DOI QR코드

DOI QR Code

Heterologous Expression of a Thermostable α-Galactosidase from Parageobacillus thermoglucosidasius Isolated from the Lignocellulolytic Microbial Consortium TMC7

  • Wang, Yi (Institute of Agricultural Products Processing and Nuclear Agriculture Technology Research, Hubei Academy of Agricultural Sciences) ;
  • Wang, Chen (Institute of Agricultural Products Processing and Nuclear Agriculture Technology Research, Hubei Academy of Agricultural Sciences) ;
  • Chen, Yonglun (Institute of Agricultural Products Processing and Nuclear Agriculture Technology Research, Hubei Academy of Agricultural Sciences) ;
  • Cui, MingYu (Institute of Agricultural Products Processing and Nuclear Agriculture Technology Research, Hubei Academy of Agricultural Sciences) ;
  • Wang, Qiong (Institute of Agricultural Products Processing and Nuclear Agriculture Technology Research, Hubei Academy of Agricultural Sciences) ;
  • Guo, Peng (Institute of Agricultural Products Processing and Nuclear Agriculture Technology Research, Hubei Academy of Agricultural Sciences)
  • 투고 : 2022.01.20
  • 심사 : 2022.05.10
  • 발행 : 2022.06.28

초록

α-Galactosidase is a debranching enzyme widely used in the food, feed, paper, and pharmaceuticals industries and plays an important role in hemicellulose degradation. Here, T26, an aerobic bacterial strain with thermostable α-galactosidase activity, was isolated from laboratory-preserved lignocellulolytic microbial consortium TMC7, and identified as Parageobacillus thermoglucosidasius. The α-galactosidase, called T26GAL and derived from the T26 culture supernatant, exhibited a maximum enzyme activity of 0.4976 IU/ml when cultured at 60℃ and 180 rpm for 2 days. Bioinformatics analysis revealed that the α-galactosidase T26GAL belongs to the GH36 family. Subsequently, the pET-26 vector was used for the heterologous expression of the T26 α-galactosidase gene in Escherichia coli BL21 (DE3). The optimum pH for α-galactosidase T26GAL was determined to be 8.0, while the optimum temperature was 60℃. In addition, T26GAL demonstrated a remarkable thermostability with more than 93% enzyme activity, even at a high temperature of 90℃. Furthermore, Ca2+ and Mg2+ promoted the activity of T26GAL while Zn2+ and Cu2+ inhibited it. The substrate specificity studies revealed that T26GAL efficiently degraded raffinose, stachyose, and guar gum, but not locust bean gum. This study thus facilitated the discovery of an effective heat-resistant α-galactosidase with potent industrial application. Meanwhile, as part of our research on lignocellulose degradation by a microbial consortium, the present work provides an important basis for encouraging further investigation into this enzyme complex.

키워드

과제정보

This work was supported by the Applied Basic Research Frontier Foundation of Wuhan, China (2020020601012265), Major Technological Innovation Project of Hubei Province, China (2019ABA114), Natural Science Foundation of Hubei Province, China (2019CFB588), and Special Funds for Local Science and Technology Development guided by the central government of China (2019ZYYD030).

