title: Expression and characterization of hyperthermotolerant xylanases, SyXyn11P and SyXyn11E, in Pichia pastoris and Escherichia coli.
abstract: Both Syxyn11P and Syxyn11E, two codon-optimized genes encoding glycoside hydrolase (GH) family 11 hyperthermotolerant xylanases (designated SyXyn11P and SyXyn11E), were synthesized and inserted into pPIC9K(M) and pET-28a(+) vectors, respectively. The resulting recombinant expression vectors, pPIC9K(M)-Syxyn11P and pET-28a(+)-Syxyn11E, were transformed into Pichia pastoris GS115 and Escherichia coli BL21, respectively. The maximum activities of two recombinant xylanases (reSyXyn11P and reSyXyn11E) expressed in P. pastoris and E. coli reached 30.9 and 17.8 U/ml, respectively. The purified reSyXyn11P and reSyXyn11E displayed the same pH optimum at 6.5 and pH stability at a broad range of 4.5-9.0. The temperature optimum and stability of reSyXyn11P were 85 and 80 °C, higher than those of reSyXyn11E, respectively. Their activities were not significantly affected by metal ions tested and EDTA, but strongly inhibited by Mn(2+) and Ag(+). The K m and V max of reSyXyn11P toward birchwood xylan were 4.3 mg/ml and 694.6 U/mg, whose K m was close to that (4.8 mg/ml), but whose V max was much higher than that (205.6 U/mg) of reSyXyn11E. High-performance liquid chromatography analysis indicated that xylobiose and xylotriose as the major products were excised from insoluble corncob xylan by reSyXyn11P.
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, People's Republic of China.
authors.forename: Jianfang
authors.initials: J
authors.lastname: Li
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhang
authors.forename: Minchen
authors.initials: M
authors.lastname: Wu
authors.forename: Chunjuan
authors.initials: C
authors.lastname: Wang
authors.forename: Yunhai
authors.initials: Y
authors.lastname: Dong
authors.forename: Lijuan
authors.initials: L
authors.lastname: Zhu
authors.forename: Peng
authors.initials: P
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005813:Genes, Synthetic; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases