Hongge Chen

Terminal Amino Acids Disturb Xylanase Thermostability and Activity

Background: Unlike the α-helix/β-strand, the non-regular region contains amino acid defined as disordered residue (DR); its effect on enzyme structure and function is elusive. Results: Terminal DR deletions significantly increased xylanase thermostability and activity. Conclusion: Terminal DRs disturb xylanase thermostability and activity. Significance: DR deletion increased regular secondary structural content, and hence, led to slow decreased ΔG0 in the thermal denaturation process, and ultimately, enhanced enzyme thermostability. Protein structure is composed of regular secondary structural elements (α-helix and β-strand) and non-regular region. Unlike the helix and strand, the non-regular region consists of an amino acid defined as a disordered residue (DR). When compared with the effect of the helix and strand, the effect of the DR on enzyme structure and function is elusive. An Aspergillus niger GH10 xylanase (Xyn) was selected as a model molecule of (β/α)8 because the general structure consists of ∼10% enzymes. The Xyn has five N-terminal DRs and one C-terminal DR, respectively, which were deleted to construct three mutants, XynΔN, XynΔC, and XynΔNC. Each mutant was ∼2-, 3-, or 4-fold more thermostable and 7-, 4-, or 4-fold more active than the Xyn. The N-terminal deletion decreased the xylanase temperature optimum for activity (Topt) 6 °C, but the C-terminal deletion increased its Topt 6 °C. The N- and C-terminal deletions had opposing effects on the enzyme Topt but had additive effects on its thermostability. The five N-terminal DR deletions had more effect on the enzyme kinetics but less effect on its thermo property than the one C-terminal DR deletion. CD data showed that the terminal DR deletions increased regular secondary structural contents, and hence, led to slow decreased Gibbs free energy changes (ΔG0) in the thermal denaturation process, which ultimately enhanced enzyme thermostabilities.

Domain-swapping of mesophilic xylanase with hyper-thermophilic glucanase

BackgroundDomain fusion is limited at enzyme one terminus. The issue was explored by swapping a mesophilic Aspergillus niger GH11 xylanase (Xyn) with a hyper-thermophilic Thermotoga maritima glucanase (Glu) to construct two chimeras, Xyn-Glu and Glu-Xyn, with an intention to create thermostable xylanase containing glucanase activity.ResultsWhen expressed in E. coli BL21(DE3), the two chimeras exhibited bi-functional activities of xylanase and glucanase. The Xyn-Glu Xyn moiety had optimal reaction temperature (Topt) at 50 °C and thermal in-activation half-life (t1/2) at 50 °C for 47.6 min, compared to 47 °C and 17.6 min for the Xyn. The Glu-Xyn Xyn moiety had equivalent Topt to and shorter t1/2 (5.2 min) than the Xyn. Both chimera Glu moieties were more thermostable than the Glu, and the three enzyme Topt values were higher than 96 °C. The Glu-Xyn Glu moiety optimal pH was 5.8, compared to 3.8 for the Xyn-Glu Glu moiety and the Glu. Both chimera two moieties cooperated with each other in degrading substrates.ConclusionsDomain-swapping created different effects on each moiety properties. Fusing the Glu domain at C-terminus increased the xylanase thermostability, but fusing the Glu domain at N-terminus decreased the xylanase thermostability. Fusing the Xyn domain at either terminus increased the glucanase thermostability, and fusing the Xyn domain at C-terminus shifted the glucanase pH property 2 units higher towards alkaline environments. Fusing a domain at C-terminus contributes more to enzyme catalytic activity; whereas, fusing a bigger domain at N-terminus disturbs enzyme substrate binding affinity.

Non-Structured Amino-Acid Impact on GH11 Differs from GH10 Xylanase

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.

Effect of Codon Message on Xylanase Thermal Activity

Background: Because it is known for degeneracy, the effect of codon on enzyme thermal property is elusive. Results: Three purine-rich codons correlate positively with xylanase Topt, and two pyridine-rich codons correlate negatively. Two positive codons have A-ends. One negative codon has a C-end. Conclusion: Codons have effects on enzyme thermal property. Significance: The effect of codon message is lost when thermal property is analyzed at the residual level. Because the genetic codon is known for degeneracy, its effect on enzyme thermal property is seldom investigated. A dataset was constructed for GH10 xylanase coding sequences and optimal temperatures for activity (Topt). Codon contents and relative synonymous codon usages were calculated and respectively correlated with the enzyme Topt values, which were used to describe the xylanase thermophilic tendencies without dividing them into two thermophilic and mesophilic groups. After analyses of codon content and relative synonymous codon usages were checked by the Bonferroni correction, we found five codons, with three (AUA, AGA, and AGG) correlating positively and two (CGU and AGC) correlating negatively with the Topt value. The three positive codons are purine-rich codons, and the two negative codons have A-ends. The two negative codons are pyridine-rich codons, and one has a C-end. Comparable with the codon C- and A-ending features, C- and A-content within mRNA correlated negatively and positively with the Topt value, respectively. Thereby, codons have effects on enzyme thermal property. When the issue is analyzed at the residual level, the effect of codon message is lost. The codons relating to enzyme thermal property are selected by thermophilic force at nucleotide level.

Molecular Cloning and Characterizations of Xylanase Inhibitor Protein from Wheat (Triticum Aestivum)

Xylanase inhibitor proteins (XIPs) were regarded to inhibit the activity of xylanases during baking and gluten-starch separation processes. To avoid the inhibition to xylanases, it is necessary to define the conditions under which the inhibition takes place. In this study, we cloned the XIP gene from 2 different variety of Triticum aestivum, that is, Zhengmai 9023 and Zhengmai 366, and investigated the properties of XIP protein expressed by Pichia pastoris. The results showed that the 2 XIP genes (xip-9023 and xip-366) were highly homologous with only 3 nucleotide differences. XIP-9023 showed the optimal inhibition pH and temperature were 7 °C and 40 °C, respectively. Inhibition of xylanase by XIP-9023 reached the maximum in 40 min. At 50% inhibition of xylanase, the molar ratio of inhibitor: xylanase was 26:1. XIP-9023 was active to various fungal xylanases tested as well as to a bacterial xylanase produced by Paenibacillus sp. isolated from cow rumen.

Investigating the expression of F10 and G11 xylanases in Aspergillus niger A09 with qPCR

There exist significant differences between the 2 main types of xylanases, family F10 and G11. A clear understanding of the expression pattern of microbial F10 and G11 under different culture conditions would facilitate better production and industrial application of xylanase. In this study, the fungal xylanase producer Aspergillus niger A09 was systematically investigated in terms of induced expression of xylanase F10 and G11. Results showed that carbon and nitrogen sources could influence xylanase F10 and G11 transcript abundance, with G11 more susceptible to changes in culture media composition. The most favorable carbon and nitrogen sources for high G11 and low F10 production by A. niger A09 were xylan (2%) and (NH4)2C2O4 (0.3%), respectively. Following cultivation at 33 °C for 60 h, the highest xylanase activity (1132 IU per gram of wet mycelia) was observed. On the basis of differential gene expression of F10 and G11, as well as their different properties, we deduced that the F10 protein initially targeted xylan and hydrolyzed it into fragments including xylose, after which xylose acted as the inducer of F10 and G11 gene expression. These speculations also accounted for our failure to identify conditions favoring the high production of F10 but a low production of G11.