참고문헌

  1. Taha M, Foda M, Shahsavari E, Aburto-Medina A, Adetutu E, Ball A. 2016. Commercial feasibility of lignocellulose biodegradation: possibilities and challenges. Curr. Opin. Biotechnol. 38: 190-197. https://doi.org/10.1016/j.copbio.2016.02.012
  2. Kaushal G, Kumar J, Sangwan RS, Singh SP. 2018. Metagenomic analysis of geothermal water reservoir sites exploring carbohydrate-related thermozymes. Int. J. Biol. Macromol. 119: 882-895. https://doi.org/10.1016/j.ijbiomac.2018.07.196
  3. Wang C, Dong D, Wang H, Muller K, Qin Y, Wang H, Wu W. 2016. Metagenomic analysis of microbial consortia enriched from compost: new insights into the role of actinobacteria in lignocellulose decomposition. Biotechnol. Biofuels 9: 22. https://doi.org/10.1186/s13068-016-0440-2
  4. Desiderato JG, Alvarenga DO, Constancio MT, Alves L, Varani AM. 2018. The genome sequence of Dyella jiangningensis FCAV SCS01 from a lignocellulose-decomposing microbial consortium metagenome reveals potential for biotechnological applications. Genet. Mol. Biol. 41: 507-513. https://doi.org/10.1590/1678-4685-gmb-2017-0155
  5. Wang Y, Wang C, Chen Y, Chen B, Guo P, Cui Z. 2021. Metagenomic insight into lignocellulose degradation of the thermophilic microbial consortium TMC7. J. Microbiol. Biotechnol. 31: 1123-1133. https://doi.org/10.4014/jmb.2106.06015
  6. Juturu V, Wu JC. 2013. Insight into microbial hemicellulases other than xylanases: a review. J. Chem. Technol. Biotechnol. 88: 353-363. https://doi.org/10.1002/jctb.3969
  7. Xie J, Wang B, He Z, Pan L. 2020. A thermophilic fungal GH36 α-galactosidase from Lichtheimia ramosa and its synergistic hydrolysis of locust bean gum. Carbohydr. Res. 491: 107911. https://doi.org/10.1016/j.carres.2020.107911
  8. Bhatia S, Singh A, Batra N, Singh J. 2020. Microbial production and biotechnological applications of α-galactosidase. Int. J. Biol. Macromol. 150: 1294-1313. https://doi.org/10.1016/j.ijbiomac.2019.10.140
  9. Xu Y, Wang YH, Liu TQ, Zhang H, Zhang H, Li J. 2018. The GlaA signal peptide substantially increases the expression and secretion of α-galactosidase in Aspergillus niger. Biotechnol. Lett. 40: 949-955. https://doi.org/10.1007/s10529-018-2540-5
  10. Patil AGG, Kumar SKP, Mulimani VH, Veeranagouda Y, Lee K. 2010. α-galactosidase from Bacillus megaterium VHM1 and its application in removal of flatulence-causing factoers from soymilk. J. Microbiol. Biotechnol. 20: 1546-1554. https://doi.org/10.4014/jmb.0912.12012
  11. Lee J, Park I, Cho J. 2013. Immobilization of the Antarctic Bacillus sp. LX-1 alpha-galactosidase on eudragit L-100 for the production of a functional feed additive. Asian-Australas. J. Anim. Sci. 26: 552-557. https://doi.org/10.5713/ajas.2012.12557
  12. Zhang J, Song G, Mei Y, Li R, Zhang H, Liu Y. 2019. Present status on removal of raff inose family oligosaccharides-a review. Czech J. Food Sci. 37: 141-154. https://doi.org/10.17221/472/2016-cjfs
  13. Gao H, Li S, Tan Y, Ji S, Wang Y, Bao G, Bao G, Xu L, Gong F. 2013. Application of α-N-acetylgalactosaminidase and α-galactosidase in AB to O red blood cells conversion. Artif. Cells Nanomed. Biotechnol. 41: 32-36. https://doi.org/10.3109/10731199.2012.724422
  14. Aguilar-Moncayo M, Takai T, Higaki K, Mena-Barragan T, Hirano Y, Yura K, et al. 2012. Tuning glycosidase inhibition through aglycone interactions: pharmacological chaperones for Fabry disease and GM 1 gangliosidosis. Chem. Commun. 48: 6514-6516. https://doi.org/10.1039/c2cc32065g
  15. Katrolia P. 2013. Biotechnological potential of microbial α-galactosidases. Crit. Rev. Biotechnol. 34: 307-317. https://doi.org/10.3109/07388551.2013.794124
