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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.


Cheng Y.-S.,National Taiwan University | Ko T.-P.,Academia Sinica, Taiwan | Wu T.-H.,Genozyme Biotechnology Inc. | Ma Y.,CAS Tianjin Institute of Industrial Biotechnology | And 6 more authors.
Proteins: Structure, Function and Bioinformatics | Year: 2011

Cellulases have been used in many applications to treat various carbohydrate-containing materials. Thermotoga maritima cellulase 12A (TmCel12A) belongs to the GH12 family of glycoside hydrolases. It is a β-1,4-endoglucanase that degrades cellulose molecules into smaller fragments, facilitating further utilization of the carbohydrate. Because of its hyperthermophilic nature, the enzyme is especially suitable for industrial applications. Here the crystal structure of TmCel12A was determined by using an active-site mutant E134C and its mercury-containing derivatives. It adopts a β-jellyroll protein fold typical of the GH12-family enzymes, with two curved β-sheets A and B and a central active-site cleft. Structural comparison with other GH12 enzymes shows significant differences, as found in two longer and highly twisted β-strands B8 and B9 and several loops. A unique Loop A3-B3 that contains Arg60 and Tyr61 stabilizes the substrate by hydrogen bonding and stacking, as observed in the complex crystals with cellotetraose and cellobiose. The high-resolution structures allow clear elucidation of the network of interactions between the enzyme and its substrate. The sugar residues bound to the enzyme appear to be more ordered in the -2 and -1 subsites than in the +1, +2 and -3 subsites. In the E134C crystals the bound -1 sugar at the cleavage site consistently show the α-anomeric configuration, implicating an intermediate-like structure. Proteins 2011; © 2011 Wiley-Liss, Inc.


Wu T.-H.,National Taiwan University | Chen C.-C.,CAS Tianjin Institute of Industrial Biotechnology | Cheng Y.-S.,Genozyme Biotechnology Inc. | Cheng Y.-S.,AsiaPac Biotechnology Co. | And 11 more authors.
Journal of Biotechnology | Year: 2014

Escherichia coli phytase (EcAppA) which hydrolyzes phytate has been widely applied in the feed industry, but the need to improve the enzyme activity and thermostability remains. Here, we conduct rational design with two strategies to enhance the EcAppA performance. First, residues near the substrate binding pocket of EcAppA were modified according to the consensus sequence of two highly active Citrobacter phytases. One out of the eleven mutants, V89T, exhibited 17.5% increase in catalytic activity, which might be a result of stabilized protein folding. Second, the EcAppA glycosylation pattern was modified in accordance with the Citrobacter phytases. An N-glycosylation motif near the substrate binding site was disrupted to remove spatial hindrance for phytate entry and product departure. The de-glycosylated mutants showed 9.6% increase in specific activity. On the other hand, the EcAppA mutants that adopt N-glycosylation motifs from CbAppA showed improved thermostability that three mutants carrying single N-glycosylation motif exhibited 5.6-9.5% residual activity after treatment at 80. °C (1.8% for wild type). Furthermore, the mutant carrying all three glycosylation motifs exhibited 27% residual activity. In conclusion, a successful rational design was performed to obtain several useful EcAppA mutants with better properties for further applications. © 2014 Elsevier B.V.


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.


Cheng Y.-S.,Genozyme Biotechnology Inc. | Cheng Y.-S.,AsiaPac Biotechnology Co. | Huang C.-H.,CAS Tianjin Institute of Industrial Biotechnology | Chen C.-C.,CAS Tianjin Institute of Industrial Biotechnology | And 9 more authors.
Biochimica et Biophysica Acta - Proteins and Proteomics | Year: 2014

The thermostable 1,3-1,4-β-glucanase PtLic16A from the fungus Paecilomyces thermophila catalyzes stringent hydrolysis of barley β-glucan and lichenan with an outstanding efficiency and has great potential for broad industrial applications. Here, we report the crystal structures of PtLic16A and an inactive mutant E113A in ligand-free form and in complex with the ligands cellobiose, cellotetraose and glucotriose at 1.80 Å to 2.25 Å resolution. PtLic16A adopts a typical β-jellyroll fold with a curved surface and the concave face forms an extended ligand binding cleft. These structures suggest that PtLic16A might carry out the hydrolysis via retaining mechanism with E113 and E118 serving as the nucleophile and general acid/base, respectively. Interestingly, in the structure of E113A/1,3-1,4-β- glucotriose complex, the sugar bound to the - 1 subsite adopts an intermediate-like (α-anomeric) configuration. By combining all crystal structures solved here, a comprehensive binding mode for a substrate is proposed. These findings not only help understand the 1,3-1,4-β-glucanase catalytic mechanism but also provide a basis for further enzymatic engineering. © 2013 Elsevier B.V.


PubMed | CAS Tianjin Institute of Industrial Biotechnology, Academia Sinica, Taiwan, Genozyme Biotechnology Inc. and Jiangnan University
Type: | Journal: Enzyme and microbial technology | Year: 2016

Ganoderma lucidum is a saprotrophic white-rot fungus which contains a rich set of cellulolytic enzymes. Here, we screened an array of potential 1,4--endoglucanases from G. lucidum based on the gene annotation library and found that one candidate gene, GlCel5A, exhibits CMC-hydrolyzing activity. The recombinant GlCel5A protein expressed in Pichia pastoris is able to hydrolyze CMC and -glucan but not xylan and mannan. The enzyme exhibits optimal activity at 60C and pH 3-4, and retained 50% activity at 80 and 90C for at least 15 and 10min. The crystal structure of GlCel5A and its complex with cellobiose, solved at 2.7 and 2.86 resolution, shows a classical (/)8 TIM-barrel fold as seen in other members of glycoside hydrolase family 5. The complex structure contains a cellobiose molecule in the +1 and +2 subsites, and reveals the interactions with the positive sites of the enzyme. Collectively, the present work provides the first comprehensive characterization of an endoglucanase from G. lucidum that possesses properties for industrial applications, and strongly encourages further studying in the cellulolytic enzyme system of G. lucidum.


