Novel Properties for Endoglucanase Acquired by Cell-Surface Display Technique
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In order to improve the stability of endoglucanase under thermal and acidic conditions, the endoglucanase gene was fused to the N-terminus of the Saccharomyces cerevisiae pir gene, encoding the cell wall protein PIR. The fusion gene was transformed into Pichia pastoris GS115 for expression. A resulting strain with high expression and high activity was identified by examining resistance to Geneticin 418, Congo red staining, and quantitative analysis of enzyme activity. SDS-PAGE analysis revealed that the endoglucanase was successfully displayed on the yeast cell surface. The displayed endoglucanase (DEG) showed maximum activity towards sodium carboxyl methyl cellulose at approximately 275 IU/g cell dry weight. DEG exhibited greater than 60% residual activity in the pH range 2.5-8.5, higher than free endoglucanase (FEG), which had 40% residual activity at the same pH range. The highest tolerated temperature for DEG was 70ºC, much higher than that of FEG, which was approximately 50ºC. Moreover, DEG showed 91.1% activity at 65ºC for 120 min, while FEG only kept 77.8% residual activity over the same period. The half-life of DEG was 270 min at 65ºC, compared with only 150 min for FEG. DEG could be used repeatedly at least three times. These results suggest that the DEG has broad applications as a yeast whole-cell biocatalyst, due to its novel properties of high catalytic efficiency, acid-thermal stabilities, and reusability.
Abstract
In order to improve the stability of endoglucanase under thermal and acidic conditions, the endoglucanase gene was fused to the N-terminus of the Saccharomyces cerevisiae pir gene, encoding the cell wall protein PIR. The fusion gene was transformed into Pichia pastoris GS115 for expression. A resulting strain with high expression and high activity was identified by examining resistance to Geneticin 418, Congo red staining, and quantitative analysis of enzyme activity. SDS-PAGE analysis revealed that the endoglucanase was successfully displayed on the yeast cell surface. The displayed endoglucanase (DEG) showed maximum activity towards sodium carboxyl methyl cellulose at approximately 275 IU/g cell dry weight. DEG exhibited greater than 60% residual activity in the pH range 2.5-8.5, higher than free endoglucanase (FEG), which had 40% residual activity at the same pH range. The highest tolerated temperature for DEG was 70ºC, much higher than that of FEG, which was approximately 50ºC. Moreover, DEG showed 91.1% activity at 65ºC for 120 min, while FEG only kept 77.8% residual activity over the same period. The half-life of DEG was 270 min at 65ºC, compared with only 150 min for FEG. DEG could be used repeatedly at least three times. These results suggest that the DEG has broad applications as a yeast whole-cell biocatalyst, due to its novel properties of high catalytic efficiency, acid-thermal stabilities, and reusability.
참고문헌 (27)
- Bai Y, "Cloning of endo-β-glucanase I gene and expression in Pichia pastoris", Front. Agric. China, 5, pp.196~200 (2011)
- Bai Y., "DNA shuffling of Thermoascus aurantiacus endo-β-glucanase I gene and the study of enzymic properties", 석사, pp.2011
- Bhat MK, "Cellulases and related enzymes in biotechnology", Biotechnol. Adv, 18, pp.355~383 (2000)
- Ecker M, "Pir proteins of Saccharomyces cerevisiae are attached to β-1,3-glucan by a new protein–carbohydrate linkage", J. Biol. Chem, 281, pp.11523~11529 (2006)
- Gai SA, "Yeast surface display for protein engineering and characterization", Curr. Opin. Struct. Biol, 17, pp.467~473 (2001)
