Signal peptide of Aureobasidium pullulans xylanase: use for extracellular production of a fungal xylanase by Escherichia coli.
An extracellular xylanase XynI of glycoside hydrolase family 11 from the dimorphic fungus Aureobasidium pullulans ATCC 20524 possesses an N-terminal extension of 34 amino acids (Ohta et al., J. Biosci. Bioeng. 92:262-270, 2001). The N-terminal extension includes three sites (Ala-X-Ala-X-Ala-X-Ala) that are potentially cleavable by signal peptidase I of Escherichia coli. The A. pullulans xynI signal sequence was fused in frame to the mature protein region of the equivalent xylanase gene xynA from the filamentous fungus Penicillium citrinum. The gene fusion xynI::A was inserted into the plasmid pET-26b(+) to yield pEXP401. An E. coli BL21(DE3) transformant harboring the pEXP401 exhibited xylanase activity (per ml of the culture) of 16.8 U in the fraction of culture supernatant as well as 4.29 U in the fraction of cell-free extract after 12 h of growth with isopropyl-β-D-thiogalactopyranoside at 30°C. N-terminal amino acid sequence analysis of the secreted recombinant proteins revealed cleavage at four distinct sites within the N-terminal extension of XynI, two of which conformed to the Ala-X-Ala motif prior to the cleavage site. The XynA proteins secreted into the culture medium showed high specific activities from 506 to 651 U/mg, which were twofold higher than that of the native enzyme.
Ohta
Kazuyoshi
K
Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, University of Miyazaki, 1-1 Gakuen Kibanadai Nishi, Miyazaki, 889-2192, Japan. k.ohta@cc.miyazaki-u.ac.jp
Tanaka
Hidenori
H
Yamakawa
Daisuke
D
Hamasuna
Hironori
H
Fujimoto
Hirohisa
H
D001203:Ascomycota / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D043364:Endo-1,4-beta Xylanases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics; D010407:Penicillium / Q000201:enzymology / Q000235:genetics; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D021382:Protein Sorting Signals / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics; D014169:Transformation, Bacterial
D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases; D012697:Serine Endopeptidases; C026515:type I signal peptidase