The 2,835-bp open reading frame of ckxgl74A (Locus_tag CALKRO_RS04315) with a natural carbohydrate module (CBM3b) from thermophilic anaerobic microorganism Caldicellulosiruptor kronotskyensis encodes a calculated 104-kDa of GH74 xyloglucanase Ckxgl74A. The purified recombinant Ckxgl74A expressed in Escherichia coli BL21 (DE3) revealed its optimal pH of 4.5 and temperature of 80 °C. The Ckxgl74A was stable over a temperature no more than 70 °C and a pH range of 4.5 to 5.0. Kinetic experiments with xyloglucan as a substrate gave a Km of 2.29 ± 0.04 mg mL-1, Vmax of 22.98 ± 0.02 mol mg-1 min-1, and kcat of 66.98 ± 0.01 s-1. Its activity could be activated by Ca2+ approximately two folds, while being significantly inhibited by Cu2+. These results showed that Ckxgl74A could be utilized in acid condition and possessed a good thermostability.
Characterization of a Xyloglucananse in Biodegradation of Woody Plant Xyloglucan from Caldicellulosiruptor kronotskyensis
Hao Shi,a Jiannan Guo,a Xing Yan,a Ge Cui,a Zhongbiao Tan,a Xiaoyan Zhu,a Jia Zhou,a Shuai He,a Tao Wang,b and Xiangqian Li a,*
The 2,835-bp open reading frame of ckxgl74A (Locus_tag CALKRO_RS04315) with a natural carbohydrate module (CBM3b) from thermophilic anaerobic microorganism Caldicellulosiruptor kronotskyensis encodes a calculated 104-kDa of GH74 xyloglucanase Ckxgl74A. The purified recombinant Ckxgl74A expressed in Escherichia coli BL21 (DE3) revealed its optimal pH of 4.5 and temperature of 80 °C. The Ckxgl74A was stable over a temperature no more than 70 °C and a pH range of 4.5 to 5.0. Kinetic experiments with xyloglucan as a substrate gave a Km of 2.29 ± 0.04 mg mL-1, Vmax of 22.98 ± 0.02 μmol mg-1 min-1, and kcat of 66.98 ± 0.01 s-1. Its activity could be activated by Ca2+ approximately two folds, while being significantly inhibited by Cu2+. These results showed that Ckxgl74A could be utilized in acid condition and possessed a good thermostability.
Keywords: Acid resistance; Hemicellulose; Thermal stability; Xyloglucanase
Contact information: a: Jiangsu Provincial Engineering Laboratory for Biomass Conversion and Process Integration, Huaiyin Institute of Technology, Huaian 223003, China; b: Department of Microbiology, The University of Georgia, Athens, GA 30602, USA; * Corresponding author: email@example.com
Hemicellulose is a kind of component commonly found in the plant cell wall (Gupta et al. 2020). It is a polysaccharide composed of a variety of monosaccharides, mainly including xylan, mannan, arabinan, xyloglucan, etc., and they are able to be hydrolyzed into five- and six- fermentable carbon sugars, such as xylose, mannose, arabinose, and galactose. The hydrolysis application of hemicellulose polysaccharides is very extensive in energy and food industries (Balat 2011; Limayem and Ricke 2012; Keshwani and Cheng 2019).
