N- and C-terminal truncations of a GH10 xylanase significantly increase its activity and thermostability but decrease its SDS resistance

Abstract

XynII from Volvariella volvacea has high sodium dodecyl sulfate (SDS) resistance, with the potential for industrial applications under harsh conditions. It consists of a single glycoside hydrolase family 10 (GH10) catalytic domain but contains an additional unique 10 and 4 amino acid residues at the N- and C-terminus, respectively. In this study, five XynII derivatives with N- and/or C-terminus deletions were constructed to determine the effects of these regions on enzyme activity, substrate specificity, thermostability, and SDS resistance. Our results revealed that N- and/or C-terminal truncations significantly increased enzyme activity and thermostability, but reduced SDS resistance. Specifically, the XynIIΔNC4 mutant had 2.53-fold more catalytic efficiency (k cat/K m) towards beechwood xylan than wild-type and 3.0-fold more thermostability (t 1/2 [55°C]). XynIIΔNC4 displayed 3.33-, 4.38-, 1.37-, and 1.98-fold more activity against xylotriose, xylotetraose, xylopentaose, and xylohexaose, respectively, than XynII did. However, its half-life (t 1/2) in 4 % SDS was only 1.72 h, while that of XynII was 4.65 h. Circular dichroism analysis revealed that deletion of N- and C-terminal segments caused minor changes in secondary structure. Our observations suggest that the extra N- and C-terminal segments in wild-type XynII evolved to strengthen the interaction between these regions of the protein, making the local structure more rigid and reducing structural flexibility. In this way, N- and C-terminal truncations increased the thermostability and activity of XynII on different xylans and linear xylooligosaccharides, but reduced its resistance to SDS.

Characterization of the Wild-Type and Truncated Forms of a Neutral GH10 Xylanase from Coprinus cinereus: Roles of C-Terminal Basic Amino Acid-Rich Extension in Its SDS Resistance, Thermostability, and Activity

“…CcXyn and CcXy-nΔ5C (0.028 U each) were incubated with various XOS (each 50 μg) at 50ºC in 0.1 M phosphate/citrate buffer (1.5 ml, pH 7.5) for 24 h. After incubation, the reactions were stopped by boiling, and the end products of hydrolysis were then analyzed at 30ºC using a Carbo-Pac PA200 column (3 × 250 mm) fitted to an ICS-3000 high-performance anion exchange chromatography system (Dionex, USA) with pulsed amperometric detection (HPAEC-PAD) as described as previously [18]. The hydrolysis products of CcXyn and CcXyn-Δ5C towards beechwood xylan were determined under the standard conditions at 60ºC for up to 24 h, and ampicillin (25 μg/ml) and zeocin (25 μg/ml) were added in the reaction mixture to avoid microbial contamination.…”

“…On the other hand, CcXyn also lacks an N-terminal extension rich in Pro and Phe residues as XynII has. This may explain the different effect of C-terminal extension on SDS resistance between the two enzymes [18]. The elevated SDS resistance of CcXyn-Δ5C implied that the corresponding factors may reside in its core domain [32].…”

“…Consequently, less attention has been paid to the role of the N-or/and C-terminal sequences in protein structure and function. However, recent studies have proposed that the terminal sequences have significant effect on protein secretion, folding, and function, particularly for proteins with a single domain [11][12][13][14][15][16][17][18].…”

“…We previously reported a SDS-resistant xylanase (XynII) from V. volvacea. We proposed that the extreme high SDS resistance of XynII might partially be attributed to its N-and C-terminal extensions, because deletion of the N-and C-terminal extensions significantly reduced its SDS resistance although the XynII activity and thermostability were improved [18]. Unlike XynII, deletion of the C-terminal extension in CcXyn significantly enhanced its SDS tolerance.…”

Effect of introducing disulfide bridges in C-terminal structure on the thermostability of xylanase XynZF-2 from <i>Aspergillus niger</i>

“…As an important stable structure in proteins(Zheng et al 2016), disulfide bonds serve the function 15 of maintaining protein stability in the form which can keep the bridge formation in the peptide residues 16 as a complex biological reactions (Chen et al 2017; Kisiela et al 2018; Li et al 2017; Nakamura et al 17 2018; Xie et al 2018). There exists a very stable core region of xylanase, so we can start this work in 18 the C-terminal or the N-terminal which is far away from the core region (Letian et al 2014).…”