Date Published: April 2, 2018
Publisher: Springer Berlin Heidelberg
Author(s): Yanbo Hu, Xuecui Yan, Han Zhang, Jiaqi Liu, Feng Luo, Yingying Cui, Weiyang Wang, Yifa Zhou.
The discovery and creation of biocatalysts for plant biomass conversion are essential for industrial demand and scientific research of the plant cell wall. α-1,2 and α-1,3-l-arabinofuranosidases are debranching enzymes that catalyzing hydrolytic release of α-l-arabinofuranosyl residues in plant cell wall. Gene database analyses shows that GH62 family only contains specific α-l-arabinofuranosidases that play an important role in the degradation and structure of the plant cell wall. At present, there are only 22 enzymes in this group has been characterized. In this study, we cloned a novel α-1,3-arabinofuranosidase gene (poabf62a) belonging to glycoside hydrolase family 62 from Penicillium oxalicum sp. 68 and expressed it in Pichia pastoris. The molecular mass of recombinant PoAbf62A was estimated to be 32.9 kDa. Using p-nitrophenyl-α-l-arabinofuranoside (pNPαAbf) as substrate, purified PoAbf62A exhibited an optimal pH of 4.5 and temperature of 35 °C. Results of methylation and 13C NMR analyses showed that PoAbf62A was exclusively α-1,3-arabinofuranosidase, specific for cleavage of α-1,3-arabinofuranosyl residues, and with the absence of activity towards α-1,2-arabinofuranose and α-1,5-arabinofuranose. Therefore, PoAbf62A exhibits high activity on sugar beet arabinan and wheat arabinoxylan, because their branched side chain are decorated with α-1,3-arabinofuranose. On the other hand, there is a lack of activity with linear-α-l-1,5-arabinan and xylan that only contained α-l-1,5-arabinofuranose or β-1,4-xylose. The α-1,3-arabinofuranosidase activity identified here provides a new biocatalytic tool to degrade hemicellulose and analyze the structure of plant cell walls.
Glycoside hydrolases are a broad group of enzymes that hydrolyse the glycosidic bond of saccharides and their derivatives (Scigelova et al. 1999). Presently, about 148 glycoside hydrolase families have been reported, based on sequence and structural similarities in the CAZy database (Lombard et al. 2014). There are widely used in the bio-processing industry and for the structural analysis of polysaccharides from the plant cell wall because of high efficiency, specificity, and low pollution (Himmel et al. 2007). The α-l-arabinofuranosidases (ABFs) are enzymes that hydrolyze terminal non-reducing end α-l-arabinofuranoside residues in α-l-arabinosides. They are classified into families GH2, GH3, GH10, GH43, GH51, GH54 and GH62, with the ABFs of families GH43 and GH62 belonging to GH-F that have a five-bladed β propeller arrangement and an inverting mechanism of hydrolysis (Vandermarliere et al. 2009; Wang et al. 2014; Maehara et al. 2014). The GH62 family only contains α-l-arabinofuranosidases of fungal and bacterial origin. Based on ABFs’ substrate specificity, they can be divided into type A and B (Pitson et al. 1996). ABFs of type A only act on arabinoxylooligosaccharides and p-nitrophyl-α-l-arabinofuranoside, whereas ABFs of type B have equivalent activities on oligosaccharide and polysaccharides (Van Laere et al. 1997). All GH62 family belong to type B, because they are more active on polymeric arabinoxylan that is singly or doubly substituted with α-l-1,2-Araf and α-l-1,3-Araf residues (Kaur et al. 2015; Hashimoto et al. 2011). They have little activity on low molecular-weight substrates, such as unbranch arabinan and pNPαAraf (Wilkens et al. 2017; Sakamoto et al. 2011). ABFs have become known from their various industrial applications, such as biotransformation of plant residues, food processing, and bleaching of pulp (Saha 2000; Numan and Bhosle 2006). Aside from this, ABFs can completely degrade hemicelluloses and pectins in combination with other accessory hemicellulases and pectic enzymes (Margolles-Clark et al. 1996; Spagna et al. 1998). The ABFs from GH62 family are promising candidates for use in the structural analysis of polysaccharides and in regulating the chemical xylan to saccharify xylan for bio-refinery applications.
Our present study reports on the ability of the recombinant enzyme PoAbf62A of the GH62 family from P. oxalicum sp. 68 to hydrolyze arabinose-containing polysaccharides. PoAbf62A exhibits a high specifity for α-1,3-l-arabinan and a much lower activity towards linear-1,5-α-l-arabinan. And the optimal reaction conditions of recombinant PoAbf62A was pH 4.5 and temperature of 35 °C, which showed better adaptability in an acid environment, likely because fungi prefer to grow under mildly acid conditions. This actually makes recombinant PoAbf62A more valuable in application with fruit juices and drinks that clarify under acid condition.