Xyloglucan consists of highly recalcitrant and substituted polysaccharides found in the primary walls of vascular plants (Vieira et al. 2021). It is tightly connected to the rest of the hemicellulose through hydrogen bonding to form a network structure, supporting the stability of plant cell walls (Park and Cosgrove 2015). Xyloglucan is composed of β-1, 4-glucosidic bonds to form the main chain and is further substituted with xylosyl residues on the branch chain (Pauly and Keegstra 2016; Ray et al. 2004). It consists of a β-1,4-linked glucan backbone that is further substituted with xylosyl residues. In addition to the strength of the plant cell wall, xyloglucan can assist in gastroenteritis and acute diarrhea (Gnessi et al. 2015; Condratovici et al. 2016). Xyloglucanase, as one of the hydrolases, hydrolyzes β-1,4-glucosidic bonds in the main chain of xyloglucan to release oligosaccharides. Its activity can be found among enzymes that are members of glycoside hydrolase 5, 12, 16, 44, and 74, mainly from microbial and vegetative sources (Attia and Brumer 2016; Gloster et al. 2007). Among microorganisms, xyloglucanase is mainly produced by certain bacteria and fungi (Arnal et al. 2019; Berezina et al. 2021). In the existing reports on xyloglucanase, the optimal reaction conditions for xyloglucanase produced by Paenibacillus xylanilyticus were at 50 °C (Ishida et al. 2007), pH 7.0, and the enzyme activity decreased faster when the pH was lower than 5.0 and the temperature was higher than 50 °C, similar to the xyloglucanase from Myceliophthora thermophila (Berezina et al. 2021). The study of xyloglucanase produced from Streptomyces lividans showed that the optimal temperature was 50 to 55 °C with a pH of 7.5 to 9.0, while the optimal reaction conditions for xyloglucanase from Phanerochaete chrysosporium were a temperature of 55 °C and a pH of 6.0 (Wang et al. 2016). The reported optimal pH for those xyloglucanases was mostly neutral and alkaline and had almost no ability and tendency to hydrolyze hemicellulose substrates under higher temperature and lower acidic conditions.
The production and utilization of hemicellulose resources generally have been carried out under high temperature and acidic conditions, one of the benefits being to prevent the contamination of microorganisms such as bacteria. The application of xyloglucanase with excellent heat and acid resistance in bioenergy and other industrial fields has become one of the main research directions. Therefore, this study reports the expression and enzymatic properties of the thermostable and acidic xyloglucanase derived from Caldicellulosiruptor kronotskyensis including its kinetic parameters, optimum temperature and pH, and substrate specificity.
Bacterial strains, plasmids, and general growth conditions
Caldicellulosiruptor kronotskyensis 18902 was purchased from DSMZ (Braunschweig, Germany). Plasmid pET-28a was used for cloning and expression. Escherichia coli DH5α from Sigma-Aldrich (St. Louis, MO, USA) was selected as the cloning host, while 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).
Primers were synthesized by Generay Biotech (Shanghai, China). The DNA polymerase was purchased from Takara (Dalian, China). The DNA endonucleases, and ligase were purchased from New England Biolabs (Beijing, China). Substrate xyloglucan (from tamarind seed) was purchased from Neogen Bio-scientific Technology Co., Ltd. (Shanghai, China). β-D-Glucan (from barley) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
Genomic DNA of C. kronotskyensis and plasmid pET-28a were used as templates, and the following primers (Table 1) were used for gene cloning. Primers F1 and R1 were used to ckXgl74A gene amplification, while F2 and R2 for pET-28a amplification. Then, primers F2 and R1 were used to amplify pET-28a-ckXgl74A fused gene using mixed ckXgl74A and pET28a products above with same concentration as templates using DNA polymerase. The polymerase chain reaction (PCR) was conducted as follows: pre-denaturation at 94 °C for 4 min; 30 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 3 min; then extensions at 72 °C for 10 min. A Biomiga Gel/PCR Kit and a Biomiga Plaminiprep Kit (Shanghai, China) were used for the purification of PCR fragments and recombinant vectors respectively. T4-ligase was used to construct the circular pET-28a-ckXgl74A, and then the recombinant plasmid was confirmed by sequencing by Sangon Biotech (Shanghai, China). The DNA transformation was conducted via electroporation by using GenePulser Xcell (Bio-Rad, Hercules, CA, USA).
Table 1. Nucleotide Sequences of Used Primers
Protein expression and purification
Relevant recombinant E. coli BL21 was cultured on a kanamycin plate at 37 °C for 12 h. Then, the single colony was selected and cultured overnight in a 20 mL LB medium flask containing kanamycin of 50 μg mL-1, and then it was inoculated in the 1000 mL flask with 200 mL LB medium. After about 2 to 3 h incubation, the cells were grown to an OD600 of about 0.6, and then 0.5 mM isopropyl β-D-thiogalactoside (IPTG) (Sangon Biotech, Shanghai, China) was added to induce the protein expression. The cells were collected by centrifugation at 8000 rpm, 4 °C for 20 min. After that, cell pellets were resuspended and centrifuged again. Then, the crude enzyme solution was crushed for 25 min by sonication for 4 s, intermittently for 2s, and at 45% power. The crude mixture was centrifuged at 11000 rpm for 20 min at 4 °C.
