AsiaPac Biotechnology Co.

Dongguan, China

AsiaPac Biotechnology Co.

Dongguan, China
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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.


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.


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. | 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.


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, Urbana University, AsiaPac Biotechnology Co. and Academia Sinica, Taiwan
Type: Journal Article | Journal: Angewandte Chemie (International ed. in English) | Year: 2016

The structure of MoeN5, a unique prenyltransferase involved in the biosynthesis of the antibiotic moenomycin, is reported. MoeN5 catalyzes the reaction of geranyl diphosphate (GPP) with the cis-farnesyl group in phosphoglycolipid 5 to form the (C25) moenocinyl-sidechain-containing lipid 7. GPP binds to an allylic site (S1) and aligns well with known S1 inhibitors. Alkyl glycosides, glycolipids, can bind to both S1 and a second site, S2. Long sidechains in S2 are bent and co-locate with the homoallylic substrate isopentenyl diphosphate in other prenyltransferases. These observations support a MoeN5 mechanism in which 5 binds to S2 with its C6-C11 group poised to attack C1 in GPP to form the moenocinyl sidechain, with the more distal regions of 5 aligning with the distal glucose in decyl maltoside. The results are of general interest because they provide the first structures of MoeN5 and a structural basis for its mechanism of action, results that will facilitate the design of new antibiotics.


PubMed | Genozyme Biotechnology Inc., CAS Tianjin Institute of Industrial Biotechnology, AsiaPac Biotechnology Co., National Taiwan University and 2 more.
Type: Journal Article | Journal: Biochimica et biophysica acta | Year: 2014

The thermostable 1,3-1,4--glucanase PtLic16A from the fungus Paecilomyces thermophila catalyzes stringent hydrolysis of barley -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 to 2.25 resolution. PtLic16A adopts a typical -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--glucotriose complex, the sugar bound to the -1 subsite adopts an intermediate-like (-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--glucanase catalytic mechanism but also provide a basis for further enzymatic engineering.


PubMed | CAS Tianjin Institute of Industrial Biotechnology, Urbana University, AsiaPac Biotechnology Co., Tianjin University of Science and Technology and Academia Sinica, Taiwan
Type: Journal Article | Journal: Angewandte Chemie (International ed. in English) | Year: 2016

We report the first X-ray structure of the unique head-to-middle monoterpene synthase, lavandulyl diphosphate synthase (LPPS). LPPS catalyzes the condensation of two molecules of dimethylallyl diphosphate (DMAPP) to form lavandulyl diphosphate, a precursor to the fragrance lavandulol. The structure is similar to that of the bacterial cis-prenyl synthase, undecaprenyl diphosphate synthase (UPPS), and contains an allylic site (S1) in which DMAPP ionizes and a second site (S2) which houses the DMAPP nucleophile. Both S-thiolo-dimethylallyl diphosphate and S-thiolo-isopentenyl diphosphate bind intact to S2, but are cleaved to (thio)diphosphate, in S1. His78 (Asn in UPPS) is essential for catalysis and is proposed to facilitate diphosphate release in S1, while the P1 phosphate in S2 abstracts a proton from the lavandulyl carbocation to form the LPP product. The results are of interest since they provide the first structure and structure-based mechanism of this unusual prenyl synthase.


Sun H.,Chinese Academy of Sciences | Zhao P.,Chinese Academy of Sciences | Ge X.,Jiangnan University | Xia Y.,Jiangnan University | And 3 more authors.
Applied Biochemistry and Biotechnology | Year: 2010

Raw starch degrading enzymes (RSDE) refer to enzymes that can directly degrade raw starch granules below the gelatinization temperature of starch. These promising enzymes can significantly reduce energy and simplify the process in starch industry. RSDE are ubiquitous and produced by plants, animals, and microorganisms. However, microbial sources are the most preferred one for large-scale production. During the past few decades, RSDE have been studied extensively. This paper reviews the recent development in the production, purification, properties, and application of microbial RSDE. This is the first review on microbial RSDE to date. © 2009 Humana Press.

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