Abstract
Caldicellulosiruptor bescii is a cellulolytic bacterium that secretes multifunctional glycoside hydrolases for efficient hydrolysis of lignocellulose into fermentable sugars. Additionally, some abundant noncatalytic proteins accompanying multifunctional glycoside hydrolases are also secreted by C. bescii, but its function has not yet been demonstrated. In this study, noncatalytic protein Athe_0181 and multifunctional glycoside hydrolases CbMan5C/Cel5A were expressed and purified from Escherichia coli BL21(DE3). Effective binding capacity of Athe_0181 to lignocellulose was displayed, and it showed preferential affinity to rice straw. Athe_0181 was shown to be a cellulase synergistic protein. It exhibited high synergistic activity of 523% in the presence of 25 μg/mL of CbMan5C/Cel5A with microcrystalline cellulose as the substrate. The structure-modifying activity of Athe_0181 to microcrystalline cellulose was demonstrated by scanning electron microscopy and X-ray diffraction analysis. These characteristics demonstrated that Athe_0181 played a role in the synergism of glycoside hydrolases from C. bescii for efficient hydrolysis of lignocellulose.
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Functional Characterization of a Noncatalytic Protein, Athe_0181, from Caldicellulosiruptor bescii in Promoting Lignocellulose Hydrolysis
Xiaoyan Zhu,a,b,* Shanshan Xin,b Hao Ding,b Yang Yang,b Yiling Chen,b Xiangqian Li,a,b* Hao Shi,a,b Zhongbiao Tan,a,b Jia Zhou,a,b and Pei Liu a,b
Caldicellulosiruptor bescii is a cellulolytic bacterium that secretes multifunctional glycoside hydrolases for efficient hydrolysis of lignocellulose into fermentable sugars. Additionally, some abundant noncatalytic proteins accompanying multifunctional glycoside hydrolases are also secreted by C. bescii, but its function has not yet been demonstrated. In this study, noncatalytic protein Athe_0181 and multifunctional glycoside hydrolases CbMan5C/Cel5A were expressed and purified from Escherichia coli BL21(DE3). Effective binding capacity of Athe_0181 to lignocellulose was displayed, and it showed preferential affinity to rice straw. Athe_0181 was shown to be a cellulase synergistic protein. It exhibited high synergistic activity of 523% in the presence of 25 μg/mL of CbMan5C/Cel5A with microcrystalline cellulose as the substrate. The structure-modifying activity of Athe_0181 to microcrystalline cellulose was demonstrated by scanning electron microscopy and X-ray diffraction analysis. These characteristics demonstrated that Athe_0181 played a role in the synergism of glycoside hydrolases from C. bescii for efficient hydrolysis of lignocellulose.
DOI: 10.15376/biores.17.2.3067-3081
Keywords: Athe_0181; CbMan5C/Cel5A; Glycoside hydrolase; Lignocellulose hydrolysis; Noncatalytic protein; Synergism
Contact information: a: Jiangsu Provincial Engineering Laboratory for Biomass Conversion and Process Integration, Huaiyin Institute of Technology, Huaian 223003, China; b: School of Life Science and Food Engineering, Huaiyin Institute of Technology, Huaian 223003, China;
*Corresponding author: zhuxyle@163.com; lixq2002@126.com
INTRODUCTION
Lignocellulose is the most abundant and renewable bioresource for producing biofuels and biochemicals, and microbial glycoside hydrolases (GHs) are essential in the conversion process (Liu et al. 2021). However, complex structure and the recalcitrance of lignocellulose play a role as barriers for enzymatic hydrolysis (Kahn et al. 2020). Caldicellulosiruptor bescii, an anaerobic and Gram-positive thermophile, is able to grow at temperatures up to 90 °C (the optimal growth temperature is 78 °C), and it can efficiently degrade crystalline cellulose, xylan, and untreated biomass (Poudel et al. 2018). The secretome of C. bescii is more efficient than Trichoderma reesei in hydrolyzing microcrystalline cellulose, untreated timothy grass, and rice straw (Kanafusa-Shinkai et al. 2013). Six multifunctional GHs, namely CelA (Athe_1867, CbCel9A/Cel48A), CelB (Athe_1859, CbMan5B/Cel44A), CelC (Athe_1857, CbXyn10C/Cel48B), CelD (Athe_1866, CbMan5C/Cel5A), CelE (Athe_1865, CbCel9B/Man5A), and CelF (Athe_1860, CbXyl74A/Cel48C), play essential roles in the deconstruction of lignocellulose (Dam et al. 2011; Ye et al. 2012; Xue et al. 2015; Conway et al. 2018). The multi-domain architecture of these multifunctional GHs are commonly two or three cellulose binding carbohydrate binding module (CBM) domains surrounded by two catalytic GH domains. Most of multifunctional GHs or single domain of multifunctional GHs have been characterized (Su et al. 2012; Ye et al. 2012; Yi et al. 2013; Xue et al. 2015; Rong et al. 2016; Chu et al. 2019).
Proteomic studies revealed that C. bescii possesses not only an array of abundant GHs, but also some noncatalytic proteins binding to carbohydrate substrate (Lochner et al. 2011; Yokoyama et al. 2014; Poudel et al. 2018). Only few studies on these noncatalytic proteins were characterized. The binding properties of noncatalytic proteins Athe_0847 and Athe_0597 were investigated, and both proteins showed the highest binding affinity for the plant cell wall among the insoluble polysaccharides. The binding of these noncatalytic proteins might be necessary for efficient utilization of polysaccharides by C. bescii at high temperatures (Yokoyama et al. 2014). Tāpirins (Athe_1870) showed the binding ability to microcrystalline cellulose, switchgrass, poplar, and filter paper (Lee et al. 2019). Type IV pilus (T4P) was demonstrated to play a role in attachment to crystalline cellulose and xylan (Khan et al. 2020). CbHsp18 from C. bescii was reported to enhance the hydrolysis activity and thermostability of glycoside hydrolases CbCelA-TM1(GH9-CBM3C), CbXyn10A and CbCdx1A (Su et al. 2012). Noncatalytic protein Athe_0181 of unknown function were identified in the C. bescii proteins binding to the cell walls of timothy grass. It is one of the most abundant proteins in the extracellular secretome of C. bescii specific for growth on complex substrates (xylan, switchgrass, and Avicel), which likely have crucial roles in the deconstruction or utilization of complex substrates (Poudel et al. 2018). Synergistic proteins without significant hydrolytic activity on cellulose, such as swollenin and EXLX1, could act as accessory or helper agents to promote the efficiency of enzymatic hydrolysis of lignocellulose (Kim et al. 2014). Briefly, the current authors propose that the function of Athe_0181 may work as a synergistic protein to GHs of C. bescii in the hydrolysis of lignocellulose.
In this study, Athe_0181 and CbMan5C/Cel5A were expressed and purified from Escherichia coli BL21(DE3). Then, the lignocellulose binding capacity of Athe_0181 and the synergism of Athe_0181 to CbMan5C/Cel5A hydrolyzing lignocellulose, as well as the structure-modifying activity of Athe_0181 to microcrystalline cellulose were determined.
EXPERIMENTAL
Materials
Bacterial strains, plasmids, and regents
Plasmid pET22b (+) and pET-28a (+) were used for expression. E. coli BL21 (DE3) was utilized as the expression host. The components of the Luria-Bertani (LB) medium used for culture were from Oxoid Ltd. (Basingstoke, England). Additionally, 3,5-dinitrosalicylic acid (DNS), microcrystalline cellulose, and protein marker were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Isopropyl β-D-1-thiogalactopyranoside and (IPTG) and carboxymethyl cellulose (CMC-Na) were provided by Sigma Aldrich (St. Louis, MO, USA). Konjac glucomannan and cellobiose were purchased from Megazyme (Bray, Ireland). Lastly, a His-Bind Purification Kit was procured from Novagen (Beijing, China).
Methods
Protein expression and purification
The amino acid sequences of Athe_0181 (GenBank Accession No. ACM59333) and CbMan5B/Cel44A (GenBank Accession No. ACM60954) were analyzed by signalP-5.0 Server. Proteins homologous to Athe_0181 were obtained by Basic Local Alignment Search Tool (BLAST, programwww.ncbi.nlm.nih.gov/BLAST/). Athe_0181 was reported as unsuccessfully expressed in E. coli, probably as a result of its cytotoxicity (Yokoyama et al. 2014). PET22b (+) was employed as the expression plasmid in this study. Gene Athe_0181 and CbMan5C/Cel5A via codon optimization were synthesized by Shanghai Generay Biotech Co., Ltd. (Shanghai, China) and connected to the pET22b (+) and pET28a (+), respectively, and the recombinant plasmids pET22b (+)-0181 and pET28a (+)-CbMan5C/Cel5A were obtained. The recombinant plasmids were transformed into E. coli BL21(DE3), which were then spread on LB plates containing ampicillin at 80 μg/mL. After overnight culture at 37 °C, the monoclonal cells were inoculated into LB liquid medium (80 μg/mL ampicillin) and grown at 37 ℃ (200 rpm) overnight, and then were transferred to fresh liquid LB medium (80 μg/mL ampicillin). Protein expression was induced by adding IPTG at a final concentration of 0.05 to 0.5 mM when OD600nm of cultures reached 0.6-0.8, and then grown at 16 °C for 6 to 12 h. The E. coli cells were harvested by centrifugation (5000 × g for 10 min at 4 °C) and the recombinant proteins were purified as described below.
The cells were crushed by sonication and then were centrifuged at 10000 × g for 30 min at 4 °C. The crude enzyme solution was filtered through 0.45 μm membrane filter. The target proteins were purified using His-Bind Purification Kit with 1mL nickel affinity column chromatography, eluted with different concentrations of imidazole (20 to 400 mM) in 20 mM Tris-HCl buffer (150 mM NaCl, pH 7.5) at a flow rate of 1 mL/min. The molecular mass of protein and purity were determined by 12% sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) kit (Sangon Biotech, Shanghai, China). The protein concentration was assayed by a Bradford Assay Kit (Sangon Biotech, Shanghai, China). The purified proteins were used in the subsequent assays. All assays were performed in triplicate, and error bars represented the standard error of the mean.
The binding capacity of Athe_0181
Athe_0181 (300 μg/mL, final concentration) was incubated with lignocellulose containing microcrystalline cellulose, rice straw, and palm kernel meal (20 mg/mL, final concentration) in 50 mM citrate buffer (pH 5.0) for 24 h at 70 °C. After centrifugation, protein unbound to the lignocellulose in the supernatant were assayed by a Bradford Assay Kit. The amount of bound protein was calculated as by subtracting the unbound from the total amount of protein in the supernatant from the total protein (Lin et al. 2013).
Synergism of Athe_0181 and CbMan5C/Cel5A in hydrolysis of lignocellulose
Synergism reactions were performed with 25 to 300 μg/mL of CbMan5C/Cel5A and 25 to 300 μg/mL of Athe_0181 incubated in 50 mM citrate buffer (pH 5.0). Lignocellulose (20 mg/mL) containing microcrystalline cellulose, rice straw, and palm kernel meal were substrates. The reactions were performed at 70 °C for 4 to 48 h. The reaction mixtures with inactive protein were defined as the control. The released reducing sugars were quantified using the DNS method (Xia et al. 2019). Synergistic activity was calculated according to Eq. 1 (Meng et al. 2020),
Synergistic activity (%) = (B / A – 1) × 100 (1)
where A represents the amount of reducing sugars (μg/mL) released by CbMan5C/Cel5A alone, and B represents the amount of reducing sugars (μg/mL) released by CbMan5C/Cel5A and Athe_0181.
Scanning electron microscopy
Microcrystalline cellulose (20 mg/mL) was treated with 300 mg/mL of Athe_0181 at 70 °C for 48 h with samples only treated with 50 mM citrate buffer (pH 5.0) as the control, and then washed by deionized water. The morphology of microcrystalline cellulose was observed by scanning electron microscopy (SEM) (Sigma-500, ZEISS, Oberkochen, Germany).
X-ray diffraction (XRD)
The XRD patterns of microcrystalline cellulose treated by the same method as SEM, were measured by a smartlab X-ray diffractometer (Rigaku Corp., Tokyo, Japan) with the CuKα radiation generated at 40 kV and 150 mA. The scans were conducted in a 2θ range, from 5° to 50°, at a scanning rate of 2° per min. The cellulose crystallinity index (CrI) was calculated according to Eq. 2,
CrI = (Itotal – Iam) / Itotal (2)
where Itotal is the scattered intensity at the main peak (around 2θ = 22.5°) and Iam is the scattered intensity due to the amorphous portion (around 2θ = 18°) (Zhe et al. 2018).
RESULTS AND DISCUSSION
Protein Expression and Purification
In this study, Athe_0181 was abundantly expressed in soluble form in E. coli BL21 (DE3) (Fig. 1A).
