Characterization of the Wild-Type and Truncated Forms of a Neutral GH10 Xylanase fromCoprinus cinereus: Roles of C-Terminal Basic Amino Acid-Rich Extension in Its SDS Resistance, Thermostability, and Activity.
A neutral xylanase (CcXyn) was identified fromCoprinus cinereus. It has a single GH10 catalytic domain with a basic amino acid-rich extension (PVRRK) at the C-terminus. In this study, the wild-type (CcXyn) and C-terminus-truncated xylanase (CcXyn-Δ5C) were heterologously expressed inPichia pastorisand their characteristics were comparatively analyzed with aims to examine the effect of this extension on the enzyme function. The circular dichorism analysis indicated that both enzymes in general had a similar structure, but CcXyn-Δ5C contained less α-helices (42.9%) and more random coil contents (35.5%) than CcXyn (47.0% and 32.8%, respectively). Both enzymes had the same pH (7.0) and temperature (45°C) optima, and similar substrate specificity on different xylans. They all hydrolyzed beechwood xylan primarily to xylobiose and xylotriose. The amounts of xylobiose and xylotriose accounted for 91.5% and 92.2% (w/w) of total xylooligosaccharides (XOS) generated from beechwood by CcXyn and CcXyn-Δ5C, respectively. However, truncation of the C-terminal 5-amino-acids extension significantly improved the thermostability, SDS resistance, and pH stability at pH 6.0-9.0. Furthermore, CcXyn-Δ5C exhibited a much lowerKmvalue than CcXyn (0.27 mg/ml vs 0.83 mg/ml), and therefore, the catalytic efficiency of CcXyn-Δ5C was 2.4-times higher than that of CcXyn. These properties make CcXyn-Δ5C a good model for the structure-function study of (α/β)8-barrel-folded enzymes and a promising candidate for various applications, especially in the detergent industry and XOS production.
College of Chemical Engineering, Nanjing Forestry University, Nanjing, Jiangsu 210037, P.R. China.
Hang
H
Hu
College of Chemical Engineering, Nanjing Forestry University, Nanjing, Jiangsu 210037, P.R. China.
Kaixiang
K
Chen
College of Chemical Engineering, Nanjing Forestry University, Nanjing, Jiangsu 210037, P.R. China.
Lulu
L
Li
College of Chemical Engineering, Nanjing Forestry University, Nanjing, Jiangsu 210037, P.R. China.
Liangkun
L
Long
College of Chemical Engineering, Nanjing Forestry University, Nanjing, Jiangsu 210037, P.R. China.
Shaojun
S
Ding
D000595:Amino Acid Sequence; D024361:Amino Acids, Basic / Q000737:chemistry*; D003302:Coprinus / Q000201:enzymology* / Q000235:genetics; D004271:DNA, Fungal; D004187:Disaccharides / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D057075:Enzyme Assays; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D016681:Genome, Fungal; D005965:Glucuronates / Q000032:analysis / Q000378:metabolism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D009844:Oligosaccharides / Q000032:analysis / Q000378:metabolism; D010843:Pichia / Q000201:enzymology; D016415:Sequence Alignment; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013379:Substrate Specificity; D014312:Trisaccharides / Q000378:metabolism; D014990:Xylans / Q000378:metabolism; D014994:Xylose / Q000032:analysis / Q000378:metabolism
D024361:Amino Acids, Basic; D004271:DNA, Fungal; D004187:Disaccharides; D005656:Fungal Proteins; D005965:Glucuronates; D008670:Metals; D009844:Oligosaccharides; D014312:Trisaccharides; D014990:Xylans; C570991:xylooligosaccharide; C515044:xylotriose; D012967:Sodium Dodecyl Sulfate; D004492:Edetic Acid; D014994:Xylose; D043364:Endo-1,4-beta Xylanases; C004173:xylobiose
C-terminal extension; Coprinus cinereus; GH10 xylanase; SDS resistance; catalytic activity; thermostability