The secondary substrate binding site of the Pseudoalteromonas haloplanktis GH8 xylanase is relevant for activity on insoluble but not soluble substrates

Sven Cuyvers, Emmie Dornez, Jan A. Delcour, Christophe M. Courtin*

Laboratory of Food Chemistry and Biochemistry & Leuven Food Science and Nutrition Research Centre (LFoRCe), Katholieke Universiteit Leuven, Kasteelpark Arenberg 20 - box 2463, B-3001 Leuven, Belgium.

*Corresponding author: Christophe Courtin - Tel: + 3216321917 - Fax: + 3216321997 - E-mail address:


SupplementaryTable - Summary of template DNA and oligonucleotide primers used in site-directed mutagenesis for the genetic engineering of the Pseudoalteromonas haloplanktis xylanase (XPH).

Template DNA / Primers (5' → 3')
forward primer / reverse primer
XPH
W249A / pEXP5-CT-xph / GTTAGCCCGCATGCGAACTTACCTAC / GTAGGTAAGTTCGCATGCGGGCTAAC
N250A / pEXP5-CT-xph / GTTAGCCCGCATTGGGCCTTACCTACATTTTTATC / GATAAAAATGTAGGTAAGGCCCAATGCGGGCTAAC
Y315A / pEXP5-CT-xph / CTGGGCTTTTTAAGTGCTGCAAAAACAAAC / GTTTGTTTTTGCAGCACTTAAAAAGCCCAG
W249A-Y315A / pEXP5-CT-xph_W249A / CTGGGCTTTTTAAGTGCTGCAAAAACAAAC / GTTTGTTTTTGCAGCACTTAAAAAGCCCAG
N307Q / pEXP5-CT-xph / CATAAAAGTGCAGTTCAGAAAGCACTGGGC / GCCCAGTGCTTTCTGAACTGCACTTTTATG
K308Y / pEXP5-CT-xph / GTGCAGTTAATTACGCACTGGGCTTTTTAAG / CTTAAAAAGCCCAGTGCGTAATTAACTGCAC
N357Q / pEXP5-CT-xph / GCTTCAACACAGGCTGGGCAAG / CTTGCCCAGCCTGTGTTGAAGC


Supplementary Fig.1 - Enzyme stability in the conditions of the activity assays. The stability of XPH and its mutant variants is tested by measurement of the activity on Xylazyme AX after an additional 60min pre-incubation at 30°C. The amount of Xylazyme AX breakdown for each enzyme in a control experiment is used as a reference activity (100%). Standard deviations are shown as error bars. Measurements were done on three independent enzyme dilutions for each condition. Bars with the same letter above a not significantly different from each other according to Tukey’s tests (p-value < 0.05) performed with Statistical Analysis System software 9.2 (SAS Institute, Cary, NC, USA).

Supplementary Fig.2 - Temperature optimum of P.haloplanktis xylanase (XPH) mutants with a modified secondary binding site. Breakdown of Xylazymearabinoxylan (AX) was determined at different temperatures in a 1.00nM enzyme solution in sodium phosphate buffer (200mM, pH 7.0) with 0.5mg/mL bovine serum albumin for 60min. Values are shown relative to the amount of Xylazyme AX breakdown by the wild-type enzyme at 30°C (100%).