Fig. 1. SDS-PAGE analysis of the expressed and purified Athe_0181: A: the expression of Athe_0181. Lane 1: cell lysate without induction; Lanes 2 through 5: cell lysate with induction (concentration of IPTG was 0.5 mM, 0.3 mM, 0.1 mM, and 0.05 mM, respectively); and Lane 6: protein molecular mass marker. B: the purification of Athe_0181. Lane 1: protein molecular mass marker; Lane 2: the purified proteins eluted with 50 mM imidazole; Lane 3: the purified proteins eluted with 20 mM imidazole; and Lane 4: the flow through fluid via nickel affinity column
The theoretical molecular mass of Athe_0181 without signal sequence is 62.15 kDa, which was consistent with the results of SDS-PAGE (Fig. 1). The purification process was conducted as shown in Table 1, which shows that the yield and purification fold were 47.9% and 11.6, respectively. The purified fraction of lane 2 (Fig. 1B) was further characterized.
Table 1. Purification Process of Athe_0181
The cellulolytic activity of Athe_0181 was investigated and no traceable amount of reducing sugar was detected when CMC-Na, cellobiose, konjac glucomannan, microcrystalline cellulose, palm kernel meal, and rice straw were incubated with Athe_0181 (data not shown). These findings indicated that Athe_0181 was definitely a noncatalytic protein to lignocellulose. Athe_0181 is annotated as extracellular solute-binding protein in databases. It contains an SBP bacterial family 1 (SBP bac 1) domain and UgpB domain (Yokoyama et al. 2014)). Extracellular solute-binding proteins from Caldicellulosiruptor changbaiensis, Caldicellulosiruptor saccharolyticus, and Caldicellulosiruptor acetigenus all showed the highest identity to Athe_0181 (83.5% amino acid sequence similarity). With the exception of the genus Caldicellulosiruptor, extracellular solute-binding protein from Treponema sp. displayed the highest identity of 44.2% to Athe_0181.
Binding Capacity and Synergistic Activity of Athe_0181
Although Athe_0181 was discovered as the plant cell wall-binding protein (Yokoyama et al. 2014), the binding capacity had not been characterized. It exhibited a binding capacity of 9.19, 9.77, and 13.2 μg/mg-substrate to microcrystalline cellulose, palm kernel meal, and rice straw, respectively (Fig. 2A). Athe_0181 displayed more preferential affinity to rice straw and the protein binding percentage was 88.2%.
The synergistic activity of Athe_0181 to CbMan5C/Cel5A was investigated. CbMan5C/Cel5A has been confirmed as a multimodular GH, which has a mannanase module at the N terminus and a cellulase module at the C terminus (Xue et al. 2015). The cellulase is capable of hydrolyzing soluble substrates such as CMC, barley β-Glucan, locust bean gum galactomannan, and 1,4-β-D-mannan (Conway et al. 2018). As shown in Fig. 2B, CbMan5C/Cel5A also showed hydrolysis activity to insoluble substrates, and Athe_0181 acted as helpers to promote CbMan5C/Cel5A activity. The amount of reducing sugar produced from microcrystalline cellulose, palm kernel meal, and rice straw by using a binary of 100 μg/mL of CbMan5C/Cel5A and 300μg/mL of Athe_0181 was greatly increased compared to CbMan5C/Cel5A alone, and synergistic activity reached 87.0%, 80.1%, and 73.2%, respectively. These results indicated that Athe_0181 worked as a synergistic protein to CbMan5C/Cel5A.
Fig. 2. The binding capacity and synergism of Athe_0181 and CbMan5C/Cel5A in hydrolysis of lignocellulose: A: the lignocellulose bound protein and B: synergism of Athe_0181 and CbMan5C/Cel5A in hydrolysis of lignocellulose (MC, PKM, and RS represent microcrystalline cellulose, palm kernel meal, and rice straw, respectively)
Synergism of Athe_0181 and CbMan5C/Cel5A in Hydrolysis of Microcrystalline Cellulose
Synergism of Athe_0181 to CbMan5C/Cel5A hydrolyzing microcrystalline cellulose was further investigated. It could facilitate the hydrolysis of microcrystalline cellulose, with a yield of 237 μg/mL of reducing sugars at 12 h, which was much more than CbMan5C/Cel5A alone (121 μg/mL) (Fig. 3), and the synergistic activity was 95.1%. The synergistic activity declined slightly with the extension of time, but the synergistic activity still achieved 53.8%, which was similar to the synergistic activity (51.5%) of swollenin POSWOI from Penicillium oxalicum at 48 h (Kang et al. 2013).
