Heterologous Expression of a Thermostable α-Galactosidase from Parageobacillus thermoglucosidasius Isolated from the Lignocellulolytic Microbial Consortium TMC7
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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 60oC 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 60oC. In addition, T26GAL demonstrated a remarkable thermostability with more than 93% enzyme activity, even at a high temperature of 90oC. 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 heatresistant 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.
Abstract
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 60oC 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 60oC. In addition, T26GAL demonstrated a remarkable thermostability with more than 93% enzyme activity, even at a high temperature of 90oC. 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 heatresistant 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.
참고문헌 (45)
- Taha M, "Commercial feasibility of lignocellulose biodegradation : possibilities and challenges", Curr. Opin. Biotechnol, 38, pp.190~197 (2016)
- Kaushal G, "Metagenomic analysis of geothermal water reservoir sites exploring carbohydraterelated thermozymes", Int. J. Biol. Macromol, 119, pp.882~895 (2018)
- Wang C, "Metagenomic analysis of microbial consortia enriched from compost : new insights into the role of actinobacteria in lignocellulose decomposition", Biotechnol. Biofuels, 9, pp.22~ (2016)
- Desiderato JG, "The genome sequence of Dyella jiangningensis FCAV SCS01 from a lignocellulose-decomposing microbial consortium metagenome reveals potential for biotechnological applications", Genet. Mol. Biol, 41, pp.507~513 (2018)
- Wang Yi, "Metagenomic Insight into Lignocellulose Degradation of the Thermophilic Microbial Consortium TMC7", Journal of Microbiology and Biotechnology, 31(8), pp.1123~1133 (2021), 한국미생물·생명공학회[DOI]
- Juturu V, "Insight into microbial hemicellulases other than xylanases : a review", J. Chem. Technol. Biotechnol, 88, pp.353~363 (2013)
- Xie J, "A thermophilic fungal GH36 α-galactosidase from Lichtheimia ramosa and its synergistic hydrolysis of locust bean gum", Carbohydr. Res, 491, pp.107911~ (2020)
- Bhatia S, "Microbial production and biotechnological applications of α-galactosidase", Int. J. Biol. Macromol, 150, pp.1294~1313 (2020)
- Xu Y, "The GlaA signal peptide substantially increases the expression and secretion of α-galactosidase in Aspergillus niger", Biotechnol. Lett, 40, pp.949~955 (2018)
- Patil, Aravind Goud G., "α-Galactosidase from Bacillus megaterium VHM1 and Its Application in Removal of Flatulence-Causing Factors from Soymilk", Journal of Microbiology and Biotechnology, 20(11), pp.1546~1554 (2010), 한국미생물·생명공학회
- 이재구, "Immobilization of the Antarctic Bacillus sp. LX-1 α-Galactosidase on Eudragit L-100 for the Production of a Functional Feed Additive", Animal Bioscience, 26(4), pp.552~557 (2013), 아세아·태평양축산학회[DOI]
- Zhang J, "Present status on removal of raffinose family oligosaccharides–a review", Czech J. Food Sci, 37, pp.141~154 (2019)
- Gao H, "Application of α-N-acetylgalactosaminidase and α-galactosidase in AB to O red blood cells conversion", Artif. Cells Nanomed. Biotechnol, 41, pp.32~36 (2013)
- Aguilar-Moncayo M, "Tuning glycosidase inhibition through aglycone interactions : pharmacological chaperones for Fabry disease and GM 1 gangliosidosis", Chem. Commun, 48, pp.6514~6516 (2012)
- Katrolia P, "Biotechnological potential of microbial α-galactosidases", Crit. Rev. Biotechnol, 34, pp.307~317 (2013)
- Dongdong Zhang, "Characterization of a Thermophilic Lignocellulose-Degrading Microbial Consortium with High Extracellular Xylanase Activity", Journal of Microbiology and Biotechnology, 28(2), pp.305~313 (2018), 한국미생물·생명공학회
- Bradford MM, "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding", Anal. Biochem, 72, pp.248~254 (1976)
- Semenova EM, "Geobacillus proteiniphilus sp. nov., a thermophilic bacterium isolated from a high-temperature heavy oil reservoir in China", Int. J. Syst. Evol. Microbiol, 69, pp.3001~3008 (2019)
