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Zheng H.,Key Laboratory of Industrial Fermentation Microbiology | Zheng H.,Tianjin University of Science and Technology | Liu Y.,Key Laboratory of Industrial Fermentation Microbiology | Liu Y.,Tianjin Key Laboratory of Industrial Microbiology | And 9 more authors.
Journal of Microbiology and Biotechnology | Year: 2012

High levels of xylanase activity (143.98 IU/ml) produced by the newly isolated Paenibacillus campinasensis G1-1 were detected when it was cultivated in a synthetic medium. A thermostable xylanase, designated XynG1-1, from P. campinasensis G1-1 was purified to homogeneity by Octyl-Sepharose hydrophobic-interaction chromatography, Sephadex G75 gel-filter chromatography, and Q-Sepharose ion-exchange chromatography, consecutively. By multistep purification, the specific activity of XynG1-1 was up to 1,865.5 IU/mg with a 9.1-fold purification. The molecular mass of purified XynG1-1 was about 41.3 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Sequence analysis revealed that XynG1-1 containing 377 amino acids encoded by 1,134 bp genomic sequences of P. campinasensis G1-1 shared 96% homology with XylX from Paenibacillus campinasensis BL11 and 77%~78% homology with xylanases from Bacillus sp. YA- 335 and Bacillus sp. 41M-1, respectively. The activity of XynG1-1 was stimulated by Ca 2+, Ba 2+, DTT, and β- mercaptoethanol, but was inhibited by Ni 2+, Fe 2+, Fe 3+, Zn 2+, SDS, and EDTA. The purified XynG1-1 displayed a greater affinity for birchwood xylan, with an optimal temperature of 60°C and an optimal pH of 7.5. The fact that XynG1-1 is cellulose-free, thermostable (stability at high temperature of 70°C~80°C), and active over a wide pH range (pH 5.0~9.0) suggests that the enzyme is potentially valuable for various industrial applications, especially for pulp bleaching pretreatment. © The Korean Society for Microbiology and Biotechnology. Source


Zheng H.,Key Laboratory of Industrial Fermentation Microbiology | Zheng H.,Tianjin University of Science and Technology | Zheng H.,Chinese Academy of Agricultural Sciences | Liu Y.,Key Laboratory of Industrial Fermentation Microbiology | And 12 more authors.
Journal of Industrial Microbiology and Biotechnology | Year: 2014

The extreme process condition of high temperature and high alkali limits the applications of most of natural xylanases in pulp and paper industry. Recently, various methods of protein engineering have been used to improve the thermal and alkalic tolerance of xylanases. In this work, directed evolution and site-directed mutagenesis were performed to obtain a mutant xylanase improved both on alkali stability and thermostability from the native Paenibacillus campinasensis Family-11 xylanase (XynG1-1). Mutant XynG1-1B43 (V90R/P172H) with two units increased in the optimum pH (pH 7.0-pH 9.0) and significant improvement on alkali stability was selected from the second round of epPCR library. And the further thermoduric mutant XynG1-1B43cc16 (V90R/P172H/T84C- T182C/D16Y) with 10 C increased in the optimum temperature (60-70 C) was then obtained by introducing a disulfide bridge (T84C-T182C) and a single amino acid substitution (D16Y) to XynG1-1B43 using site-directed mutagenesis. XynG1-1B43cc16 also showed higher thermostability and catalytic efficiency (k cat /K m ) than that of wild-type (XynG1-1) and XynG1-1B43. The attractive improved properties make XynG1-1B43cc16 more suitable for bioleaching of cotton stalk pulp under the extreme process condition of high temperature (70 C) and high alkali (pH 9.0). © 2013 Society for Industrial Microbiology and Biotechnology. Source


Zheng H.,Key Laboratory of Industrial Fermentation Microbiology | Zheng H.,Tianjin University of Science and Technology | liu Y.,Key Laboratory of Industrial Fermentation Microbiology | liu Y.,Tianjin Key Laboratory of Industrial Microbiology | And 10 more authors.
Bioresource Technology | Year: 2012

A xylanase gene (xynG1-1) from Paenibacillus campinasensis G1-1 was expressed in Bacillus megaterium MS941 and a high level of extracellular xylansae activity (304.26. IU/mL) was achieved after induction with 0.5% xylose. The purified recombinant xylanase (XynG1-1R) revealed optimal activity at 60. °C and pH 7.0 and retained 79% and 81% activity after incubation without substrate at 60. °C, pH 5.0 and pH 8.0 for 3. h, respectively. Application of XynG1-1R (15. IU/g pulp) to cotton stalk pulp bleaching increased brightness by 3.65% over that of the control without the xylanase and reduced the need for chlorine compounds by 50% without loss of brightness and pulp fiber qualities. When XynG1-1R (80. IU/g paper sludge) was used in combination with mixed cellulolytic enzymes, the saccharification efficiency of recycled paper sludge was increased by 10%. These results indicated that XynG1-1R is a promising candidate for various industrial applications such as biobleaching and bioenergy conversion. © 2012 Elsevier Ltd. Source

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