Characterization of a GH Family 43 E-Xylosidase Having a Novel Carbohydratebinding Module from Paenibacillus xylaniclasticus Strain TW1

 Paenibacillus xylaniclasticus strain TW1, a gram-positive facultative anaerobic bacterium, was isolated as a xylanolytic microorganism from the wastes of a pineapple processing factory. A gene encoding one of its xylanolytic enzymes, a E-xylosidase, was cloned and sequenced. Sequence analysis revealed that this E-xylosidase, named PxXyl43A, was composed of a glycoside hydrolase (GH) family 43 subfamily 12 catalytic module and an unknown function module (UM). The full-length PxXyl43A (PxXyl43A) was heterologously expressed in Escherichia coli and purified. Recombinant PxXyl43A exhibited hydrolysis activity against both p-nitrophenyl-E-D-xylopyranoside (pNPX) and p-nitrophenyl-D-L-arabinofuranoside at specific activity of 250 and 310 mU/mg, respectively. the optimal reaction pH and temperature for pNPX hydrolysis were 7.1 and 54oC, respectively. At pH 7.0 and 54oC, the Km and kcat for pNPX were 1.2 mM and 2.8 ±0.15, respectively. It was also discovered that the recombinant unknown function module of PxXyl43A (PxXyl43A-UM) could bind to insoluble xylans like birchwood xylan and oat spelt xylan, whereas it did not bind to cellulosic substrates such as ball-milled cellulose, carboxymethyl cellulose or lichenan. The PxXyl43A-UMʼs binding constant value Ka for oat spelt xylan was 2.0 x 10-5 M-1. These results suggest that PxXyl43A possesses a novel carbohydrate-binding module, named as CBM91, specific for xylan-containing polysaccharide.