PubMed | CAS Tianjin Institute of Industrial Biotechnology, National Taiwan University, Academia Sinica, Taiwan and Genozyme Biotechnology Inc.
Type: | Journal: Enzyme and microbial technology | Year: 2015

A thermophilic glycoside hydrolase family 16 (GH16) -1,3-1,4-glucanase from Clostridium thermocellum (CtLic16A) holds great potentials in industrial applications due to its high specific activity and outstanding thermostability. In order to understand its molecular machinery, the crystal structure of CtLic16A was determined to 1.95 resolution. The enzyme folds into a classic GH16 -jellyroll architecture which consists of two -sheets atop each other, with the substrate-binding cleft lying on the concave side of the inner -sheet. Two Bis-Tris propane molecules were found in the positive and negative substrate binding sites. Structural analysis suggests that the major differences between the CtLic16A and other GH16 -1,3-1,4-glucanase structures occur at the protein exterior. Furthermore, the high catalytic efficacy and thermal profile of the CtLic16A are preserved in the enzyme produced in Pichia pastoris, encouraging its further commercial applications.


PubMed | CAS Tianjin Institute of Industrial Biotechnology, Genozyme Biotechnology Inc. and Academia Sinica, Taiwan
Type: Journal Article | Journal: Applied microbiology and biotechnology | Year: 2015

XynCDBFV from Neocallimastix patriciarum is among the most effective xylanases and holds great potentials in a wide variety of industrial applications. In the present study, several active site residues were modified referring to the instrumental information of the complex structure of XynCDBFV and xylooligosaccharides. Among the 12 single active site mutants, W125F and F163W show increased activity comparing to the wild-type protein. The double mutant W125F/F163W was then generated which displayed nearly 20 % increase in the enzyme activity. Although W125F/F163W showed 5 C reduction in the optimal temperature, it still preserves similar thermostability and is more active than the wild-type enzyme at temperatures lower than 60 C. These properties make the double mutant a suitable candidate for commercial applications that involve lower operating temperatures. Furthermore, we investigated the effect of N-glycosylation on the thermostability of XynCDBFV when expressed in the yeast strain Pichia pastoris for industrial use. Two potential glycosylation sites (Asn-37 and Asn-88) were examined, and their roles in enzyme performance were validated. We found that the N-glycosylations of XynCDBFV are related to both catalytic activity and heat stability, with Asn-37 motif playing a dominant role. Collectively, the enzymatic properties of XynCDBFV were improved by molecular engineering, and the influences of N-glycosylations on the enzyme have been clearly elucidated herein.


PubMed | CAS Tianjin Institute of Industrial Biotechnology, Genozyme Biotechnology Inc. and Jiangnan University
Type: Journal Article | Journal: Biochemical and biophysical research communications | Year: 2016

Eukaryotic 1,4--endoglucanases (EC 3.2.1.4) have shown great potentials in many commercial applications because they effectively catalyze hydrolysis of cellulose, the main component of the plant cell wall. Here we expressed a glycoside hydrolase family (GH) 5 1,4--endoglucanase from Aspergillus niger (AnCel5A) in Pichia pastoris, which exhibits outstanding pH and heat stability. In order to further investigate the molecular mechanism of AnCel5A, apo-form and cellotetraose (CTT) complex enzyme crystal structures were solved to high resolution. AnCel5A folds into a typical (/)8-TIM barrel architecture, resembling other GH5 members. In the substrate binding cavity, CTT is found to bind to -4 - -1 subsites, and several polyethylene glycol molecules are found in positive subsites. In addition, several unique N-glycosylation motifs that may contribute to protein higher stability were observed from crystal structures. These results are of great importance for understanding the molecular mechanism of AnCel5A, and also provide guidance for further applications of the enzyme.


PubMed | CAS Tianjin Institute of Industrial Biotechnology, Genozyme Biotechnology Inc. and Jiangnan University
Type: Journal Article | Journal: Acta crystallographica. Section F, Structural biology communications | Year: 2015

Cellulose is the most abundant renewable biomass on earth, and its decomposition has proven to be very useful in a wide variety of industries. Endo-1,4--D-glucanase (EC 3.2.1.4; endoglucanase), which can catalyze the random hydrolysis of -1,4-glycosidic bonds to cleave cellulose into smaller fragments, is a key cellulolytic enzyme. An endoglucanase isolated from Aspergillus aculeatus F-50 (FI-CMCase) that was classified into glycoside hydrolase family 12 has been found to be effectively expressed in the industrial strain Pichia pastoris. Here, recombinant FI-CMCase was crystallized. Crystals belonging to the orthorhombic space group C222, with unit-cell parameters a = 74.2, b = 75.1, c = 188.4, were obtained by the sitting-drop vapour-diffusion method and diffracted to 1.6 resolution. Initial phase determination by molecular replacement clearly shows that the crystal contains two protein molecules in the asymmetric unit. Further model building and structure refinement are in progress.

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