Identification and characterization of Tswo gene
The ORF of putative Tswo consisted of six exons, encoding a deduced protein (TlSWO) consisting of 503 amino acids and a signal peptide at the cleavage site between amino acids 20 and 21 (SignalP 4.1 server). The cloning sequence of putative TlSWO was submitted to NCBI GenBank as MT180127. The predicted product shows the highest sequence similarity to the amino acid sequence of the known swollenin which are from Aspergillus fumigatus or Penicillium oxalicum. Further analysis by PROSITE (http://br.expasy.org/prosite) demonstrated that TlSWO consists of three domains, fungal-type carbohydrate-binding module family 1 (CBM1) (amino acids 23−59), family 45 endoglucanase-like domain of expansin (Expansin_EG45) (amino acids 206−388) and a cellulose-binding-like domain of expansin (Expansin_CBD) (amino acids 400−492), which are typical of the swollenins from fungi (Fig. 1). In the CBM1 of TlSWO, six cysteines were highly reserved same as GH6 cellobiohydrolase. Disulfide bond prediction by DiANNA (DiANNA 1.1 web server) showed that there are three disulfide bonds in the CBM1 of TlSWO (Cys4-Cys21, Cys11-Cys28, Cys22-Cys28). CBM1 and Expansin_EG45 are connected by a Serine -Threonine rich linker domain. Although the function of linker has been well studied in cellulases, it was not clear if linker play the same role in swollenins as that of cellulases. Sequence alignment of swollenins showed that TlSWO maintained the conserved HMD (histidine, methionine, aspartic acid) catalytic motif of GH45 cellulase (HFD, histidine, phenylalanine, aspartic acid), which is part of the active site (Fig. 1). In HFD, aspartic acid is the proton donor during the catalytic process in GH45 cellulase. However, the other catalytic active site aspartic acid is absent in swollenin and part of GH45 cellulases. In the C-terminal region of TlSWO, Expansin_CBD is homology to pollen allergen. There are a few conserved aromatic amino acids in the sequence, i.e. Y400, Y401, F402, W429, Y447, W450, Y496 and F503, which may play a key role in binding substrates (Fig. 1).
Expression of TlSWO in P. pastoris GS115 and T. reesei AST1116
The expression was done using the aox1 promoter and eno promoter in P. pastoris and T. reesei,respectively. Following high-throughput fungal expression and screening, a large set of colonies resulting in SDS bands after transformation were identified. The results revealed that recombinant TlSWO (483 amino acid residues, 51.1 kDa and a theoretical isoelectric point of 4.45) was successfully expressed using P. pastoris and T. reesei (Figure S1). The purified swollenin protein migrated as an about ∼80 kDa protein in SDS-PAGE. Since the difference between the SDS-band and the theoretical molecular weight, the single band was analyzed by MALDI-TOF MS and the trypsin-digested peptide sequences were matched to the deduced amino acid sequences of TlSWO (Figure S2). Sequence prediction suggested that there are five N-glycan sites on TlSWO (Asn35, Asn154, Asn249, Asn366 and Asn436). After Endo H digestion, TlSWO decreased in molecular weights to ∼72 kDa༌still higher than calculated value (Figure S1A). We speculate that the rest of the molecular weights increase is caused by heavily O-glycan glycosylation in the linker region which is rich in serines and threonines.
Activity of TlSWO on different substrates
The cellulolytic activity of TlSWO was measured with lichenan, barly β-glucan, CMC-Na, laminarin, avicel, glucomannan, and the xylanase activity was measured with birchwood xylan, and the mannase activity was measured with locust bean gum. All the reaction were carried out overnight. As a result, TlSWO only showed significant activity on lichenan, barly β-glucan, glucomannan, CMC-Na and a very low activty on laminarin. However, we didn’t observe any reducing sugars released from the other substrates. We further examined the specific activites of TlSWO with lichenan, barly β-glucan, CMC-Na as the substrate. TlSWO showed the highest activity on lichenan (9.0 ± 0.100 U/mg) and barley β-glucan (8.9 ± 0.100 U/mg) which were followed by CMC-Na (2.3 ± 0.002 U/mg). On the other hand, only slight activity against laminarin (0.79 ± 0.002 U/mg) was found (Fig. 2). These findings suggest that TlSWO mainly acts on cellulose rich substrates and showed a preference on the substrates that contain β-1,3 − 1,4 linkages.
Mode of action of TlSWO
The mode of action of TlSWO was also assessed using lichenan,barly β-glucan༌CMC-Na and laminarin as substrates. As a result, CMC-Na was hydrolyzed to cellobiose and small amount of cellotriose (Fig. 3). Analysis of the hydrolysis products of lichenan, barly β-glucan showed that TlSWO preferentially hydrolyzed these two substrates to products with different degrees of polymerization, including cellobiose and cellopentose, followed by cellohexose and cellotetrose (Fig. 3). We detected no sugar release after incubating TlSWO with laminarin (Fig. 3). These results suggested that TlSWO may have a mixed function of endo-cellulase and exo-cellulase.
Effect of temperature and pH on TlSWO
The effect of pH and temperature on TlSWO activity were further investigated with lichenan as substrate. As a result, we determined that the optimum pH is 4.0 but the enzyme showed activity over a broad pH range of 2.0–12.0 (Fig. 4A). As for pH stability, TlSWO retained more than 80% of its activity in pH range 2.0–9.0 after incubation at 37 ℃ for 1 h, but lost 30% of its activity when incubated at pH 10.0–12.0 (Fig. 4B). In addition, TlSWO was optimally activity at 50 ℃ and retained more than 90% activity in the range of 40–60 ℃, but rapidly lost its activity above 70 ℃ (Fig. 4C). As we can see from Fig. 4D, TlSWO was stable at a temperature of 37 ℃ and 50 ℃ after incubation for 1 h, however, when the temperature was raised to 70 ℃, the activity was dramatically decreased to 40% after incubation for 10 min.
