title: Novel Properties for Endoglucanase Acquired by Cell-Surface Display Technique.
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 70°C, much higher than that of FEG, which was approximately 50°C. Moreover, DEG showed 91.1% activity at 65°C for 120 min, while FEG only kept 77.8% residual activity over the same period. The half-life of DEG was 270 min at 65°C, 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.
authors.affiliation: College of Landscape, Agriculture University of Hebei, Baoding 071001, P.R. China.
authors.forename: Baosheng
authors.initials: B
authors.lastname: Shi
authors.affiliation: College of Food Science and Technology, Agricultural University of Hebei, Baoding 071001, P.R. China.
authors.forename: Xiaojing
authors.initials: X
authors.lastname: Ke
authors.affiliation: College of Food Science and Technology, Agricultural University of Hebei, Baoding 071001, P.R. China.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Yu
authors.affiliation: College of Food Science and Technology, Agricultural University of Hebei, Baoding 071001, P.R. China.
authors.forename: Jing
authors.initials: J
authors.lastname: Xie
authors.affiliation: College of Biology Science and Engineering, Hebei University of Science and Technology, Shijiazhuang 050000, P.R. China.
authors.forename: Yingmin
authors.initials: Y
authors.lastname: Jia
authors.affiliation: College of Food Science and Technology, Agricultural University of Hebei, Baoding 071001, P.R. China.
authors.forename: Runfang
authors.initials: R
authors.lastname: Guo
mesh_terms: D061505:Cell Surface Display Techniques; D002480:Cellulase / Q000235:genetics / Q000378:metabolism*; D003224:Congo Red / Q000378:metabolism; D004795:Enzyme Stability; D015870:Gene Expression; D005839:Gentamicins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013696:Temperature; D014170:Transformation, Genetic
chemical_list: D005839:Gentamicins; D011993:Recombinant Fusion Proteins; D003224:Congo Red; C010680:antibiotic G 418; D002480:Cellulase
keywords: Pichia pastoris; acid-thermal stabilities; cell-surface display; endoglucanase; reusability