  16. Zhang D, Wang Y, Zhang C, Zheng D, Guo P, Cui Z. 2018. Characterization of a thermophilic lignocellulose-degrading microbial consortium with high extracellular xylanase activity. J. Microbiol. Biotechnol. 28: 305-313. https://doi.org/10.4014/jmb.1709.09036
  17. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. https://doi.org/10.1006/abio.1976.9999
  18. Semenova EM, Sokolova DS, Grouzdev DS, Poltaraus AB, Vinokurova NG, Tourova TP, et al. 2019. Geobacillus proteiniphilus sp. nov., a thermophilic bacterium isolated from a high-temperature heavy oil reservoir in China. Int. J. Syst. Evol. Microbiol. 69: 3001-3008. https://doi.org/10.1099/ijsem.0.003486
  19. Ademark P, Larsson M, Tjerneld F, Stalbrand H. 2001. Multiple α-galactosidases from Aspergillus niger: purification, characterization and substrate specificities. Enzyme Microb. Technol. 29: 441-448. https://doi.org/10.1016/S0141-0229(01)00415-X
  20. Maruta A, Yamane M, Matsubara M, Suzuki S, Nakazawa M, Ueda M, et al. 2017. A novel α-galactosidase from Fusarium oxysporum and its application in determining the structure of the gum arabic side chain. Enzyme Microb. Technol. 103: 25-33. https://doi.org/10.1016/j.enzmictec.2017.04.006
  21. Wang W. 2015. The Molecular Detection of Corynespora Cassiicola on Cucumber by PCR Assay Using DNAman Software and NCBI. 9th International Conference on Computer and Computing Technologies in Agriculture (CCTA). pp. 248-258. 10.1007/978-3-319-48354-2_26. hal-01614171.
  22. Armenteros JJA, Tsirigos KD, Sonderby CK, Petersen TN, Winther O, Brunak S, et al. 2019. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat. Biotechnol. 37l: 420-423.
  23. Bateman A, Coin L, Durbin R, Finn RD, Hollich V, Griffiths-Jones S, et al. 2004. The Pfam protein families database. Nucleic Acids Res. 32(Database issue): D138-141. https://doi.org/10.1093/nar/gkh121
  24. Guex N, Peitsch MC. 1997. SWISS-MODEL and the Swiss-Pdb viewer: an environment for comparative protein modeling. Electrophoresis 18: 2714-2723. https://doi.org/10.1002/elps.1150181505
  25. Ulya M, Oesman F, Iqbalsyah TM. 2019. Low molecular weight alkaline thermostable α-amylase from Geobacillus sp. nov. Heliyon 5: e02171. https://doi.org/10.1016/j.heliyon.2019.e02171
  26. Semenova EM, Sokolova DS, Grouzdev DS, Poltaraus AB, Vinokurova NG, Tourova TP, et al. 2019. Geobacillus proteiniphilus sp. nov., a thermophilic bacterium isolated from a high-temperature heavy oil reservoir in China. Int. J. Syst. Evol. Microbiol. 69: 3001-3008. https://doi.org/10.1099/ijsem.0.003486
  27. Johnson M, Zaretskaya I, Raytselis Y, Merezhuk Y, McGinnis S, Madden TL. 2008. NCBI BLAST: a better web interface. Nucleic Acids Res. 36: 5-9.
  28. Merceron R, Foucault M, Haser R, Mattes R, Watzlawick H, Gouet P. 2012. The molecular mechanism of thermostable α-galactosidases AgaA and AgaB explained by X-ray crystallography and mutational studies. J. Biol. Chem. 287: 39642-39652. https://doi.org/10.1074/jbc.M112.394114
  29. Zhang B, Chen Y, Li Z, Lu W, Cao Y. 2011. Cloning and functional expression of α-galactosidase cDNA from Penicillium janczewskii zaleski. Biologia 66: 205-212. https://doi.org/10.2478/s11756-011-0014-5
  30. Cao Y, Wang Y, Meng K, Bai Y, Shi P, Luo H, et al. 2009. A novel protease-resistant α-galactosidase with high hydrolytic activity from Gibberella sp. F75: gene cloning, expression, and enzymatic characterization. Appl. Microbiol. Biotechnol. 83: 875-884. https://doi.org/10.1007/s00253-009-1939-2