The target protein was purified using Novagen’s His Bind Purifucation Kit (Beijing, China), eluted with different concentrations of imidazole (50 mM, 200 mM, and 400 mM) in 20 mM Tris-HCl buffer (0.5 M NaCl and pH 7.9). The molecular mass of protein and purity were determined by 12% SDS-PAGE gel using a protein marker from Thermo Fisher Scientific Inc. (Shanghai, China). After this, the obtained recombinant enzymes were finally stored in 30% glycerol with 1 mM of Dithiothreitol (DTT) in -20 °C freezer.
Enzyme activity determination
For the enzyme activity determination, 100 μL of the corresponding pH buffer, 90 μl of 0.5% (w/v) xyloglucan (in water), and 10 μL of the appropriately substituted enzyme solution were added to the 200 μL reaction system, and then incubated at 80 °C for 10 min. Subsequently, 300 μL of 3, 5-dinitrosalicylic acid (DNS) (Miller 1959) was added to stop the reaction, and then the mixture was boiled for 5 min. The reducing sugar absorbance was measured at 540 nm. One unit of enzyme activity was defined as the amount of enzyme to release 1 μmol of reducing sugar per min. All assays were conducted in triplicate.
The optimum temperature was measured by setting a reaction temperature of 50 to 90 °C (5 °C intervals) in 200 μL system containing 50 mM citrate buffer at pH 4.5 for 10 min. The optimum pH measurement conditions were set at an optimum reaction temperature of 80 °C at 3.0 to 8.0 (0.5 intervals in 50 mM citrate buffer). The highest enzyme activity at each temperature or pH was set to 100%.
The pH stability was determined by adding 10 μL of enzyme and 90 μL of a buffer of different pHs to a 200 μL reaction system, incubating in a water bath at 40 °C for 2 h; then 100 μL of 0.5% xyloglucan was added to start the reaction by incubating at 80 °C for 10 min. Thermal stability was investigated by adding 10 μL of enzyme and 90 μL (pH 4.5) buffer to a 200 μL reaction system, incubating at 50 to 90 °C (10 °C intervals) for 0.5 to 2 h (0.5 h intervals).
The 100 μL 0.1% to 0.5% substrate (0.1% intervals), 90 μL of a pH 4.5 citrate-sodium citrate buffer, and 10 μL of an appropriately substituted enzyme solution were added to the reaction system, and the process was carried out at 80 °C water bath for 10 min. Kinetic parameters, Km, Vmax, and kcat, were deduced form double reciprocal Lineweaver-Burk mapping.
RESULTS AND DISCUSSION
Protein Expression, Purification, and Sequences Alignment
In this study, the ckxgl74A gene was successfully constructed and expressed in E. coli BL21 (DE3) with unique characteristics. The theoretical protein of ckXgl74A was 104 kDa, which was consistent with the results of the protein electropherogram (Fig. 1). The purification process was conducted as shown in Table 2, and the purified fraction of lane 4 (Fig. 1) was used to characterize the enzyme activity. Multiple sequence alignment of xyloglucanases obtained from CAZy (http://www.cazy.org/) was processed using Clustal X2 (data not shown). Based on amino acid similarity, CkXgl74A was judged to be a typical member of GH74 xyloglucanases relative to other Caldicellulosiruptor homologs (Arnal et al. 2019; Conway et al. 2018).
