PMID- 28521816 OWN - NLM STAT- MEDLINE DCOM- 20171116 LR - 20190112 IS - 1472-6750 (Electronic) IS - 1472-6750 (Linking) VI - 17 IP - 1 DP - 2017 May 18 TI - Biochemical characterization of the xylan hydrolysis profile of the extracellular endo-xylanase from Geobacillus thermodenitrificans T12. PG - 44 LID - 10.1186/s12896-017-0357-2 [doi] LID - 44 AB - BACKGROUND: Endo-xylanases are essential in degrading hemicellulose of various lignocellulosic substrates. Hemicellulose degradation by Geobacillus spp. is facilitated by the hemicellulose utilization (HUS) locus that is present in most strains belonging to this genus. As part of the HUS locus, the xynA gene encoding an extracellular endo-xylanase is one of the few secreted enzymes and considered to be the key enzyme to initiate hemicellulose degradation. Several Geobacillus endo-xylanases have been characterized for their optimum temperature, optimum pH and generation of degradation products. However, these analyses provide limited details on the mode of action of the enzymes towards various substrates resulting in a lack of understanding about their hydrolytic potential. RESULTS: A HUS-locus associated gene (GtxynA1) from the thermophile Geobacillus thermodenitrificans T12 encodes an extracellular endo-xylanase that belongs to the family 10 glycoside hydrolases (GH10). The GtxynA1 gene was cloned and expressed in Escherichia coli. The resulting endo-xylanase (termed GtXynA1) was purified to homogeneity and showed activity between 40 degrees C and 80 degrees C, with an optimum activity at 60 degrees C, while being active between pH 3.0 to 9.0 with an optimum at pH 6.0. Its thermal stability was high and GtXynA1 showed 85% residual activity after 1 h of incubation at 60 degrees C. Highest activity was towards wheat arabinoxylan (WAX), beechwood xylan (BeWX) and birchwood xylan (BiWX). GtXynA1 is able to degrade WAX and BeWX producing mainly xylobiose and xylotriose. To determine its mode of action, we compared the hydrolysis products generated by GtXynA1 with those from the well-characterized GH10 endo-xylanase produced from Aspergillus awamori (AaXynA). The main difference in the mode of action between GtXynA1 and AaXynA on WAX is that GtXynA1 is less hindered by arabinosyl substituents and can therefore release shorter oligosaccharides. CONCLUSIONS: The G. thermodenitrificans T12 endo-xylanase, GtXynA1, shows temperature tolerance up to 80 degrees C and high activity to a variety of xylans. The mode of action of GtXynA1 reveals that arabinose substituents do not hamper substrate degradation by GtXynA1. The extensive hydrolysis of branched xylans makes this enzyme particularly suited for the conversion of a broad range of lignocellulosic substrates. FAU - Daas, Martinus J A AU - Daas MJA AD - Laboratory of Microbiology, Wageningen University, Stippeneng 4, Wageningen, 6708 WE, The Netherlands. FAU - Martinez, Patricia Murciano AU - Martinez PM AD - Laboratory of Food Chemistry, Wageningen University, Bornse Weilanden 9, Wageningen, 6708 WG, The Netherlands. FAU - van de Weijer, Antonius H P AU - van de Weijer AHP AD - Laboratory of Microbiology, Wageningen University, Stippeneng 4, Wageningen, 6708 WE, The Netherlands. FAU - van der Oost, John AU - van der Oost J AD - Laboratory of Microbiology, Wageningen University, Stippeneng 4, Wageningen, 6708 WE, The Netherlands. FAU - de Vos, Willem M AU - de Vos WM AD - Laboratory of Microbiology, Wageningen University, Stippeneng 4, Wageningen, 6708 WE, The Netherlands. FAU - Kabel, Mirjam A AU - Kabel MA AD - Laboratory of Food Chemistry, Wageningen University, Bornse Weilanden 9, Wageningen, 6708 WG, The Netherlands. FAU - van Kranenburg, Richard AU - van Kranenburg R AUID- ORCID: 0000-0001-5856-1546 AD - Laboratory of Microbiology, Wageningen University, Stippeneng 4, Wageningen, 6708 WE, The Netherlands. r.vankranenburg@wur.nl. AD - Corbion, Arkelsedijk 46, Gorinchem, 4206 AC, The Netherlands. r.vankranenburg@wur.nl. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20170518 PL - England TA - BMC Biotechnol JT - BMC biotechnology JID - 101088663 RN - 0 (Monosaccharides) RN - 0 (Oligosaccharides) RN - 0 (Recombinant Proteins) RN - 0 (Xylans) RN - EC 3.2.1.8 (Endo-1,4-beta Xylanases) SB - IM MH - Chromatography, Gel MH - Chromatography, High Pressure Liquid MH - Cloning, Molecular MH - Endo-1,4-beta Xylanases/genetics/*metabolism MH - Enzyme Stability MH - Geobacillus/*enzymology MH - Hydrogen-Ion Concentration MH - Hydrolysis MH - Monosaccharides/analysis MH - Oligosaccharides/analysis MH - Recombinant Proteins/biosynthesis/isolation & purification MH - Substrate Specificity MH - Temperature MH - Xylans/analysis/*metabolism PMC - PMC5437666 OTO - NOTNLM OT - Biotechnology OT - Endo-xylanase OT - Geobacillus OT - Thermophile OT - Xylan EDAT- 2017/05/20 06:00 MHDA- 2017/11/29 06:00 PMCR- 2017/05/18 CRDT- 2017/05/20 06:00 PHST- 2016/12/11 00:00 [received] PHST- 2017/03/25 00:00 [accepted] PHST- 2017/05/20 06:00 [entrez] PHST- 2017/05/20 06:00 [pubmed] PHST- 2017/11/29 06:00 [medline] PHST- 2017/05/18 00:00 [pmc-release] AID - 10.1186/s12896-017-0357-2 [pii] AID - 357 [pii] AID - 10.1186/s12896-017-0357-2 [doi] PST - epublish SO - BMC Biotechnol. 2017 May 18;17(1):44. doi: 10.1186/s12896-017-0357-2.