Molecular cloning and characterization of the novel acidic xylanase XYLD from Bispora sp. MEY-1 that is homologous to family 30 glycosyl hydrolases

Abstract

We cloned and sequenced a xylanase gene named xylD from the acidophilic fungus Bispora sp. MEY-1 and expressed the gene in Pichia pastoris. The 1,422-bp full-length complementary DNA fragment encoded a 457-amino acid xylanase with a calculated molecular mass of 49.8 kDa. The mature protein of XYLD showed high sequence similarity to both glycosyl hydrolase (GH) families 5 and 30 but was more homologous to members of GH 30 based on phylogenetic analysis. XYLD shared the highest identity (49.9%) with a putative endo-1,6-beta-D-glucanase from Talaromyces stipitatus and exhibited 21.1% identity and 34.3% similarity to the well-characterized GH family 5 xylanase from Erwinia chrysanthemi. Purified recombinant XYLD showed maximal activity at pH 3.0 and 60 degrees C, maintained more than 60% of maximal activity when assayed at pH 1.5-4.0, and had good thermal stability at 60 degrees C and remained stable at pH 1.0-6.0. The enzyme activity was enhanced in the presence of Ni(2+) and beta-mercaptoethanol and inhibited by some metal irons (Hg(2+), Cu(2+), Pb(2+), Mn(2+), Li(+), and Fe(3+)) and sodium dodecyl sulfate. The specific activity of XYLD for beechwood xylan, birchwood xylan, 4-O-methyl-D-glucuronoxylan, and oat spelt xylan was 2,463, 2,144, 2,020, and 1,429 U mg(-1), respectively. The apparent K (m) and V (max) values for beechwood xylan were 5.6 mg ml(-1) and 3,622 micromol min(-1) mg(-1), respectively. The hydrolysis products of different xylans were mainly xylose and xylobiose.

Modular Glucuronoxylan-Specific Xylanase with a Family CBM35 Carbohydrate-Binding Module

“…Recently, a GH30 enzyme active on both glucuronoxylans and arabinoxylans from the fungus Bispora sp. MEY-1 has been identified as a xylanase (29). However, the Bispora enzyme has been classified in subgroup G of the GH30 family, while the glucurunoxylan-specific xylanases are classified in subgroup H (39).…”

Consolidation of glycosyl hydrolase family 30: A dual domain 4/7 hydrolase family consisting of two structurally distinct groups

“…In doing so, it was confirmed [7,[9][10][11] that five groups of enzymes currently assigned in GH family 5 are more similar to GH30 enzymes. Subgroup E contains two similar sequences with endo-b-1,6-galactanase activity [39,40], subgroup G has a recently characterized xylanase activity [41] and subgroup H contains the reassigned glucuronoxylan xylanohydrolase enzymes [4,5,42]. Subgroups D and F contain no biochemically characterized enzymes.…”

“…Subgroup E is identified by two biochemically characterized enzymes with endo-b-1,6-galactanase activity [39,40]. A recent report [41] assigned an acidic b-1,4-xylanase function to a fungal enzyme that shares approximately 50% identity with a subgroup G enzyme, also of fungal origin (ACC B8M989). No information is available for the sparsely populated subgroup F. Two subgroup H members are characterized having glucuronoxylan xylanohydrolase activity and structure models are available for these same enzymes [4][5][6]11,42].…”

“…In contrast, subgroups G and H both have assigned xylanase activity. However, subgroup H xylanases are fundamentally unique in comparison to subgroup G having an enzymatic activity which relies upon recognition of an a-1,2-linked 4-O-methyl glucuronate moiety [4,5,41].…”

Cloning and expression of a family 10 xylanase gene (Aoxyn10) from Aspergillus oryzae in Pichia pastoris

“…Some xylanase genes from filamentous fungi, such as Aspergillus niger (Ruanglek et al, 2007), A. terreus (Chantasingh et al, 2006), Penicillium thiersii (Cui et al, 2009) and Fusarium oxysporum (Moukouli et al, 2011), have been cloned and expressed in heterologous cells. Based on the structure alignment and hydrophobic cluster analysis, xylanases have been mainly classified into GH families 10 and 11 (Collins et al, 2005), while enzymes with xylanolytic activity were also widespread in GH families 5, 7, 8, 16, 26, 30, 43, 52 and 62 (http://www.cazy.org/fam/acc_GH.html) (Luo et al, 2010). Compared to GH family 11 counterparts, the family 10 xylanases exhibited higher molecular weight ( 30 kDa) and lower substrate specificity, and also had catalytic activities towards some cellulosic substrates, such as aryl cellobioside (Biely et al, 1997).…”