Fig. 1. SDS-PAGE of ckXgl74A. Lane M: marker; lane 1: the extract of recombinant E. coli BL21 (DE3) (10 μL); lane 2: the fractions purified via nickel affinity column chromatography (50 mM imidazole) (10 μL); lane 3: the purified proteins via nickel affinity column chromatography (200 mM imidazole) (10 μL); lane 4: the purified proteins via nickel affinity column chromatography (400 mM imidazole) (10 μL)
Table 2. Purification Process of CkXgl74A
The optimum temperature for CkXgl74A was 80 °C (Fig. 2a). The results showed that the activity was relatively stable below 80 °C (Fig. 2b). At 50 and 60 °C, the relative activity was maintained for more than 90% within 1 h; however, it decreased rapidly when incubating for 1 to 2 h, maintaining only about 40% activity. The recombinant CkXgl74A also showed that it was very unstable above 80 °C. The optimum pH value of the recombinant enzyme was 4.5 (Fig. 2c), and it showed good pH stability at pH 4.5 to 5.5 (Fig. 2d), remaining above 70% activity after incubation for 2 h. This establishes that the xyloglucanase has good acid resistance under pH 4.5. These results showed that recombinase CkXgl74A possessed good high-temperature catalysis and acidic resistance.
Fig. 2. Temperature and pH on CkXgl74A activity. a) Temperature optimum (pH 4.5, 50 °C to 95 °C for 10 min); b) Thermal stability (pH 4.5, 50 °C to 90 °C for 0, 30, 60, 90, and 120 min); c) pH optimum (pH 3 to 8, 80 °C for 10 min); and d) pH stability (pH 4.5 to 9, 40 °C for 2 h)
Compared to the xyloglucanases from P. xylanilyticus and Streptomyces lividans (Ishida et al. 2007; Wang et al. 2016), CkXgl74A from C. kronotskyensis in this study showed higher temperature optima and stability at an acidic condition. Generally, fermentation processes such as beer are usually carried out under acidic condition and saccharification at relatively high temperature (65 °C for 1 h) (Tokpohozin et al. 2019). Therefore, this recombinant xyloglucanase could be used in the fermentation processes of bioethanol, beer, and cereals, generating more fermentable sugars.
Effect of cations on CkXgl74A activity
When the concentration of the metal ion was 1 or 10 mM, it was found that Fe2+, Fe3+, and Ca2+ could obviously activate the enzyme activity, while Mg2+, Mn2+, and Cu2+ could significantly inhibit the enzyme activity. Besides, the enzyme activity could be significantly increased to 180% by adding 10 mM of Ca2+ (Fig. 3), which was similar with other studies (Wong et al. 2010).
Fig. 3. Effects of cations on CkXgl74A activity. The rection was conducted using 0.5% xyloglucan and 4 μg of purified enzyme in 50 mM citrate buffer at pH 4.5 for 10 min. Values shown were the mean of triplicate experiments.
Substrate specificity and enzyme kinetics
The recombinant enzyme had no substrate activity on CMC-Na, filter paper, xylan, and β-D-glucan (Table 3). The enzyme was found to have a Km of 2.29 ± 0.04 mg mL-1, kcat was 6.69 ± 0.01 s-1, and Vmax was 22.98 ± 0.02 μmol mg-1 min-1 (Table 4). These results showed that the Km value was smaller than xyloglucanase XEG5 (2.0 mg mL-1) (Yaoi et al. 2005).
Table 3. Substrate Specificity
Table 4. Kinetic Parameters
Application of Xyloglucanase
Xyloglucanase is very useful in biodegradation of xyloglucan. It hydrolyzes the xyloglucan, one component of hemicellulose in plant cell, to liberate fermentable sugars (Pauly and Keegstra 2016; Arnal et al. 2019; Berezina et al. 2021). Furthermore, it can be used to cooperate with cellulase, xylanase, and glucanase in degradation of cellulose, hemicellulose, and starch. CkXgl74A was only active to xyloglucan, and with its temperature and acidic stability, it should be a potential candidate in process of saccharification in industrial application.
- The recombinant pET-28a harboring the CkXgl74A gene (2835 bp) was introduced into E. coli BL21 (DE3) for expression. After sonication and centrifugation of cell debris, cell extracts were purified via a nickel column. The molecular weight was 104 kDa, which was consistent with theoretical molecular weight.
