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Expression of an endo-β-1,4-glucanase gene from orpinomyces PC-2 in Pichia pastoris.

Jin X, Meng N, Xia LM - Int J Mol Sci (2011)

Bottom Line: The most favorable methanol addition concentration was discussed and given as 1.0%.After methanol induction for 96 h, the endo-β-1,4-glucanase activity reached 72.5 IU mL(-1).The endo-β-1,4-glucanase secreted by recombinant P. pastoris represents an attractive potential for both academic research and textile industry application.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering and Bioengineering, Zhejiang University, Hangzhou 310027, China; E-Mails: guyin018@gmail.com (X.J.); mengnan17@163.com (N.M.).

ABSTRACT
The endo-β-1,4-glucanase gene celE from the anaerobic fungus Orpinomyces PC-2 was placed under the control of an alcohol oxidase promoter (AOX1) in the plasmid pPIC9K, and integrated into the genome of a methylotrophic yeast P. pastoris GS115 by electroporation. The strain with highest endo-β-1,4-glucanase activity was selected and designed as P. pastoris egE, and cultivated in shaking flasks. The culture supernatant was assayed by SDS-polyacrylamide gel electrophoresis and showed a single band at about 52 kDa. Furthermore, the recombinant P. pastoris egE was proved to possess the ability to utilize sodium carboxymethyl cellulose as a carbon source. The recombinant endoglucanase produced by P. pastoris showed maximum activity at pH 6.0 and temperature 45 °C, indicating it was a mesophilic neutral endo-β-1,4-glucanase, suitable for denim biofinishing/washing. Further research was carried out in suitable fermentation medium in shaking flasks. The most favorable methanol addition concentration was discussed and given as 1.0%. After methanol induction for 96 h, the endo-β-1,4-glucanase activity reached 72.5 IU mL(-1). This is the first report on expression and characterization of endo-β-1,4-glucanase from Orpinomyces in P. pastoris. The endo-β-1,4-glucanase secreted by recombinant P. pastoris represents an attractive potential for both academic research and textile industry application.

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Effect of methanol addition concentration on CelE enzyme production in P. pastoris GS115 transformed with pPICE plasmid. The enzyme activity was detected in the liquid BCG medium. Methanol was supplemented every 24 h to a final concentration of 0.5% (squares), 1.0% (circles) or 1.5% (up-triangles). The BMMY medium induced by 0.5% methanol was taken for comparison (down-triangles). Data shown are the means of three independent experiments for each strain.
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f4-ijms-12-03366: Effect of methanol addition concentration on CelE enzyme production in P. pastoris GS115 transformed with pPICE plasmid. The enzyme activity was detected in the liquid BCG medium. Methanol was supplemented every 24 h to a final concentration of 0.5% (squares), 1.0% (circles) or 1.5% (up-triangles). The BMMY medium induced by 0.5% methanol was taken for comparison (down-triangles). Data shown are the means of three independent experiments for each strain.

Mentions: Recombinant P. pastoris strain egE was cultivated in BCG medium in shaking flasks. Meanwhile, different methanol addition concentrations were discussed, and the endo-β-1,4-glucanase activity in supernatants was assayed every 12 h after methanol induction (Figure 4). We noticed that the enzyme production had barely changed when cells were induced by 0.5% methanol in BMMY medium and BCG medium, reaching a level of 57.3 IU mL−1 and 54.4 IU mL−1 respectively, after 96 h induction, demonstrating that the BCG medium was also appropriate for enzyme production. It should be emphasized here that the BMMY medium is a defined medium in common use, while the BCG medium is a complex medium designed in this study, with corn steep liquor and glucose as the main components. Like BMMY medium, BCG medium contains 100 mM potassium phosphate buffer. The enzyme production was increased significantly more rapidly in BMMY medium than in BCG medium in the first 12–24 h, while the EG accumulation in the broth became closer later. In this sense, the BCG medium could replace the BMMY medium. It is possible that the glucose in BCG medium may prime the cells at the early stage for increased production of the heterologous enzyme at later stages. This was consistent with determination of cell number: about 1.5 times more cells were grown in BCG medium than in BMMY medium at 24 h. Although glucose would repress the synthesis of methanol metabolizing enzymes [39], it had been already exhausted in the first 24–30 h induction time, having little impact on enzyme expression. Besides, the minimal media FBSH has also been tried for induction but was found to have lower enzyme production (39.5 IU mL−1) after induction by 0.5% methanol for 96 h.