Fig. 3. Synergism of Athe_0181 and CbMan5C/Cel5A in hydrolysis of microcrystalline cellulose
Effect of Athe_0181 and CbMan5C/Cel5A Amount on Synergism in Hydrolysis of Microcrystalline Cellulose
The mixing ratio of synergistic protein to GHs is thought to be essential to exhibit synergism (Kim et al. 2014). Here, the relationship between the amount of Athe_0181 and the synergistic effect on CbMan5C/Cel5A hydrolyzing microcrystalline cellulose was studied (Fig. 4A). The reducing sugar yield was increased significantly with Athe_0181 concentration (0 to 50 μg/mL) (P<0.05), and the highest production was 299 μg/mL at an Athe_0181 concentration of 50 μg/mL and was 2.43 times of CbMan5C/Cel5A (100 μg/mL) in the absence of Athe_0181. With Athe_0181 concentration increased, no obvious change of reducing sugar yield was observed (P >0.05). This indicated that the synergistic effect is saturated, which is similar to the results of POSWOI and BsEXLX1 (Kang et al. 2013; Zhang et al. 2021b). Athe_0181 was shown to have strong binding capacity to the microcrystalline cellulose (Fig. 2), so there may be competition for substrate sites between Athe_0181 and CbMan5C/Cel5A.
As shown in Fig. 4B, the amount of reducing sugar rose as the concentration of CbMan5C/Cel5A rose from 25 to 300 μg/mL,and in coordination with Athe_0181, the production of reducing sugar was enhanced significantly (P <0.05). With the assistance of 50 μg/mL of Athe_0181, the reducing sugar yield increased from 19.3 to 120 μg/mL in presence of 25 μg/mL of CbMan5C/Cel5A. The highest synergistic activity reached 523%. While the higher concentration, such as 200 μg/mL of CbMan5C/Cel5A, resulted in the synergistic activity of 112.0%, indicating that Athe_0181 was more collaborative at a lower concentration of cellulase, which was consistent with the behavior of BsEXLX1 (Zhang et al. 2021b).
Fig. 4. Effect of Athe_0181 and CbMan5C/Cel5A amount on synergism in the hydrolysis of microcrystalline cellulose hydrolysis: A: effect of Athe_0181 amount and B: effect of CbMan5C/Cel5A amount
SEM Analysis of Microcrystalline Cellulose
Synergistic proteins are known to be capable of loosening or disrupting the packaging or changing morphology of the plant cell wall and polysaccharides. For example, the enlargement of fibers was observed, and microcrystalline cellulose (Avicel) was disrupted into smaller particles with the treatment of swollenin SWO and TlSWO, respectively (Xiao et al. 2020; Zhang et al. 2021a). However, some synergistic proteins were reported to have no cellulose disruption activity, such as swollenin Swo2 (Zhou et al. 2011). In the current study, microphotographs of the Athe_0181-treated microcrystalline cellulose were taken using SEM (Fig. 5). The results showed that microcrystalline cellulose treated with Athe_0181 (Figs. 5D, E, and F) was less interconnected and dense, and the surface was rougher, compared to microcrystalline cellulose treated with citrate buffer (Figs. 5A, B, and C).
Fig. 5. The SEM images of microcrystalline cellulose (A, D: 30 KX; B, E: 10KX; C, F: 500X). A through C: citrate buffer-treated and D through E: citrate buffer containing Athe_0181-treated
XRD Analysis of Microcrystalline Cellulose
The XRD patterns of microcrystalline cellulose were analyzed (Fig.6). Athe_0181- treated sample and the control sample presented the typical cellulose type I crystal structure (Yang et al. 2020). This suggested that Athe_0181 did not alter the crystal structure. The scattered intensity presented the difference between the buffer and Athe_0181-treated microcrystalline cellulose. Furthermore, Itotal and CrI of microcrystalline cellulose treated with Athe_0181 was decreased and CrI was reduced by 1.65%.