- The temperature and pH optima of CkXgl74A were 80 °C and 4.5, respectively. The CkXgl74A showed good thermal and acidic stability, and it could remain above 60% activity after a 1 h incubation at 50, 60, and 70 °C. Besides, it could maintain over 70% activity after incubation for 2 h at pH 4.5, 5.0, and 5.5.
- CkXgl74A was only active toward xyloglucan, and it was significantly activated by Fe2+, Fe3+, and Ca2+, and strongly inhibited by Mg2+, Mn2+, and Cu2+. The kinetic parameters of α-glucosidase were a Km of 2.29 ± 0.04 mg mL-1, Vmax of 22.98 ± 0.02 μmol mg-1 min-1, and a kcat of 6.69 ± 0.01 s-1.
- These characteristics of ckXgl74A revealed that it may possess desirable industrial applications in the fermentation process of bioethanol, beer, and other bioproducts.
This work was ﬁnancially supported by the Primary Research and Development Plan of Jiangsu Province (Grant No. BE2020392), the Universities Natural Science Research Project of Jiangsu Province (Grant No. 19KJA430016), and Jiangsu Provincial Engineering Laboratory for Biomass Conversion and Process Integration (Grant No. JPELBCPI2015003, JPELBCPI2016004, and JPELBCPI2018005).
Arnal, G., Stogios, P. J., Asohan, J., Attia, M. A., Skarina, T., Viborg, A. H., Henrissat, B., Savchenko, A., and Brumer, H. (2019). “Substrate specificity, regiospecificity, and processivity in glcoside hyrolase family 74,” J. Biol. Chem. 294(36), 13233-13247. DOI: 10.1074/jbc.RA119.009861
Attia, M. A., and Brumer H. (2016). “Recent structural insights into the enzymology of the ubiquitous plant cell wall glycan xyloglucan,” Curr. Opin. Struct. Biol. 40, 43-53. DOI: 10.1016/j.sbi.2016.07.005
Balat, M. (2011). “Production of bioethanol from lignocellulosic materials via the biochemical pathway: A review,” Energ. Convers. Manage. 52(2), 858-875. DOI: 10.1016/j.enconman.2010.08.013
Berezina, O. V., Rykov, S. V., Polyakova, A. K., Bozdaganyan, M. E., Sidochenko, A. V., Baudrexl, M., Schwarz, W. H., Zverlov, Vladimir, V. V., and Yarotsky, S. V. (2021). “Strategic aromatic residues in the catalytic cleft of the xyloglucanase MtXgh74 modifying thermostability, mode of enzyme action, and viscosity reduction ability,” Appl. Microbiol. Biotechnol. 105(4), 1461-1476. DOI: 10.1007/s00253-021-11106-3
Condratovici, C. P., Bacarea, V., and Pique, N. (2016). “Xyloglucan for the treatment of acute gastroenteritis in children: Results of a randomized, controlled, clinical trial,” Gastroenterol. Res. Pract. 2016, article ID 6874207. DOI: 10.1155/2016/6874207
Conway, 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 secretome,” Biotechnol. Bioeng. 115(10), 2426-2440. DOI: 10.1002/bit.26773
Gloster, T. M., Ibatullin, F. M., Macauley, K., Eklöf, J. M., Roberts, S., Turkenburg, J. P., Bjørnvad, M. E., Jørgensen, P. L., Danielsen, S., Johansen, K. S., Borchert, T. V., Wilsin, K. S., Brumer, H., and Davies, G. J. (2007). “Characterization and three-dimensional structures of two distinct bacterial xyloglucanases from families GH5 and GH12,” J. Biol. Chem. 282(26), 19177-19189. DOI: 10.1074/jbc.M700224200
Gnessi, L., Bacarea, V., Marusteri, M., and Pique, N. (2015). “Xyloglucan for the treatment of acute diarrhea: Results of a randomized, controlled, open-label, parallel group, multicentre, national clinical trial,” BMC Gastroenterol. 15, article number 153. DOI: 10.1186/s12876-015-0386-z
