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Expression of the Thermobifida fusca xylanase Xyn11A in Pichia pastoris and its characterization.

ABSTRACT: Xylan is a major component of plant cells and the most abundant hemicellulose. Xylanases degrade xylan into monomers by randomly cleaving ?-1,4-glycosidic bonds in the xylan backbone, and have widespread potential applications in various industries. The purpose of our study was to clone and express the endoxylanase gene xynA of Thermobifida fusca YX in its native form and with a C-terminal histidine (His) tag in Pichia pastoris X-33. We analyzed and compared these two forms of the protein and examined their potential applications in various industries.The xynA gene from T. fusca YX was successfully cloned and expressed using P. pastoris X-33. We produced a recombinant native form of the protein (rXyn11A) and a C-terminal His-tagged form of the desired protein (rXyn11A-(His)6). The specific activities of rXyn11A and rXyn11A-(His)6 in culture supernatants approached 149.4 and 133.4 U/mg, respectively. These activities were approximately 4- and 3.5-fold higher than those for the non-recombinant wild-type Xyn11A (29.3 U/mg). Following purification, the specific activities of rXyn11A and rXyn11A-(His)6 were 557.35 and 515.84 U/mg, respectively. The specific activity of rXyn11A was 8% higher than that of rXyn11A-(His)6. Both recombinant xylanases were optimally active at 80°C and pH 8.0, and exhibited greater than 60% activity between pH 6-9 and 60-80°C. They exhibited similar pH stability, while rXyn11A exhibited better thermostability; N-glycosylation enhanced the thermostability of both recombinant xylanases. The products of beechwood xylan hydrolyzed by both xylanases included xylobiose, xylotriose, xylotetraose and xylopentaose.The C-terminal His tag had adverse effects when added to the Xyn11A protein. The thermostability of both recombinant xylanases was enhanced by N-glycosylation. Their stabilities at a high pH and temperature indicate their potential for application in various industries.

SUBMITTER: Zhao L

PROVIDER: S-EPMC4369062 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Expression of the Thermobifida fusca xylanase Xyn11A in Pichia pastoris and its characterization.

Zhao Longmei L Geng Jiang J Guo Yaoqi Y Liao Xiudong X Liu Xuhui X Wu Rujuan R Zheng Zhaojun Z Zhang Rijun R

BMC biotechnology 20150318

<h4>Background</h4>Xylan is a major component of plant cells and the most abundant hemicellulose. Xylanases degrade xylan into monomers by randomly cleaving β-1,4-glycosidic bonds in the xylan backbone, and have widespread potential applications in various industries. The purpose of our study was to clone and express the endoxylanase gene xynA of Thermobifida fusca YX in its native form and with a C-terminal histidine (His) tag in Pichia pastoris X-33. We analyzed and compared these two forms of ...[more]

PMID: 25887328

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Project description:Xylanases have the potential to be used as bio-deinking and bio-bleaching materials and their application will decrease the consumption of the chlorine-based chemicals currently used for this purpose. However, xylanases with specific properties could act effectively, such as having significant thermostability and alkali resistance, etc. In this study, we found that TfXyl10A, a xylanase from Thermobifida fusca, was greatly induced to transcript by microcrystalline cellulose (MCC) substrate. Biochemical characterization showed that TfXyl10A is optimally effective at temperature of 80 °C and pH of 9.0. After removing the carbohydrate-binding module (CBM) and linker regions, the optimum temperature of TfXyl10A-CD was reduced by 10°C (to 70°C), at which the enzyme's temperature tolerance was also weakened. While truncating only the CBM domain (TfXyl10AdC) had no significant effect on its thermostability. Importantly, polysaccharide-binding experiment showed that the auxiliary domain CBM2 could specifically bind to cellulose substrates, which endowed xylanase TfXyl10A with the ability to degrade xylan surrounding cellulose. These results indicated that TfXyl10A might be an excellent candidate in bio-bleaching processes of paper industry. In addition, the features of active-site architecture of TfXyl10A in GH10 family were further analyzed. By mutating each residue at the -2 and -1 subsites to alanine, the binding force and enzyme activity of mutants were observably decreased. Interestingly, the mutant E51A, locating at the distal -3 subsite, exhibited 90% increase in relative activity compared with wild-type (WT) enzyme TfXyl10A-CD (the catalytic domain of TfXyl110A). This study explored the function of a GH10 xylanase containing a CBM2 domain and the contribution of amino acids in active-site architecture to catalytic activity. The results obtained provide guidance for the rational design of xylanases for industrial applications under high heat and alkali-based operating conditions, such as paper bleaching.

Dataset Information

Expression of the Thermobifida fusca xylanase Xyn11A in Pichia pastoris and its characterization.

Publications

Expression of the Thermobifida fusca xylanase Xyn11A in Pichia pastoris and its characterization.

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