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Dongguan, China

Han X.,CAS Tianjin Institute of Industrial Biotechnology | Gao J.,CAS Tianjin Institute of Industrial Biotechnology | Shang N.,CAS Tianjin Institute of Industrial Biotechnology | Huang C.-H.,CAS Tianjin Institute of Industrial Biotechnology | And 9 more authors.
Proteins: Structure, Function and Bioinformatics | Year: 2013

Xylanases are capable of decomposing xylans, the major components in plant cell wall, and releasing the constituent sugars for further applications. Because xylanase is widely used in various manufacturing processes, high specific activity, and thermostability are desirable. Here, the wild-type and mutant (E146A and E251A) catalytic domain of xylanase from Thermoanaerobacterium saccharolyticum JW/SL-YS485 (TsXylA) were expressed in Escherichia coli and purified subsequently. The recombinant protein showed optimal temperature and pH of 75°C and 6.5, respectively, and it remained fully active even after heat treatment at 75°C for 1 h. Furthermore, the crystal structures of apo-form wild-type TsXylA and the xylobiose-, xylotriose-, and xylotetraose-bound E146A and E251A mutants were solved by X-ray diffraction to high resolution (1.32-1.66 Å). The protein forms a classic (β/α)8 folding of typical GH10 xylanases. The ligands in substrate-binding groove as well as the interactions between sugars and active-site residues were clearly elucidated by analyzing the complex structures. According to the structural analyses, TsXylA utilizes a double displacement catalytic machinery to carry out the enzymatic reactions. In conclusion, TsXylA is effective under industrially favored conditions, and our findings provide fundamental knowledge which may contribute to further enhancement of the enzyme performance through molecular engineering. © 2013 Wiley Periodicals, Inc. Source


Cheng Y.-S.,National Taiwan University | Ko T.-P.,Academia Sinica, Taiwan | Huang J.-W.,Genozyme Biotechnology Inc. | Wu T.-H.,National Taiwan University | And 10 more authors.
Applied Microbiology and Biotechnology | Year: 2012

Cellulase 12A from Thermotoga maritima (TmCel12A) is a hyperthermostable ß-1,4-endoglucanase. We recently determined the crystal structures of TmCel12A and its complexes with oligosaccharides. Here, by using sitedirected mutagenesis, the role played by Arg60 and Tyr61 in a unique surface loop of TmCel12A was investigated. The results are consistent with the previously observed hydrogen bonding and stacking interactions between these two residues and the substrate. Interestingly, the mutant Y61G had the highest activity when compared with the wild-type enzyme and the other mutants. It also shows a wider range of working temperatures than does the wild type, along with retention of the hyperthermostability. The kcat and Km values of Y61G are both higher than those of the wild type. In conjunction with the crystal structure of Y61G-substrate complex, the kinetic data suggest that the higher endoglucanase activity is probably due to facile dissociation of the cleaved sugar moiety at the reducing end. Additional crystallographic analyses indicate that the insertion and deletion mutations at the Tyr61 site did not affect the overall protein structure, but local perturbations might diminish the substrate-binding strength. It is likely that the catalytic efficiency of TmCel12A is a subtle balance between substrate binding and product release. The activity enhancement by the single mutation of Y61G provides a good example of engineered enzyme for industrial application. © Springer-Verlag 2011. Source


Jiang T.,CAS Tianjin Institute of Industrial Biotechnology | Chan H.-C.,CAS Tianjin Institute of Industrial Biotechnology | Huang C.-H.,CAS Tianjin Institute of Industrial Biotechnology | Ko T.-P.,Academia Sinica, Taiwan | And 4 more authors.
Biochemical and Biophysical Research Communications | Year: 2013

β-Glucanases have been utilized widely in industry to treat various carbohydrate-containing materials. Recently, the Podospora anserina β-glucanase 131A (PaGluc131A) was identified and classified to a new glycoside hydrolases GH131 family. It shows exo-β-1,3/exo-β-1,6 and endo-β-1,4 glucanase activities with a broad substrate specificity for laminarin, curdlan, pachyman, lichenan, pustulan, and cellulosic derivatives. Here we report the crystal structures of the PaGluc131A catalytic domain with or without ligand (cellotriose) at 1.8. Å resolution. The cellotriose was clearly observed to occupy the +1 to +3 subsites in substrate binding cleft. The broadened substrate binding groove may explain the diverse substrate specificity. Based on our crystal structures, the GH131 family enzyme is likely to carry out the hydrolysis through an inverting catalytic mechanism, in which E99 and E139 are supposed to serve as the general base and general acid. © 2013 Elsevier Inc. Source


Cheng Y.-S.,Genozyme Biotechnology Inc. | Cheng Y.-S.,AsiaPac Biotechnology Co. | Chen C.-C.,CAS Tianjin Institute of Industrial Biotechnology | Huang C.-H.,CAS Tianjin Institute of Industrial Biotechnology | And 5 more authors.
Journal of Biological Chemistry | Year: 2014

Background: Thermophilic xylanases are valuable in many industrial applications. Results: The structures of a xylanase XynCDBFV and its complex with xylooligosaccharides were determined, and its N-terminal region (NTR) contributes to thermostability. Conclusion: NTR may stabilize the overall protein folding of XynCDBFV. Significance: The structural and functional investigation of unprecedented NTR of XynCDBFV provides a new insight into the molecular basis of thermophilic xylanases. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc. Source


Huang J.-W.,Genozyme Biotechnology Inc. | Huang J.-W.,AsiaPac Biotechnology Co. | Cheng Y.-S.,National Taiwan University | Ko T.-P.,Academia Sinica, Taiwan | And 13 more authors.
Applied Microbiology and Biotechnology | Year: 2012

1,3-1,4-β-d-Glucanase has been widely used as a feed additive to help non-ruminant animals digest plant fibers, with potential in increasing nutrition turnover rate and reducing sanitary problems. Engineering of enzymes for better thermostability is of great importance because it not only can broaden their industrial applications, but also facilitate exploring the mechanism of enzyme stability from structural point of view. To obtain enzyme with higher thermostability and specific activity, structure-based rational design was carried out in this study. Eleven mutants of Fibrobacter succinogenes 1,3-1,4-β-d-glucanase were constructed in attempt to improve the enzyme properties. In particular, the crude proteins expressed in Pichia pastoris were examined firstly to ensure that the protein productions meet the need for industrial fermentation. The crude protein of V18Y mutant showed a 2 °C increment of Tm and W203Y showed ∼30% increment of the specific activity. To further investigate the structure-function relationship, some mutants were expressed and purified from P. pastoris and Escherichia coli. Notably, the specific activity of purified W203Y which was expressed in E. coli was 63% higher than the wild-type protein. The double mutant V18Y/W203Y showed the same increments of Tm and specific activity as the single mutants did. When expressed and purified from E. coli, V18Y/W203Y showed similar pattern of thermostability increment and 75% higher specific activity. Furthermore, the apo-form and substrate complex structures of V18Y/W203Y were solved by X-ray crystallography. Analyzing protein structure of V18Y/W203Y helps elucidate how the mutations could enhance the protein stability and enzyme activity. © 2011 Springer-Verlag. Source

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