title: Residue mutations of xylanase in Aspergillus kawachii alter its optimum pH.
abstract: Aspergillus kawachii and Aspergillus niger have been traditionally used as molds for commercial microbial fermentation because of their capability to grow in extremely acidic environments and produce acid-stable enzymes. Endo-1,4-β-xylanase cleaves the glycosidic bonds in the xylan backbone, consequently reducing the degree of polymerization of the substrate. The amino acid sequences of xylanases from A. kawachii and A. niger only differ in one amino acid residue. However, the xylanases from A. kawachii and A. niger show different optimum pH values of 2.0 and 3.0, respectively. In this study, we synthesized the A. kawachii xylanase gene (XynC) on the basis of the bias codon of yeast and mutated the gene in the dominating region related to optimum pH shifting during gene synthesis. After the overexpression of this gene in Pichia pastoris G115, the mutant (Thr64Ser) enzyme (XynC-C) showed an optimum pH of 3.8, which indicated partial alkalinity compared with the original xylanase from A. kawachii. Similar to that of the enzyme with one residue mutation (Asp48Asn), the optimum pH of the enzyme with two residue mutations (Thr64Ser and Asp48Asn) shifted to 5.0. The result indicated that mutation Asp48 was more important than mutation Thr64 in optimum pH shifting. We proposed a model that explains the lower optimum pH of XynC-C than other members of the xylanase family G. XynC-C showed similar proteolytic resistance and Km and Vmax values for beechwood xylan to other xylanases.
authors.affiliation: Shanghai Key Laboratory of Agricultural Genetics and Breeding, Agro-Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, 2901 Beidi Rd, Shanghai 201106, PR China; College of Food Science and Technology, Shanghai Ocean University, 999 Huchenghuan Rd, Shanghai 210306, PR China.
authors.forename: Jin
authors.initials: J
authors.lastname: Qiu
authors.affiliation: Shanghai Key Laboratory of Agricultural Genetics and Breeding, Agro-Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, 2901 Beidi Rd, Shanghai 201106, PR China.
authors.forename: Hongjuan
authors.initials: H
authors.lastname: Han
authors.affiliation: Shanghai Key Laboratory of Agricultural Genetics and Breeding, Agro-Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, 2901 Beidi Rd, Shanghai 201106, PR China; College of Food Science and Technology, Shanghai Ocean University, 999 Huchenghuan Rd, Shanghai 210306, PR China.
authors.forename: Baihui
authors.initials: B
authors.lastname: Sun
authors.affiliation: Shanghai Key Laboratory of Agricultural Genetics and Breeding, Agro-Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, 2901 Beidi Rd, Shanghai 201106, PR China; College of Food Science and Technology, Shanghai Ocean University, 999 Huchenghuan Rd, Shanghai 210306, PR China.
authors.forename: Lei
authors.initials: L
authors.lastname: Chen
authors.affiliation: Shanghai Key Laboratory of Agricultural Genetics and Breeding, Agro-Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, 2901 Beidi Rd, Shanghai 201106, PR China; College of Food Science and Technology, Shanghai Ocean University, 999 Huchenghuan Rd, Shanghai 210306, PR China.
authors.forename: Chengye
authors.initials: C
authors.lastname: Yu
authors.affiliation: Shanghai Key Laboratory of Agricultural Genetics and Breeding, Agro-Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, 2901 Beidi Rd, Shanghai 201106, PR China; College of Food Science and Technology, Shanghai Ocean University, 999 Huchenghuan Rd, Shanghai 210306, PR China.
authors.forename: Rihe
authors.initials: R
authors.lastname: Peng
authors.affiliation: Shanghai Key Laboratory of Agricultural Genetics and Breeding, Agro-Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, 2901 Beidi Rd, Shanghai 201106, PR China; College of Food Science and Technology, Shanghai Ocean University, 999 Huchenghuan Rd, Shanghai 210306, PR China. Electronic address: yao.quanhong65@yahoo.com.
authors.forename: Quanhong
authors.initials: Q
authors.lastname: Yao
mesh_terms: D001234:Aspergillus niger / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001665:Binding Sites; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering
chemical_list: D005656:Fungal Proteins; D043364:Endo-1,4-beta Xylanases
keywords: Aspergillus kawachii; Optimum pH; Protein structure; Xylanase