  31. Fridjonsson O, Watzlawick H, Gehweiler A, Mattes R. 1999. Thermostable α-galactosidase from Bacillus stearothermophilus NUB3621: cloning, sequencing and characterization. FEMS Microbiol. Lett. 176: 147-153. https://doi.org/10.1016/S0378-1097(99)00231-1
  32. Fredslund F, Hachem MA, Larsen RJ, Sorensen PG, Coutinho PM, Lo Leggio L, et al. 2011. Crystal structure of α-galactosidase from Lactobacillus acidophilus NCFM: insight into tetramer formation and substrate binding. J. Mol. Biol. 412: 466-480. https://doi.org/10.1016/j.jmb.2011.07.057
  33. Bruel L, Sulzenbacher G, Cervera Tison M, Pujol A, Nicoletti C, Perrier J, et al. 2011. α-Galactosidase/sucrose kinase (AgaSK), a novel bifunctional enzyme from the human microbiome coupling galactosidase and kinase activities. J. Biol. Chem. 286: 40814-40823. https://doi.org/10.1074/jbc.M111.286039
  34. Dey PM. 1984. Characteristic features of an α-galactosidase from mung beans. Eur. J. Biochem. 140: 385-390. https://doi.org/10.1111/j.1432-1033.1984.tb08113.x
  35. Liljestoum PL, Liljestrom P. 1987. Nucleotide sequence of the melA gene, coding for α-galactosidase in Escherichia coli K-12. Nucleic Acids Res. 15: 2213-2220. https://doi.org/10.1093/nar/15.5.2213
  36. Jang JM, Yang Y, Wang R, Bao H, Yuan H, Yang J. 2019. Characterization of a high performance α-galactosidase from Irpex lacteus and its usage in removal of raffinose family oligosaccharides from soymilk. Int. J. Biol. Macromol. 131: 1138-1146. https://doi.org/10.1016/j.ijbiomac.2019.04.060
  37. Junior JCB, Viana PA, de Rezende ST, Soares NDFF, Guimaraes VM. 2018. Immobilization of an alpha-galactosidase from Debaryomyces hansenni UFV-1 in cellulose film and its appilication in oligosaccharides hydrolysis. Food Bioprod. Process 111: 30-36. https://doi.org/10.1016/j.fbp.2018.06.001
  38. Huang Y, Zhang H, Ben P, Duan Y, Lu M, Li Z, et al. 2018. Characterization of a novel GH36 α-galactosidase from Bacillus megaterium and its application in degradation of raffinose family oligosaccharides. Int. J. Biol. Macromol. 108: 98-104. https://doi.org/10.1016/j.ijbiomac.2017.11.154
  39. Lee A, Choi KH, Yoon D, Kim S, Cha J. 2017. Characterization of a thermostable glycoside hydrolase family 36 α-galactosidase from Caldicellulosiruptor bescii. J. Biosci. Bioeng. 124: 289-295. https://doi.org/10.1016/j.jbiosc.2017.04.011
  40. Zhou J, Lu Q, Zhang R, Wang Y, Wu Q, Li J, et al. 2016. Characterization of two glycoside hydrolase family 36 α-galactosidases: Novel transglycosylation activity, lead-zinc tolerance, alkaline and multiple pH optima, and low-temperature activity. Food Chem. 194: 156-166. https://doi.org/10.1016/j.foodchem.2015.08.015
  41. Bhatia S, Singh A, Batra N, Singh J. 2020. Microbial production and biotechnological applications of α-galactosidase. Int. J. Biol. Macromol. 150: 1294-1313. https://doi.org/10.1016/j.ijbiomac.2019.10.140
  42. Aliyu H, Lebre P, Blom J, Cowan D, De Maayer P. 2016. Phylogenomic re-assessment of the thermophilic genus Geoobacillus. Syst. Appl. Microbiol. 39: 527-533. https://doi.org/10.1016/j.syapm.2016.09.004
  43. Liu J, Sun D, Zhu J, Liu C, Liu W. 2021. Carbohydrate-binding modules targeting branched polysaccharides: overcoming side-chain recalctirance in a non-catalytic apporoch. Bioresour. Bioprocess 8: 1-11. https://doi.org/10.1186/s40643-020-00357-z
  44. Tailford LE, Ducros VMA, Flint JE, Roberts SM, Morland C, Zechel DL, et al. 2009. Understanding how diverse β-mannanase recognize heterogeneous substrates. Biochemistry 48: 7009-7018. https://doi.org/10.1021/bi900515d
  45. Brito IL. 2021. Examining horizontal gene transfer in microbial communities. Nat. Rev. Microbiol. 19: 442-453. https://doi.org/10.1038/s41579-021-00534-7