Molecular cloning and heterologous expression of a new xylanase gene from Plectosphaerella cucumerina

Abstract

A gene encoding a new xylanase, named xynZG, was cloned by the genome-walking PCR method from the nematophagous fungus Plectosphaerella cucumerina. The genomic DNA sequence of xynZG contains a 780 bp open reading frame separated by two introns with the sizes of 50 and 46 bp. To our knowledge, this would be the first functional gene cloned from P. cucumerina. The 684 bp cDNA was cloned into vector pHBM905B and transformed into Pichia pastoris GS115 to select xylanase-secreting transformants on RBB-xylan containing plate. The optimal secreting time was 3 days at 25 degrees C and enzymatic activities in the culture supernatants reached the maximum level of 362 U ml(-1). The molecular mass of the enzyme was estimated to be 19 kDa on SDS-PAGE. The optimal pH and temperature of the purified enzyme is 6 and 40 degrees C, respectively. The purified enzyme is stable at room temperature for at least 10 h. The Km and Vmax values for birchwood xylan are 2.06 mg ml(-1) and 0.49 mmol min(-1)mg(-1), respectively. The inhibitory effects of various mental ions were investigated. It is interesting to note that Cu2+ ion, which strongly inhibits most other xylanases studied, reduces enzyme activity by only 40%. Furthermore, enzyme activity is unaffected by EDTA even at a concentration of 5 mM.

Characterization of a thermostable xylanase from an alkaliphilic Bacillus sp.

“…Previous publications reported that EDTA was an inhibitor to some xylanases (Zhang et al 2007); however, in this study, EDTA at 0.25% did not affect enzyme activity (Table 2). This suggests that xylanases may have different catalytic mechanisms, and that Xyn10 is not a metalloenzyme.…”

Thermal Stability and Thermodynamics of Xylanase from Melanocarpus albomyces in Presence of Polyols and Salts

“…As shown in Table 5 close vicinity with the active site of xylanase, and its interaction with sulfhydryl group led to strong inhibition of xylanase activity; similar observations were also reported for other xylanases (Beg et al 2001). The substantial effect of Hg 2+ (2.5%) on xylanase activity reveal the important participation of tryptophan residues in the catalytic action of enzyme; as Hg 2+ can oxidize the indole ring, it was believed to interact with aromatic ring present in tryptophan, contributing in decrease of xylanase activity Zhang et al 2007). The thiol group-bearing chemical agent β-mercaptoethanol (121.6%), the non-ionic detergent polysorbate 80 (115.2%), and the chelating agent EDTA (105.4%) all showed stimulatory effects on xylanase activity.…”

Cloning and expression of acidstable, high maltose-forming, Ca2+-independent α-amylase from an acidophile Bacillus acidicola and its applicability in starch hydrolysis

“…Further, Hg 2? is known to oxidize indole ring and to interact with aromatic ring present in tryptophan (Zhang et al 2007;Liu et al 2010). NBS exerted very strong inhibitory action as in b-D-xylanase of B. halodurans, indicating the catalytic role of tryptophan (Kumar and Satyanarayana 2011).…”