Expression of endo-1, 4-beta-xylanase from <it>Trichoderma reesei </it>in <it>Pichia pastoris </it>and functional characterization of the produced enzyme
<p>Abstract</p> <p>Background</p> <p>In recent years, xylanases have attracted considerable research interest because of their potential in various industrial applications. The yeast <it>Pichia pastoris </it>can neither utilize nor degrade xylan, but it poss...
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| Format: | Article |
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BMC
2009-06-01
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| Series: | BMC Biotechnology |
| Online Access: | http://www.biomedcentral.com/1472-6750/9/56 |
| _version_ | 1819037274561576960 |
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| author | He Jun Yu Bing Zhang Keying Ding Xuemei Chen Daiwen |
| author_facet | He Jun Yu Bing Zhang Keying Ding Xuemei Chen Daiwen |
| author_sort | He Jun |
| collection | DOAJ |
| description | <p>Abstract</p> <p>Background</p> <p>In recent years, xylanases have attracted considerable research interest because of their potential in various industrial applications. The yeast <it>Pichia pastoris </it>can neither utilize nor degrade xylan, but it possesses many attributes that render it an attractive host for the expression and production of industrial enzymes.</p> <p>Results</p> <p>The Xyn2 gene, which encodes the main <it>Trichoderma reesei </it>Rut C-30 endo-β-1, 4-xylanase was cloned into the pPICZαA vector and expressed in <it>Pichia pastoris</it>. The selected <it>P. pastoris </it>strains produced as 4,350 nkat/ml β-xylanase under the control of the methanol inducible alcohol oxidase 1 (<it>AOX1</it>) promoter. The secreted recombinant Xyn2 was estimated by SDS-PAGE to be 21 kDa. The activity of the recombinant Xyn2 was highest at 60°C and it was active over a broad range of pH (3.0–8.0) with maximal activity at pH 6.0. The enzyme was quite stable at 50°C and retained more than 94% of its activity after 30 mins incubation at this temperature. Using Birchwood xylan, the determined apparent <it>K</it><sub>m </sub>and k<sub>cat </sub>values were 2.1 mg/ml and 219.2 S<sup>-1</sup>, respectively. The enzyme was highly specific towards xylan and analysis of xylan hydrolysis products confirmed as expected that the enzyme functions as endo-xylanase with xylotriose as the main hydrolysis products. The produced xylanase was practically free of cellulolytic activity.</p> <p>Conclusion</p> <p>The <it>P. pastoris </it>expression system allows a high level expression of xylanases. Xylanase was the main protein species in the culture supernatant, and the functional tests indicated that even the non-purified enzyme shows highly specific xylanase activity that is free of cellulolytic side acitivities. Therefore, <it>P pastoris </it>is a very useful expression system when the goal is highly specific and large scale production of glycosyl hydrolases.</p> |
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| spelling | doaj.art-3bbe9348d1a940c1b21a510f66c51a632022-12-21T19:10:29ZengBMCBMC Biotechnology1472-67502009-06-01915610.1186/1472-6750-9-56Expression of endo-1, 4-beta-xylanase from <it>Trichoderma reesei </it>in <it>Pichia pastoris </it>and functional characterization of the produced enzymeHe JunYu BingZhang KeyingDing XuemeiChen Daiwen<p>Abstract</p> <p>Background</p> <p>In recent years, xylanases have attracted considerable research interest because of their potential in various industrial applications. The yeast <it>Pichia pastoris </it>can neither utilize nor degrade xylan, but it possesses many attributes that render it an attractive host for the expression and production of industrial enzymes.</p> <p>Results</p> <p>The Xyn2 gene, which encodes the main <it>Trichoderma reesei </it>Rut C-30 endo-β-1, 4-xylanase was cloned into the pPICZαA vector and expressed in <it>Pichia pastoris</it>. The selected <it>P. pastoris </it>strains produced as 4,350 nkat/ml β-xylanase under the control of the methanol inducible alcohol oxidase 1 (<it>AOX1</it>) promoter. The secreted recombinant Xyn2 was estimated by SDS-PAGE to be 21 kDa. The activity of the recombinant Xyn2 was highest at 60°C and it was active over a broad range of pH (3.0–8.0) with maximal activity at pH 6.0. The enzyme was quite stable at 50°C and retained more than 94% of its activity after 30 mins incubation at this temperature. Using Birchwood xylan, the determined apparent <it>K</it><sub>m </sub>and k<sub>cat </sub>values were 2.1 mg/ml and 219.2 S<sup>-1</sup>, respectively. The enzyme was highly specific towards xylan and analysis of xylan hydrolysis products confirmed as expected that the enzyme functions as endo-xylanase with xylotriose as the main hydrolysis products. The produced xylanase was practically free of cellulolytic activity.</p> <p>Conclusion</p> <p>The <it>P. pastoris </it>expression system allows a high level expression of xylanases. Xylanase was the main protein species in the culture supernatant, and the functional tests indicated that even the non-purified enzyme shows highly specific xylanase activity that is free of cellulolytic side acitivities. Therefore, <it>P pastoris </it>is a very useful expression system when the goal is highly specific and large scale production of glycosyl hydrolases.</p>http://www.biomedcentral.com/1472-6750/9/56 |
| spellingShingle | He Jun Yu Bing Zhang Keying Ding Xuemei Chen Daiwen Expression of endo-1, 4-beta-xylanase from <it>Trichoderma reesei </it>in <it>Pichia pastoris </it>and functional characterization of the produced enzyme BMC Biotechnology |
| title | Expression of endo-1, 4-beta-xylanase from <it>Trichoderma reesei </it>in <it>Pichia pastoris </it>and functional characterization of the produced enzyme |
| title_full | Expression of endo-1, 4-beta-xylanase from <it>Trichoderma reesei </it>in <it>Pichia pastoris </it>and functional characterization of the produced enzyme |
| title_fullStr | Expression of endo-1, 4-beta-xylanase from <it>Trichoderma reesei </it>in <it>Pichia pastoris </it>and functional characterization of the produced enzyme |
| title_full_unstemmed | Expression of endo-1, 4-beta-xylanase from <it>Trichoderma reesei </it>in <it>Pichia pastoris </it>and functional characterization of the produced enzyme |
| title_short | Expression of endo-1, 4-beta-xylanase from <it>Trichoderma reesei </it>in <it>Pichia pastoris </it>and functional characterization of the produced enzyme |
| title_sort | expression of endo 1 4 beta xylanase from it trichoderma reesei it in it pichia pastoris it and functional characterization of the produced enzyme |
| url | http://www.biomedcentral.com/1472-6750/9/56 |
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