The GH51 α-l-arabinofuranosidase from Paenibacillus sp. THS1 is multifunctional, hydrolyzing main-chain and side-chain glycosidic bonds in heteroxylans

Abstract

BackgroundConceptually, multi-functional enzymes are attractive because in the case of complex polymer hydrolysis having two or more activities defined by a single enzyme offers the possibility of synergy and reduced enzyme cocktail complexity. Nevertheless, multi-functional enzymes are quite rare and are generally multi-domain assemblies with each activity being defined by a separate protein module. However, a recent report described a GH51 arabinofuranosidase from Alicyclobacillus sp. A4 that displays both α-l-arabinofuranosidase and β-d-xylanase activities, which are defined by a single active site. Following on from this, we describe in detail another multi-functional GH51 arabinofuranosidase and discuss the molecular basis of multifunctionality.ResultsTHSAbf is a GH51 α-l-arabinofuranosidase. Characterization revealed that THSAbf is active up to 75 °C, stable at 60 °C and active over a broad pH range (4–7). THSAbf preferentially releases para-nitrophenyl from the l-arabinofuranoside (kcat/KM = 1050 s−1 mM−1) and to some extent from d-galactofuranoside and d-xyloside. THSAbf is active on 4-O-methylglucuronoxylans from birch and beechwood (10.8 and 14.4 U mg−1, respectively) and on sugar beet branched and linear arabinans (1.1 ± 0.24 and 1.8 ± 0.1 U mg−1). Further investigation revealed that like the Alicyclobacillus sp. A4 α-l-arabinofuranosidase, THSAbf also displays endo-xylanase activity, cleaving β-1,4 bonds in heteroxylans. The optimum pH for THASAbf activity is substrate dependent, but ablation of the catalytic nucleophile caused a general loss of activity, indicating the involvement of a single active center. Combining the α-l-arabinofuranosidase with a GH11 endoxylanase did not procure synergy. The molecular modeling of THSAbf revealed a wide active site cleft and clues to explain multi-functionality.ConclusionThe discovery of single active site, multifunctional enzymes such as THSAbf opens up exciting avenues for enzyme engineering and the development of new biomass-degrading cocktails that could considerably reduce enzyme production costs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0550-x) contains supplementary material, which is available to authorized users.

Screening of a Novel Glycoside Hydrolase Family 51 α-L-Arabinofuranosidase from Paenibacillus polymyxa KF-1: Cloning, Expression, and Characterization

“…THS1 and 60 • C for rAbfA from Paenibacillus sp. DG-22 [24,32]. PpAbf51b showed better stability than other published bacterial GH51 α-L-arabinofuranosidases.…”

“…The identities were approximately 26-28% to GH51 α-L-arabinofuranosidases from Thermotoga maritima (PDB ID: 3UG3), T. maritima MSB8 (PDB ID: 4ATW), Alicyclobacillus sp. A4 (KT781102), Geobacillus stearothermophilus T6 (PDB ID: 1PZ3), and Clostridium thermocellum (PDB ID: 2C7F) [20][21][22][23][24][25]. We noticed that the amino acid sequence similarities between the two α-L-arabinofuranosidases (WP_013368995.1 and WP_014599997.1) were only 27%.…”

A novel bifunctional acetyl xylan esterase/arabinofuranosidase from Penicillium chrysogenum P33 enhances enzymatic hydrolysis of lignocellulose

“…Therefore, to effectively degrade lignocellulose, fungi usually secrete a set of extracellular lignocellulolytic enzymes [ 42 ], and anaerobic bacteria have evolved cell-associated multiprotein complexes known as the cellulosome [ 43 ] or xylosome [ 44 ]. Another important strategy in lignocellulolytic microbes is the induction of multifunctional enzymes to degrade different substrates [ 7 ], and this has received significant attention of late [ 5 , 6 , 45 ]. To our knowledge, the present work is the first detailed report of a bifunctional enzyme possessing acetyl xylan esterase and α- l -arabinofuranosidase activities.…”

“…Compared with enzymes possessing a single function, multifunctional enzymes can hydrolyze a variety of different substrates simultaneously, which reduces the amount of enzyme types needed for the hydrolysis of lignocellulose, decreases their cost in biotechnological processes, and has additional advantages. In this sense, multifunctional enzymes are more intriguing than traditional enzymes, especially those involved in heteroxylan hydrolysis [ 6 , 7 ]. β- d -Xylosidase/α- l -arabinofuranosidase [ 8 ], xylanase/α- l -arabinofuranosidase [ 5 , 6 ] and xylanase/acetyl xylan esterase [ 9 – 12 ] bifunctional systems have been reported.…”

“…In this sense, multifunctional enzymes are more intriguing than traditional enzymes, especially those involved in heteroxylan hydrolysis [ 6 , 7 ]. β- d -Xylosidase/α- l -arabinofuranosidase [ 8 ], xylanase/α- l -arabinofuranosidase [ 5 , 6 ] and xylanase/acetyl xylan esterase [ 9 – 12 ] bifunctional systems have been reported. Although the bifunctional α- l -arabinofuranosidase/acetyl xylan esterase was reported in Arthrobacter sp.…”