- Ademark P, "Multiple α-galactosidases from Aspergillus niger : purification, characterization and substrate specificities", Enzyme Microb. Technol, 29, pp.441~448 (2001)
- Maruta A, "A novel α-galactosidase from Fusarium oxysporum and its application in determining the structure of the gum arabic side chain", Enzyme Microb. Technol, 103, pp.25~33 (2017)
- Weiqing Wang, "The Molecular Detection of Corynespora Cassiicola on Cucumber by PCR Assay Using DNAman Software and NCBI", Computer and Computing Technologies in Agriculture IX, pp.248~258 (2015), Springer International Publishing[DOI]
- Armenteros JJA, "SignalP 5.0 improves signal peptide predictions using deep neural networks", Nat. Biotechnol, 37, pp.420~423 (2019)
- Bateman A, "The Pfam protein families database", Nucleic Acids Res, 32, pp.138~141 (2004)
- Guex N, "SWISS-MODEL and the Swiss-Pdb viewer : an environment for comparative protein modeling", Electrophoresis, 18, pp.2714~2723 (1997)
- Ulya M, "Low molecular weight alkaline thermostable α-amylase from Geobacillus sp. nov", Heliyon, 5, pp.e02171~ (2019)
- Semenova EM, "Geobacillus proteiniphilus sp. nov., a thermophilic bacterium isolated from a high-temperature heavy oil reservoir in China", Int. J. Syst. Evol. Microbiol, 69, pp.3001~3008 (2019)
- Johnson M, "NCBI BLAST : a better web interface", Nucleic Acids Res, 36, pp.5~9 (2008)
- Merceron R, "The molecular mechanism of thermostable α-galactosidases AgaA and AgaB explained by X-ray crystallography and mutational studies", J. Biol. Chem, 287, pp.39642~39652 (2012)
- Zhang B, "Cloning and functional expression of α-galactosidase cDNA from Penicillium janczewskii zaleski", Biologia, 66, pp.205~212 (2011)
- Yanan Cao, "A novel protease-resistant α-galactosidase with high hydrolytic activity from Gibberella sp. F75: gene cloning, expression, and enzymatic characterization", Applied Microbiology and Biotechnology, 83(5), pp.875~884 (2009), Springer Science and Business Media LLC[DOI]
- Fridjonsson O, "Thermostable α-galactosidase from Bacillus stearothermophilus NUB3621 : cloning, sequencing and characterization", FEMS Microbiol. Lett, 176, pp.147~153 (1999)
- Fredslund F, "Crystal structure of α-galactosidase from Lactobacillus acidophilus NCFM : insight into tetramer formation and substrate binding", J. Mol. Biol, 412, pp.466~480 (2011)
- Bruel L, "α-Galactosidase", sucrose kinase(AgaSK), a novel bifunctional enzyme from the human microbiome coupling galactosidase and kinase activities, 2011, pp.286, 40814~40823
- Dey PM, "Characteristic features of an α-galactosidase from mung beans", Eur. J. Biochem, 140, pp.385~390 (1984)
- Liljestoüm PL, "Nucleotide sequence of the melA gene, coding for α-galactosidase in Escherichia coli K-12", Nucleic Acids Res, 15, pp.2213~2220 (1987)
- Jang JM, "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, pp.1138~1146 (2019)
- Júnior JCB, "Immobilization of an alpha-galactosidase from Debaryomyces hansenni UFV-1 in cellulose film and its appilication in oligosaccharides hydrolysis", Food Bioprod. Process, 111, pp.30~36 (2018)
- Huang Y, "Characterization of a novel GH36 α-galactosidase from Bacillus megaterium and its application in degradation of raffinose family oligosaccharides", Int. J. Biol. Macromol, 108, pp.98~104 (2018)
- Lee A, "Characterization of a thermostable glycoside hydrolase family 36 α-galactosidase from Caldicellulosiruptor bescii", J. Biosci. Bioeng, 124, pp.289~295 (2017)
- Zhou J, "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, pp.156~166 (2016)
- Bhatia S, "Microbial production and biotechnological applications of α-galactosidase", Int. J. Biol. Macromol, 150, pp.1294~1313 (2020)
- Aliyu H, "Phylogenomic re-assessment of the thermophilic genus Geoobacillus", Syst. Appl. Microbiol, 39, pp.527~533 (2016)
- Liu J, "Carbohydrate-binding modules targeting branched polysaccharides : overcoming side-chain recalctirance in a non-catalytic apporoch", Bioresour. Bioprocess, 8, pp.1~11 (2021)
- Tailford LE, "Understanding how diverse β-mannanase recognize heterogeneous substrates", Biochemistry, 48, pp.7009~7018 (2009)
- Brito IL, "Examining horizontal gene transfer in microbial communities", Nat. Rev. Microbiol, 19, pp.442~453 (2021)