Format

Send to

Choose Destination
Appl Environ Microbiol. 2017 Aug 31;83(18). pii: e00794-17. doi: 10.1128/AEM.00794-17. Print 2017 Sep 15.

Enzymatic Mechanism for Arabinan Degradation and Transport in the Thermophilic Bacterium Caldanaerobius polysaccharolyticus.

Wefers D1,2, Dong J3,2, Abdel-Hamid AM3,2, Paul HM4,3,2, Pereira GV4,3,2, Han Y3,2, Dodd D5,3,2, Baskaran R4,2, Mayer B5,3,2, Mackie RI4,3,2, Cann I1,5,3,2.

Author information

1
Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA daniel.wefers@kit.edu icann@illinois.edu.
2
Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
3
Energy Biosciences Institute, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
4
Department of Animal Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.
5
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois, USA.

Abstract

The plant cell wall polysaccharide arabinan provides an important supply of arabinose, and unraveling arabinan-degrading strategies by microbes is important for understanding its use as a source of energy. Here, we explored the arabinan-degrading enzymes in the thermophilic bacterium Caldanaerobius polysaccharolyticus and identified a gene cluster encoding two glycoside hydrolase (GH) family 51 α-l-arabinofuranosidases (CpAbf51A, CpAbf51B), a GH43 endoarabinanase (CpAbn43A), a GH27 β-l-arabinopyranosidase (CpAbp27A), and two GH127 β-l-arabinofuranosidases (CpAbf127A, CpAbf127B). The genes were expressed as recombinant proteins, and the functions of the purified proteins were determined with para-nitrophenyl (pNP)-linked sugars and naturally occurring pectin structural elements as the substrates. The results demonstrated that CpAbn43A is an endoarabinanase while CpAbf51A and CpAbf51B are α-l-arabinofuranosidases that exhibit diverse substrate specificities, cleaving α-1,2, α-1,3, and α-1,5 linkages of purified arabinan-oligosaccharides. Furthermore, both CpAbf127A and CpAbf127B cleaved β-arabinofuranose residues in complex arabinan side chains, thus providing evidence of the function of this family of enzymes on such polysaccharides. The optimal temperatures of the enzymes ranged between 60°C and 75°C, and CpAbf43A and CpAbf51A worked synergistically to release arabinose from branched and debranched arabinan. Furthermore, the hydrolytic activity on branched arabinan oligosaccharides and degradation of pectic substrates by the endoarabinanase and l-arabinofuranosidases suggested a microbe equipped with diverse activities to degrade complex arabinan in the environment. Based on our functional analyses of the genes in the arabinan degradation cluster and the substrate-binding studies on a component of the cognate transporter system, we propose a model for arabinan degradation and transport by C. polysaccharolyticusIMPORTANCE Genomic DNA sequencing and bioinformatic analysis allowed the identification of a gene cluster encoding several proteins predicted to function in arabinan degradation and transport in C. polysaccharolyticus The analysis of the recombinant proteins yielded detailed insights into the putative arabinan metabolism of this thermophilic bacterium. The use of various branched arabinan oligosaccharides provided a detailed understanding of the substrate specificities of the enzymes and allowed assignment of two new GH127 polypeptides as β-l-arabinofuranosidases able to degrade pectic substrates, thus expanding our knowledge of this rare group of glycoside hydrolases. In addition, the enzymes showed synergistic effects for the degradation of arabinans at elevated temperatures. The enzymes characterized from the gene cluster are, therefore, of utility for arabinose production in both the biofuel and food industries.

KEYWORDS:

pectic enzymes; thermophiles

PMID:
28710263
PMCID:
PMC5583487
DOI:
10.1128/AEM.00794-17
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center