- Harju S, "Rapid isolation of yeast genomic DNA: Bust n' Grab", BMC Biotechnol, 4, pp.8~14 (2004)
- Jacobs PP, "Pichia surface display: display of proteins on the surface of glycoengineered Pichia pastoris strains", Biotechnol. Lett, 30, pp.2173~2181 (2008)
- Jiang ZB, "Cell surface display of functionally active lipases from Yarrowia lipolytica in Pichia pastoris", Protein Exp. Purif, 56, pp.35~39 (2007)
- Jin Z, "Production of flavor esters catalyzed by CALB-displaying Pichia pastoris whole-cells in a batch reactor", J. Biotechnol, 159, pp.108~114 (2012)
- Kojima M, "Expression and surface display of Cellulomonas endoglucanase in the ethanologenic bacterium Zymobacter palmae", Appl. Microbiol. Biotechnol, 96, pp.1093~1104 (2012)
- Lee KM, "Purification and characterization of a thermostable endo-β-1, 4-glucanase from a novel strain of Penicillium purpurogenum", Enzyme Microb. Technol, 46, pp.206~211 (2010)
- Liu W, "Surface display of active lipase in Saccharomyces cerevisiae using Cwp2 as an anchor protein", Biotechnol. Lett, 32, pp.255~260 (2010)
- Lynd LR, "Microbial cellulose utilization: fundamentals and biotechnology", Microbiol. Mol. Biol. Rev, 66, pp.506~577 (2002)
- Matano Yi, "Display of cellulases on the cell surface of Saccharomyces cerevisiae for high yield ethanol production from high-solid lignocellulosic biomass", Bioresour. Technol, 108, pp.128~133 (2012)
- Matsui K, "Creation of a novel peptide endowing yeasts with acid tolerance using yeast cell-surface engineering", Appl. Microbiol. Biotechnol, 82, pp.105~113 (2009)
- Miller GL, "Use of dinitrosalicyclic acid reagent for determination of reducing sugar", Anal. Chem, 31, pp.426~428 (1959)
- Sutanu Samanta, "Characterization of Trichoderma reesei Endoglucanase II Expressed Heterologously in Pichia pastoris for Better Biofinishing and Biostoning", The Journal of Microbiology, 50(3), pp.518~525 (2012), 한국미생물학회[DOI]
- Sambrook J, "Molecular Cloning:A Laboratory Manual" (2002), Cold Spring Harbor Laboratory Press Cold Spring Harbor
- Shibasaki S, "Quantitative evaluation of the enhanced green fluorescent protein displayed on the cell surface of Saccharomyces cerevisiae by fluorometric and confocal laser scanning microscopic analyses", Appl. Microbiol. Biotechnol, 55, pp.471~475 (2001)
- Song HT, "Construction of a whole-cell catalyst displaying a fungal lipase for effective treatment of oily wastewaters", J. Mol. Catal. B Enzym, 71, pp.166~170 (2011)
- Sumita T, "Comparison of cell wall localization among Pir family proteins and functional dissection of the region required for cell wall binding and bud scar recruitment of Pir1p", Eukaryot. Cell, 4, pp.1872~1881 (2005)
- Tanaka T, "Recent developments in yeast cell surface display toward extended applications in biotechnology", Appl. Microbiol. Biotechnol, 95, pp.577~591 (2012)
- Wang KC, "Yeast surface display of antibodies via the heterodimeric interaction of two coiled-coil adapters", J. Immunol. Methods, 354, pp.11~19 (2010)
- Wang Q, "Construction of a novel Pichia pastoris cell-surface display system based on the cell wall protein Pir1", Curr. Microbiol, 56, pp.352~357 (2008)
- Xie J, "Construction of the general vector of Pichia pastoris cell surface display based on the anchor sequence of pir-N terminus", J. Agric. Univ. Hebei, 35, pp.81~85 (2012)
- Yang DL, "Cloning and expression of a novel thermostable cellulase from newly isolated Bacillus subtilis strain I15", Mol. Biol. Rep, 37, pp.1923~1929 (2010)
- Zhao XH, "A comparative study of β-1,4-endoglucanase (possessing β-1,4-exoglucanase activity) from Bacillus subtilis LH expressed in Pichia pastoris GS115 and Escherichia coli Rosetta (DE3)", Bioresour. Technol, 110, pp.539~545 (2012)