Expression of an endo-β-1,4-glucanase gene from orpinomyces PC-2 in Pichia pastoris.

Jin X, Meng N, Xia LM - Int J Mol Sci (2011)

Effect of methanol addition concentration on CelE enzyme production in P. pastoris GS115 transformed with pPICE plasmid. The enzyme activity was detected in the liquid BCG medium. Methanol was supplemented every 24 h to a final concentration of 0.5% (squares), 1.0% (circles) or 1.5% (up-triangles). The BMMY medium induced by 0.5% methanol was taken for comparison (down-triangles). Data shown are the means of three independent experiments for each strain.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3116196&req=5

f4-ijms-12-03366: Effect of methanol addition concentration on CelE enzyme production in P. pastoris GS115 transformed with pPICE plasmid. The enzyme activity was detected in the liquid BCG medium. Methanol was supplemented every 24 h to a final concentration of 0.5% (squares), 1.0% (circles) or 1.5% (up-triangles). The BMMY medium induced by 0.5% methanol was taken for comparison (down-triangles). Data shown are the means of three independent experiments for each strain.
Mentions: Recombinant P. pastoris strain egE was cultivated in BCG medium in shaking flasks. Meanwhile, different methanol addition concentrations were discussed, and the endo-β-1,4-glucanase activity in supernatants was assayed every 12 h after methanol induction (Figure 4). We noticed that the enzyme production had barely changed when cells were induced by 0.5% methanol in BMMY medium and BCG medium, reaching a level of 57.3 IU mL−1 and 54.4 IU mL−1 respectively, after 96 h induction, demonstrating that the BCG medium was also appropriate for enzyme production. It should be emphasized here that the BMMY medium is a defined medium in common use, while the BCG medium is a complex medium designed in this study, with corn steep liquor and glucose as the main components. Like BMMY medium, BCG medium contains 100 mM potassium phosphate buffer. The enzyme production was increased significantly more rapidly in BMMY medium than in BCG medium in the first 12–24 h, while the EG accumulation in the broth became closer later. In this sense, the BCG medium could replace the BMMY medium. It is possible that the glucose in BCG medium may prime the cells at the early stage for increased production of the heterologous enzyme at later stages. This was consistent with determination of cell number: about 1.5 times more cells were grown in BCG medium than in BMMY medium at 24 h. Although glucose would repress the synthesis of methanol metabolizing enzymes [39], it had been already exhausted in the first 24–30 h induction time, having little impact on enzyme expression. Besides, the minimal media FBSH has also been tried for induction but was found to have lower enzyme production (39.5 IU mL−1) after induction by 0.5% methanol for 96 h.

Bottom Line: The most favorable methanol addition concentration was discussed and given as 1.0%.After methanol induction for 96 h, the endo-β-1,4-glucanase activity reached 72.5 IU mL(-1).The endo-β-1,4-glucanase secreted by recombinant P. pastoris represents an attractive potential for both academic research and textile industry application.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Engineering and Bioengineering, Zhejiang University, Hangzhou 310027, China; E-Mails: guyin018@gmail.com (X.J.); mengnan17@163.com (N.M.).

ABSTRACT
The endo-β-1,4-glucanase gene celE from the anaerobic fungus Orpinomyces PC-2 was placed under the control of an alcohol oxidase promoter (AOX1) in the plasmid pPIC9K, and integrated into the genome of a methylotrophic yeast P. pastoris GS115 by electroporation. The strain with highest endo-β-1,4-glucanase activity was selected and designed as P. pastoris egE, and cultivated in shaking flasks. The culture supernatant was assayed by SDS-polyacrylamide gel electrophoresis and showed a single band at about 52 kDa. Furthermore, the recombinant P. pastoris egE was proved to possess the ability to utilize sodium carboxymethyl cellulose as a carbon source. The recombinant endoglucanase produced by P. pastoris showed maximum activity at pH 6.0 and temperature 45 °C, indicating it was a mesophilic neutral endo-β-1,4-glucanase, suitable for denim biofinishing/washing. Further research was carried out in suitable fermentation medium in shaking flasks. The most favorable methanol addition concentration was discussed and given as 1.0%. After methanol induction for 96 h, the endo-β-1,4-glucanase activity reached 72.5 IU mL(-1). This is the first report on expression and characterization of endo-β-1,4-glucanase from Orpinomyces in P. pastoris. The endo-β-1,4-glucanase secreted by recombinant P. pastoris represents an attractive potential for both academic research and textile industry application.

Show MeSH
Related in: MedlinePlus