Disruptive action of TlSWO on Avicel
The disruptive effect of TlSWO on Avicel was evaluated using light microscopy and scanning electron microscopy. The result of light microscopy analysis showed that after incubation with different amounts of TlSWO for 24 h, the physical structure of Avicel tend to be significant different with the untreated Avicel (Fig. 5). As we increased the amount of TlSWO, Avicel was disrupted to small particles. The sample that was pretreated with 300 µg of TlSWO for 12 h was taken out for further analysis using scanning electron microscope. As we can see, TlSWO created a rough and amorphic surface on Avicel compared with the unpretreated sample (Figure S3).
Synergism between TlSWO and cellulases
To test the capacity of TlSWO to enhance biomass hydrolysis by enzymatic cocktail, we hydrolyzed pretreated biomass using cellulases alone and also supplemented by TlSWO. Biomass degradation experiments were performed using β-galactosidase (EC 3.2.1.21), cellobiohydrolase (EC 3.2.1.91) and endoglucanase (EC 3.2.1.4) in the presence of TlSWO. The reactions with BSA or without TlSWO were conducted as control. The total protein was 13 mg protein/g of glucan in all the reactions. Since endoglucanase randomly cleaves the internal β-1,4-glycosidic bonds, cellobiohydrolase processively acts on the chain termini to release cellobiose, and β-glucosidase that hydrolyzes cellobiose to glucose [31], thus the production of glucose was compared in different reactions.
The results showed that TlSWO exhibited significant synergetic effects on cellobiohydrolase Cel7A when using PCS as the substrate. Initially, we measured the quantity of glucose of the reaction which contained only 13 (mg protein/g of glucan) of TlSWO. As a result, no glucose was detected in the reaction, suggesting that PCS could not be hydrolyzed by TlSWO (Fig. 6). The PCS conversion achieved in 24 h, 48 h, 72 h, 96 h and 120 h were 8.9%, 13.1%, 14.8%, 15.5% and 16.4% respectively when the reaction contained Cel7A and β-glucosidase alone, while they reached 11.2%, 18.2%, 21.6%, 23.8% and 26.4% when Cel7A and β-glucosidase was combined with 2 (mg protein/g of glucan) TlSWO. Although TlSWO action alone did not lead to any detectable levels of released glucose, the enzyme addition to Cel7A and β-glucosidase led to a significantly enhanced hydrolytic activity of the cocktail on PCS.
PASC and CNC exist in amorphous and crystalline forms, respectively, which may affect the binding of TlSWO to them. Therefore, the action of TlSWO for the two substrates were further examined. Similar to the results of PCS, TlSWO could not release any sugars from PASC and CNC when used alone (Fig. 7). When utilizing Cel7A and β-glucosidase the conversion results showed that PASC conversion rate reached 9.9%, 19.6%, 29.2%, 37.5% and 40.2% in 24 h, 48 h, 72 h, 96 h and 120 h respectively (Fig. 7A). However, when Cel7A and β-glucosidase were supplemented by TlSWO the conversion rates increased to 33.0%, 53.1%, 61.9%, 67.8% and 72.2% at the five time points respectively. Looking at the 120 h, time point the conversion rate of PASC was increased approximately 32% compared with Cel7A and β-glucosidase alone. This result suggests that TlSWO exhibited significant synergetic effects with Cel7A. In addition, the synergetic effects of TlSWO and endoglucanases were also explored during this research. When using endoglucanase and β-glucosidase alone the conversion rate was 51.4%, 65.7%, 76.5%, 82.0% and 85.6% at 24 h, 48 h, 72 h, 96 h and 120 h. When used in combination with TlSWO, the conversion rate reached to 58.6%, 73.6%, 80.4% and 85.7% in the first four time points. Although these four values were a little higher than that of endoglucanase and β-glucosidase alone, the value at 120 h was 86.4%, which was close to 85.6%. Therefore, the addition of TlSWO did not provide a significant increase of enzymatic hydrolysis of PASC when used in combination with endoglucanase.
The cellulose conversion rate of CNCs was significantly lower than that of PASC. Our results showed that on CNCs, the Cel7A and β-glucosidase achieved conversion in 24 h, 48 h, 72 h, 96 h and 120 h was 28.2%, 36.7%, 43.7%, 51.3% and 59.3% respectively (Fig. 7B). This conversion rate was also increased when TlSWO was added. The glucose yields obtained by the cocktail enzyme systems containing TlSWO and Cel7A and β-glucosidase were 31.9%, 45.1%, 54.3%, 62.4% and 68.9% respectively, higher in all cases when compare with the control. This result suggests that TlSWO showed a synergistic effect with processive cellobiohydrolases; however, when used in combination with endoglucanases, TlSWO did not present any significant synergistic effect. After 120 h of enzymatic hydrolysis, the yield of glucose released from CNCs was 16.4% when combined use of TlSWO, endoglucanase and β-glucosidase, which corresponds with the hydrolysis rate of using endoglucanase and β-glucosidase alone (14.9%). Comparing with the results of PASC, we suggest that TlSWO will act on amorphous cellulose more efficiently than the crystalline form.