 1% Avicel, respectively; f Enzyme activity of separate system; g Degree of synergism All the enzyme used in this part were all crude enzyme and the same amount of each cellulase in all enzyme systems.     Pictures of raw biomass lignocellulose and samples of enzymes hydrolyzations. Every group have five tubes, and lignocellulose were hydrolyzed by different enzyme(s) from left to right: blank, Cel-5A, Cbh-A, Bgl-16A, and three enzymes mixture. a-j presents different biomass which was marked on the picture followed the letters.

Figure 5
Sketch map of cellulases synergy effect.