Gupta, S. K., Kataki, S., Chatterjee, S., Prasad, R. K., Datta, S., Vaiarle, M. G., Sharma, S., Dwivedi, S. D., and Gupta, D. K. (2020). “Cold adaptation in bacteria with special focus on cellulase production and its potential application,” J. Clean. Prod. 258, article ID 120351. DOI: 10.1016/j.jclepro.2020.120351
Ishida, T., Yaoi, K., Hiyoshi, A., Igarashi, K., and Samejima, M. (2007). “Substrate recognition by glycoside hydrolase family 74 xyloglucanase from the basidiomycete Phanerochaete chrysosporium,” FEBS J. 274(21), 5727-5736. DOI: 10.1111/j.1742-4658.2007.06092.x
Keshwani, D. R., and Cheng, J. J. (2019). “Switchgrass for bioethanol and other value-added applications: A review,” Bioresour. Technol. 100(4), 1515-1523. DOI: 10.1016/j.biortech.2008.09.35
Limayem, A., and Ricke, S. (2012). “Lignocellulosic biomass for bioethanol production current perspectives potential issues and future prospects,” Prog. Energ. Combust. Sci. 38(4), 449-467. DOI: 10.1016/j.pecs.2012.03.002
Miller, G. L. (1959). “Use of dinitrosalicylic acid reagent for determination of reducing sugar,” Anal. Chem. 31(3), 426-428. DOI: 10.1021/ac60147a030
Park, Y. B., and Cosgrove, D. J. (2015). “Xyloglucan and its interactions with other components of the growing cell wall,” Plant Cell Physiol. 56(2), 180-194. DOI: 10.1093/pcp/pcu204
Pauly, M., and Keegstra, K. (2016). “Biosynthesis of the plant cell wall matrix polysaccharide xyloglucan,” Annu. Rev. Plant. Biol. 67, 235-259. DOI: 10.1146/annurev-arplant-043015-112222
Ray, B., Loutelier-Bourhis, C., Lange, C., Condamine, E., Driouich, A., and Lerouge, P. (2004). “Structural investigation of hemicellulosic polysaccharides from Argania spinosa: Characterisation of a novel xyloglucan motif,” Carbohydr. Res. 339(2), 201-208. DOI: 10.1016/j.carres.2003.10.011
Tokpohozin, S. E., Fischer, S., and Becker, T. (2019). “Optimization of malting conditions for two landraces of West African sorghum and influence of mash bio-acidification on saccarification improvement,” J. Cereal Sci. 85, 192-198. DOI: 10.1016/j.jcs.2018.12.011
Vieira, P. S., Bonfim, I. M., Araujo, E. A., Melo, R. R., Lima, A. R., Fessel, M. R., Paixão, D. A. A., Persinoti, G. F., Rocco, S. A., Lima, T. B., Pirolla, R. A. S., Morais, M. A. B., Correa, J. B. L., Zanphorlin, L. M., Diogo, J. A., Lima, E. A., Grandis, A., Buckeridge, M. S., Gozzo, F. C., Benedetti, I. P., Giuseppe, P. O., and Murakami, M. T. (2021). “Xyloglucan processing machinery in Xanthomonas pathogens and its role in the transcriptional activation of virulence factors,” Nat. Commun. 12(1), 4069. DOI: 10.1038/s41467-021-24277-4
Wang, R., Gong, L., 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: 10.1271/journal.pone.0156802
Wong, D. D. W. S., Chan, V. J., McCormack, A. A., and Batt, S. B. (2010). “A novel xyloglucan-specific endo-β-1,4-glucanase: Biochemical properties and inhibition studies,” Appl. Microbiol. Biotechnol. 86, 1463-1471. DOI: 10.1007/s00253-009-2364-2
Yaoi, K., Nakai, T., Kameda, Y., Hiyoshi, A., and Mitsuishi, Y. (2005). “Cloning and characterization of two xyloglucanases from Paenibacillus sp. Strain KM21,” Appl. Environ. Microbiol. 71(12), 7670-7678. DOI: 10.1128/AEM.71.12.7670-7678.2005
Article submitted: September 16, 2021; Peer review completed: November 6, 2021; Revised version received and accepted: November 24, 2021; Published: December 6, 2021.