Fig. 6. The XRD patterns of microcrystalline cellulose (Control: citrate buffer-treated; Athe_0181: citrate buffer containing Athe_0181-treated)
Enhanced catalytic efficiency of GHs by synergistic proteins could be due to different reasons such as modification of the substrate structure or the direct hydrolytic activity on the substrate (Kim et al. 2014; Georgelis et al. 2014; Xiao et al. 2020). Athe_0181 was absent of hydrolytic activity, revealing that Athe_0181 boosting the lignocellulose degradation of CbMan5C/Cel5A did not possess hydrolytic activity. The SEM and X-ray diffraction results showed that Athe_0181 demonstrated certain structure-modifying activity, which may enhance the CbMan5C/Cel5A ability for lignocellulose hydrolysis.
CONCLUSIONS
- The recombinant pET-22b (+) harboring the Athe_0181 gene was introduced into E. coli BL21 (DE3) for expression. Athe_0181 was purified via a nickel column, and it appeared to have no activity towards cellulosic substrates.
- The capacity of Athe_0181 binding to the three lignocelluloses, microcrystalline cellulose, palm kernel meal, and rice straw was 9.19, 9.77, and 13.2 μg/mg-substrate, respectively. Athe_0181 worked as the synergistic protein to CbMan5C/Cel5A and synergistic activity was 87.0%, 80.1%, and 73.2%, respectively, with the three lignocelluloses as the substrates.
- Microcrystalline cellulose was applied as the substrate, and the highest synergistic activity was observed with hydrolysis at 12 h. In the presence of 25 μg/mL of CbMan5C/Cel5A, the reducing sugar yield increased from 19.3 μg/mL to 120 μg/mL with the assistance of 50 μg/mL of Athe_0181, and the synergistic activity reached 523%.
- The SEM and X-ray diffraction results showed that Athe_0181 could modify the structure of microcrystalline cellulose, which may result in more accessibility to the lignocellulose.
- The functional characterization of Athe_0181 indicated that Athe_0181 may play a role in lignocellulose hydrolysis and contributed to better clarify the mechanisms of C. bescii in the efficient deconstruction of plant biomass.
ACKNOWLEDGMENTS
This work was financially supported by the National Natural Science Foundation of China (Grant No. 21706089) and the Universities Natural Science Research Project of Jiangsu Province (Grant No. 19KJA430016).
REFERENCES CITED
Chu, Y., Hao, Z., Wang, K., Tu, T., Huang, H., Wang, Y., Bai, Y. G., Wang, Y., Luo, H., Yao, B., et al. (2019). “The GH10 and GH48 dual-functional catalytic domains from a multimodular glycoside hydrolase synergize in hydrolyzing both cellulose and xylan,” Biotechnol. Biofuels 12, article no. 279. DOI: 10.1186/s13068-019-1617-2
Conway, J. M., Crosby, J. R., McKinley, B. S., Seals, N. L., Adams M. W. W., and Kelly, R. M. (2018). “Parsing in vivo and in vitro contributions to microcrystalline cellulose hydrolysis by multidomain glycoside hydrolases in the Caldicellulosiruptor bescii se-cretome,” Biotechnol. Bioeng. 115(10), 2426-2440. DOI: 10.1002/bit.26773
Dam, P., Kataeva, I., Yang, S., Zhou, F., Yin, Y., Chou, W., Poole, F. L., Westpheling, J., Hettich, R., Giannone, R. J., et al. (2011). “Insights into plant biomass conversion from the genome of the anaerobic thermophilic bacterium Caldicellulosiruptor bescii DSM 6725,” Nucleic Acids Res. 39(8), 3240-3254. DOI: 10.1093/nar/gkq1281
Georgelis, N., Nikolaidis, N., and Cosgrove, D. J. (2014). “Biochemical analysis of expansin-like proteins from microbes,” Carbohyd. Polym. 100(16), 17-23. DOI: 10.1016/j.carbpol.2013.04.094
Kahn, A., Moraïs, S., Chung, D., Sarai, N. S., Hengge, N. N., Kahn, A., Himmel, M. E., Bayer, E. A., and Bomble, Y. J. (2020). “Glycosylation of hyperthermostable designer cellulosome components yields enhanced stability and cellulose hydrolysis,” FEBS. J. 287(20), 4370-4388. DOI: 10.1111/febs.15251
