title: Structural modeling and further improvement in pH stability and activity of a highly-active xylanase from an uncultured rumen fungus.
abstract: Rumen fungi are a rich source of enzymes degrading lignocelluloses. XynR8 is a glycosyl hydrolase family 11 xylanase previously cloned from unpurified rumen fungal cultures. Phylogenetic analysis suggested that xynR8 was obtained from a Neocallimastix species. Recombinant XynR8 expressed in Escherichia coli was highly active and stable between pH 3.0 and 11.0, and displayed a V(max) of 66,672μmolmin(-1)mg(-1), a k(cat) of 38,975s(-1), and a K(m) of 11.20mg/mL towards soluble oat spelt xylan. Based on molecular modeling, residues N41 and N58, important in stabilizing two loops and the structure of XynR8, were mutated to D. Both mutant enzymes showed higher tolerance to pH 2.0. The V(max), k(cat) and K(m) of the N41D and N58D mutant enzymes were 79,645μmolmin(-1)mg(-1), 46,493s(-1), 29.29mg/mL, and 96,689μmolmin(-1)mg(-1), 56,503s(-1), and 21.24mg/mL, respectively. Thus, they are good candidates for application, including biofuel production.
authors.affiliation: Department of Biological Science and Technology, National Pingtung University of Science and Technology, 1, Shuehfu Rd., Neipu, Pingtung 91201, Taiwan, ROC.
authors.forename: Yo-Chia
authors.initials: YC
authors.lastname: Chen
authors.forename: Yu-Chuan
authors.initials: YC
authors.lastname: Chiang
authors.forename: Fu-Yuan
authors.initials: FY
authors.lastname: Hsu
authors.forename: Li-Chu
authors.initials: LC
authors.lastname: Tsai
authors.forename: Hsueh-Ling
authors.initials: HL
authors.lastname: Cheng
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D020088:Neocallimastix / Q000201:enzymology*; D010802:Phylogeny; D011994:Recombinant Proteins / Q000378:metabolism; D012417:Rumen / Q000382:microbiology*; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D050505:Mutant Proteins; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases