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1.
J Biotechnol ; 257: 122-130, 2017 Sep 10.
Article in English | MEDLINE | ID: mdl-28450260

ABSTRACT

Herbinix hemicellulosilytica is a newly isolated, gram-positive, anaerobic bacterium with extensive hemicellulose-degrading capabilities obtained from a thermophilic biogas reactor. In order to exploit its potential as a source for new industrial arabinoxylan-degrading enzymes, six new thermophilic xylanases, four from glycoside hydrolase family 10 (GH10) and two from GH11, three arabinofuranosidases (1x GH43, 2x GH51) and one ß-xylosidase (GH43) were selected. The recombinantly produced enzymes were purified and characterized. All enzymes were active on different xylan-based polysaccharides and most of them showed temperature-vs-activity profiles with maxima around 55-65°C. HPAEC-PAD analysis of the hydrolysates of wheat arabinoxylan and of various purified xylooligosaccharides (XOS) and arabinoxylooligosaccharides (AXOS) was used to investigate their substrate and product specificities: among the GH10 xylanases, XynB showed a different product pattern when hydrolysing AXOS compared to XynA, XynC, and XynD. None of the GH11 xylanases was able to degrade any of the tested AXOS. All three arabinofuranosidases, ArfA, ArfB and ArfC, were classified as type AXH-m,d enzymes. None of the arabinofuranosidases was able to degrade the double-arabinosylated xylooligosaccharides XA2+3XX. ß-Xylosidase XylA (GH43) was able to degrade unsubstituted XOS, but showed limited activity to degrade AXOS.


Subject(s)
Clostridiales/enzymology , Glycoside Hydrolases/metabolism , Xylans/metabolism , Xylosidases/metabolism , Carbohydrate Metabolism , Cloning, Molecular , Clostridiales/genetics , Endo-1,4-beta Xylanases/genetics , Endo-1,4-beta Xylanases/metabolism , Enzyme Activation , Glucuronates/metabolism , Glycoside Hydrolases/chemistry , Glycoside Hydrolases/genetics , Hot Temperature , Hydrogen-Ion Concentration , Hydrolysis , Oligosaccharides/metabolism , Polysaccharides/metabolism , Recombinant Proteins , Substrate Specificity , Triticum , Xylosidases/chemistry , Xylosidases/genetics , beta-Glucosidase/genetics , beta-Glucosidase/metabolism
2.
Syst Appl Microbiol ; 38(6): 424-32, 2015 Sep.
Article in English | MEDLINE | ID: mdl-26227216

ABSTRACT

Cellulolytic bacterial strains with high activity were isolated from cellulose degrading enrichment cultures derived from thermophilic biogas plants and environmental samples. The 16S rRNA gene sequences of the strains revealed >99.8% sequence identity and affiliation with the species Clostridium thermocellum. The strains differed in their ability to degrade crystalline cellulose, especially at an elevated temperature of up to 67 °C and at relatively low pH values (pH 6.5). To evaluate the influence of amino acid sequences on the discrepancies in cellulose degradation efficacy, the gene for the major cellulosomal component CelR was sequenced for all strains. The sequences were found to be almost identical (>99%). In contrast, the cellulosomal scaffoldin gene cipA showed more differences in the amino acid sequence and contained 8 or 9 cohesin modules, which indicated a different size of the cellulosome depending on the isolate. Based on MALDI-TOF MS analysis the relative abundance of important cellulosomal enzyme classes was determined. The strains with better biomass degradation properties (BC1 and NB2) had a significantly higher fraction of xylanases.


Subject(s)
Bacterial Proteins/genetics , Bioreactors/microbiology , Carrier Proteins/genetics , Cellulose/metabolism , Cellulosomes/metabolism , Clostridium thermocellum/metabolism , Environmental Microbiology , Genetic Variation , Biotransformation , Cellulosomes/chemistry , Cellulosomes/enzymology , Cellulosomes/genetics , Clostridium thermocellum/enzymology , Clostridium thermocellum/genetics , Clostridium thermocellum/isolation & purification , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , DNA, Ribosomal/chemistry , DNA, Ribosomal/genetics , Hydrogen-Ion Concentration , Molecular Sequence Data , RNA, Ribosomal, 16S/genetics , Repressor Proteins/genetics , Sequence Analysis, DNA , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Temperature
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