Kanafusa-Shinkai, S., Wakayama, J., Tsukamoto, K., Hayashi, N., Miyazaki, Y., Ohmori, H., Tajima, K., and Yokoyama, H. (2013). “Degradation of microcrystalline cellulose and non-pretreated plant biomass by a cell-free extracellular cellulase/hemicellulase system from the extreme thermophilic bacterium Caldicellulosiruptor bescii,” J. Biosci. Bioeng. 115(1), 64-70. DOI: 10.1016/j.jbiosc.2012.07.019
Kang, K., Wang, S., Lai, G., Liu, G., and Xing, M. (2013). “Characterization of a novel swollenin from Penicillium oxalicum in facilitating enzymatic saccharification of cellulose,” BMC Biotechnol. 13, article no. 42. DOI: 10.1186/1472-6750-13-42
Khan, A., Hauk, V. J., Ibrahim, M., Raffel, T. R., and Blumer-Schuette, S. E. (2020). “Caldicellulosiruptor bescii adheres to polysaccharides via a type IV pilin-dependent mechanism,” Appl. Environ. Microbiol. 86(9), article ID e00200-20. DOI: 10.1128/AEM.00200-20
Kim, I. J., Lee, H. J., Choi, I., and Kim, K. H. (2014). “Synergistic proteins for the enhanced enzymatic hydrolysis of cellulose by cellulase,” Appl. Microbiol. Biotechnol. 98(20), 8469-8480. DOI: 10.1007/s00253-014-6001-3
Lee, L. L., Hart, W. S., Lunin, V. V., Alahuhta, M., Bomble, Y. J., Himmel, M. E., Blumer-Schuette, S. E., Adams, W. W. W., and Kelly, R. M. (2019). “Comparative biochemical and structural analysis of novel cellulose binding proteins (tāpirins) from extremely thermophilic Caldicellulosiruptor species,” Appl. Environ. Microbiol. 85(3), article ID e01983-18. DOI: 10.1128/AEM.01983-18
Lin, H., Shen, Q., Zhan, J., Wang, Q., and Zhao, Y. (2013). “Evaluation of bacterial expansin EXLX1 as a cellulase synergist for the saccharification of lignocellulosic agro-industrial wastes,” PLOS One 8(9), article ID e75022. DOI: 10.1371/journal.pone.0075022
Liu, Y., Tang, Y., Gao, H., Zhang, W., Jiang, Y., Xin, F., and Jiang, M. (2021). “Challenges and future perspectives of promising biotechnologies for lignocellulosic biorefinery,” Molecules 26(17), article no. 5411. DOI: 10.3390/molecules26175411
Lochner, A., Giannone, R. J., Rodriguez, M., Shah, M. B., Mielenz, J. R., Keller, M., Antranikian, G., Graham, D. E., and Hettich, R. L. (2011). “Use of label-free quantitative proteomics to distinguish the secreted cellulolytic systems of Caldicellulosiruptor bescii and Caldicellulosiruptor obsidiansis,” Appl. Environ. Microbiol. 77(12), 4042-4054. DOI: 10.1128/AEM.02811-10
Meng, X., Ma, L., Li, T., Zhu, H., Guo, K., Liu, D., Ren, W., and Shen, Q. (2020). “The functioning of a novel protein, swollenin, in promoting the lignocellulose degradation capacity of Trichoderma guizhouense NJAU4742 from a proteomic perspective,” Bioresource Technol. 317, article ID 123992. DOI: 10.1016/j.biortech.2020.123992
Poudel, S., Giannone, R. J., Basen, M., Nookaew, I., Poole, II, F. L., Kelly, R. M., Adams, M. W. W., and Hettich, R. L. (2018). “The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate,” Biotechnol. Biofuels 11, article no. 80. DOI: 10.1186/s13068-018-1076-1
Rong, W., Li, G., Xue, X., Qin, X., Ma, R., Luo, H., Zhang, Y., Yao, B., and Su, X. (2016). “Identification of the C-terminal GH5 domain from CbCel9b/Man5a as the first glycoside hydrolase with thermal activation property from a multimodular bifunctional enzyme,” PLOS One 11(6), article ID e0156802. DOI: 10.1371/journal.pone.0156802
Su, X., Mackie, R. I., and Cann, I. K. O. (2012). “Biochemical and mutational analyses of a multidomain cellulase/mannanase from Caldicellulosiruptor bescii,” Appl. Environ. Microbiol. 78(7), 2230-2240. DOI: 10.1128/AEM.06814-11
