Abstract
The Aspergillus niger xylanase (Xyn) was used as a model to investigate impacts of un-structured residues on GH11 family enzyme, because the β-jelly roll structure has five residues (Ser1Ala2Gly3Ile4Asn5) at N-terminus and two residues (Ser183Ser184) at C-terminus that do not form to helix or strand. The N- or/and C-terminal residues were respectively deleted to construct three mutants. The optimal temperatures of XynΔN, XynΔC, and XynΔNC were 46, 50, and 46°C, and the thermostabilities were 15.7, 73.9, 15.5 min at 50°C, respectively, compared to 48°C and 33.9 min for the Xyn. After kinetic analysis, the substrate-binding affinities for birch-wood xylan decreased in the order XynΔC>Xyn>XynΔNC>XynΔN, while the Kcat values increased in the order XynΔC<XynΔNC<Xyn<XynΔN. The C-terminal deletion increased the GH11 xylanase thermostability and Topt, while the N- and NC-terminal deletions decreased its thermostability and optimal temperature. The C-terminal residues created more impact on enzyme thermal property, while the N-terminal residues created more impact on its catalytic efficiency and substrate-binding affinity. The impact of non-structured residues on GH11 xylanase was different from that of similar residues on GH10 xylanase, and the difference is attributed to structural difference between GH11 jelly-roll and GH10 (β/α)8.
Highlights
Enzyme is widely used in industrial biocatalysis, such as fermentation, bio-energy production, food, beverage, paper and pulp, etc
Homologous segments were recombined between two Streptomyces lividans xylanases [4]
The GH11 xylanase seven terminal non-structured residues were rationally deleted based on structural analysis, because the residues do not form to regular secondary structural elements
Summary
Enzyme is widely used in industrial biocatalysis, such as fermentation, bio-energy production, food, beverage, paper and pulp, etc. The higher temperatures demand for robust enzymes. Many strategies have been used to improve enzyme thermostability. Disulphide-bonds were introduced to the Trichoderma reesei xylanase and the Bacillus stearothermophilus xylanase [1,2,3]. Homologous segments were recombined between two Streptomyces lividans xylanases [4]. The N-terminus of mesophilic S. olivaceovirdis xylanase was substituted with the homologous region of thermophilic Thermomonospora fusca xylanase [5]. Disulfide-bond was introduced to the T. reesei xylanase internal region and the Thermomyces lanuginosus xylanase N-terminus [6,7]
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