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Xu W.,Henan Agricultural University | Liu Y.,Henan Agricultural University | Ye Y.,Henan Agricultural University | Liu M.,Henan Agricultural University | And 4 more authors.
Biotechnology Letters | Year: 2016

Objective: The 9_2 carbohydrate-binding module (C2) locates natively at the C-terminus of the GH10 thermophilic xylanase from Thermotoga marimita. When fused to the C-terminus, C2 improved thermostability of a GH11 xylanase (Xyn) from Aspergillus niger. However, a question is whether the C-terminal C2 would have a thermostabilizing effect when fused to the N-terminus of a catalytic module. Results: A chimeric enzyme, C2-Xyn, was created by step-extension PCR, cloned in pET21a(+), and expressed in E. coli BL21(DE3). The C2-Xyn exhibited a 2 °C higher optimal temperature, a 2.8-fold longer thermostability, and a 4.5-fold higher catalytic efficiency on beechwood xylan than the Xyn. The C2-Xyn exhibited a similar affinity for binding to beechwood xylan and a higher affinity for oat-spelt xylan than Xyn. Conclusion: C2 is a thermostabilizing carbohydrate-binding module and provides a model of fusion at an enzymatic terminus inconsistent with the modular natural terminal location. © 2016 Springer Science+Business Media Dordrecht Source

Liu L.,Henan Agricultural University | Chen L.,Henan Agricultural University | Chen L.,Key Laboratory of Enzyme Engineering of Agricultural Microbiology | Tian H.,Henan Agricultural University | And 2 more authors.
Process Biochemistry | Year: 2012

Signal peptide (SP) prediction is used, but not known correct or not. The Aspergillus niger GH10 xylanase (XynB) SP is predicted to be 1-19 (Met 1-Ser19) residue, differing from the Penicillium simplicissimum xylanase 1-25 residue SP. To determine the real SP, two types of XynB, XynΔ19 and XynΔ25, were constructed by respectively deleting the 19 (Met1-Ser19) or 25 (Met1-Arg 25) residues. The XynΔ25 had 10 °C higher Topt and 21.6-times longer thermostability than the XynΔ19 (46 vs. 36 °C and 47.6 vs. 2.2 m). When the kinetics were assayed, the XynΔ25 had ∼2.1-times higher Vmax and higher binding-affinity for xylan than the XynΔ19 (53.7 vs. 25.3 μmol/ml/m and 2.43 vs. 2.96 mg/ml). Thus, the XynB real SP is the 1-25 and not predicted 1-19 residues. The extra six N-terminal residues (Glu20Pro21Ile22Glu 23Pro24Arg25) drastically interfered with the XynΔ19 thermal activity, thermostability, and catalytic efficiency. © 2012 Elsevier Ltd. Source

Su L.-J.,Henan Agricultural University | Liu Y.-Q.,Henan Agricultural University | Liu H.,Henan Agricultural University | Wang Y.,Henan Agricultural University | And 6 more authors.
Genetics and Molecular Research | Year: 2015

Tsaitermes ampliceps (lower termites) and Mironasutitermes shangchengensis (higher termites) are highly eusocial insects that thrive on recalcitrant lignocellulosic diets through nutritional symbioses with gut dwelling prokaryotes and eukaryotes. We used denaturing gradient gel electrophoresis and a 16S rRNA clone library to investigate i) how microbial communities adapt to lignocellulosic diets with different cellulose and lignin content, ii) the differences in the dominant gut microbial communities of the 2 types of termites. The results indicated that gut microbiota composition in T. ampliceps was profoundly affected by 2-week diet shifts. Comparison of these changes indicated that Bacteroidetes and Spirochaetes act in cellulose degradation, while Firmicutes were responsible for lignin degradation. Additionally, Proteobacteria consistently participated in energy production and balanced the gut environment. Bacteroidetes may function without hindgut protozoans in higher termites. The diversity of enteric microorganisms in M. shangchengensis was higher than that in T. ampliceps, possibly because of the more complicated survival mechanisms of higher termites. © FUNPEC-RP. Source

Liu L.,Henan Agricultural University | Sun X.,Henan Agricultural University | Yan P.,Henan Agricultural University | Wang L.,Henan Agricultural University | And 3 more authors.
PLoS ONE | Year: 2012

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 Source

Wang F.,Henan Agricultural University | Wang F.,Key Laboratory of Enzyme Engineering of Agricultural Microbiology | Yin S.,Henan Agricultural University | Yin S.,Key Laboratory of Enzyme Engineering of Agricultural Microbiology | And 6 more authors.
Nongye Gongcheng Xuebao/Transactions of the Chinese Society of Agricultural Engineering | Year: 2012

To enhance the pretreatment effect for production of cellulose ethanol, the influences of water immersion and CaO treatments before steam explosion on the structure destruction of corn stalks and enzymatic saccharification were studied. Field emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR) were involved in this study. The results showed that compared to the control (corn stalks pretreated with steam explosion), corn stalk immersed with 30% water (mass ratio of water to corn stalk was 30: 100) for 5 days, or treated with 2% CaO (mass ratio of CaO to corn stalk is 2: 100) for 3days, or treated with 2% CaO and 30% water for 1 day before steam explosion enhanced the lignin degradation rate from 20.7% to 27.8%, 35.1% and 30.9%, respectively; the concentrations of reducing sugars in the three pretreatments were 3.81, 3.59 and 3.46 g/100 mL respectively; and the sugar yields in the three pretreatments were 42.2%, 39.8% and 38.3% and increased by 23.7%, 16.6% and 12.3% respectively compared with the control. Pretreatment with 30% water immersion for 5 days and 2% CaO for 3 days intensified the destruction of surface structures and the degradation of lignin, the relative crystallinity of which increased by 47.0% and 54.5% respectively compared to the control (42.6%). Pretreatment with 30% water or 2% CaO exhibited high efficiency and sugar yields. It is benefit for the promotion of this technology with low-price and non-contaminating reagents. Source

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