Su, X., Zhang J., Mackie, R. I., and Cann, I. K. O. (2012). “Supplementing with non-glycoside hydrolase proteins enhances enzymatic deconstruction of plant biomass,” PLOS One 7(8), article ID e43828. DOI: 10.1371/journal.pone.0043828
Xia, J., Yu, Y., Chen., H., Zhou, J., Tan, Z., He, S., Zhu, X., Shi, H., Liu, P., Bilal, M., et al. (2019). “Improved lignocellulose degradation efficiency by fusion of β-glucosidase, exoglucanase, and carbohydrate-binding module from Caldicellulosiruptor saccharolyticus,” BioResources 14(3), 6767-6780. DOI: 10.15376/biores.14.3.6767-6780
Xiao, Y., Poovaiah, C., Unda, F., Ritchie, L., Dombrov, M., Phalen, C., Argyros, A., and Coleman, H. (2020). “Expression of the Trichoderma reesei expansin-like protein, swollenin, in poplar results in biomass with improved sugar release by enzymatic hydrolysis,” Biomass. Bioenerg. 134, article ID 105473. DOI: 10.1016/j.biombioe.2020.105473
Xue, X., Wang, R., Tu, T., Shi, P., Ma, R., Luo, H., Yao, B., and Su, X. (2015). “The N-terminal GH10 domain of a multimodular protein from Caldicellulosiruptor bescii is a versatile xylanase/β-glucanase that can degrade crystalline cellulose,” Appl. Environ. Microbiol. 81(11), 3823-3833. DOI: 10.1128/AEM.00432-15
Yang, T., Guo, Y., Gao, N., Li, X., and Zhao, J. (2020). “Modification of a cellulase system by engineering Penicillium oxalicum to produce cellulose nanocrystal,” Carbohydr. Polym. 234(5), article ID 115862. DOI: 10.1016/j.carbpol.2020.115862
Ye, L., Su, X., Schmitz, G. E., Moon, Y. H., Zhang, J., Mackie, R. I., and Cann, I. K. O. (2012). “Molecular and biochemical analyses of the GH44 module of CbMan5B/Cel44A, a bifunctional enzyme from the hyperthermophilic bacterium Caldicellulosiruptor bescii,” Appl. Environ. Microbiol. 78(19), 7048-7059. DOI: 10.1128/AEM.02009-12
Yi, Z., Su, X., Revindran, V., Mackie, R., and Cann, I. (2013). “Molecular and biochemical analyses of CbCel9A/Cel48A, a highly secreted multi-modular cellulase by Caldicellulosiruptor bescii during growth on crystalline cellulose,” PLOS One 8(12), article ID e84172. DOI: 10.1371/journal.pone.0084172
Yokoyama, H., Yamashita, T., Morioka, R., and Ohmori, H. (2014). “Extracellular secretion of noncatalytic plant cell wall-binding proteins by the cellulolytic thermophile Caldicellulosiruptor bescii,” J. Bacteriol. 196(21), 3784-3792. DOI: 10.1128 /JB.01897-14
Zhang, H., Wang, Y., Brunecky, R., Yao, B., Xie, X., Zheng, F., and Luo, H. (2021a). “A swollenin from Talaromyces leycettanus JCM12802 enhances cellulase hydrolysis toward various substrates,” Front. Microbiol. 12, article ID 658096. DOI: 10.3389/fmicb.2021.658096
Zhang, P., Su, R., Duan, Y., Cui, M., Huang, R., Qi, W., He, Z., and Thielemans, W. (2021b). “Synergy between endo/exo-glucanases and expansin enhances enzyme adsorption and cellulose conversion,” Carbohyd. Polym. 253, article ID 117287. DOI: 10.1016/j.carbpol.2020.117287
Zhe, L., Xun, Z., Yang, G., Takabe, K., and Xu, F. (2018). “Nanocrystals of cellulose allomorphs have different adsorption of cellulase and subsequent degradation,” Ind. Crop. Prod. 112, 541-549. DOI: 10.1016/j.indcrop.2017.12.052
Zhou, Q., Lv, X., Zhang, X., Meng, X., Chen, G., and Liu, W. (2011). “Evaluation of swollenin from Trichoderma pseudokoningii as a potential synergistic factor in the enzymatic hydrolysis of cellulose with low cellulase loadings,” World. J. Microbiol. Biotechnol. 27, 1905-1910. DOI: 10.1007/s11274-011-0650-5
Article submitted: January 10, 2022; Peer review completed: April 2, 2022; Revised version received and accepted: April 13, 2022; Published: April 15, 2022.
DOI: 10.15376/biores.17.2.3067-3081