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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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Effects of pH (a) and temperature (b) on EG activity for P. pastoris transformant egE. Each value is means of three replicates. Error bars indicate standard error.
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f2-ijms-12-03366: Effects of pH (a) and temperature (b) on EG activity for P. pastoris transformant egE. Each value is means of three replicates. Error bars indicate standard error.

Mentions: At 40 °C, the effect of pH on EG activity for the hydrolysis of CMC was determined. The result showed that the optimal pH of EG was at pH 6.0 (Figure 2a) and the activity at pH 5.5 and pH 7.5 was more than 80% of that at pH 6.0. At pH 6.0, EG activities were measured at various temperatures to determine the optimal temperature. The result showed that the optimal temperature for the enzyme of interest expressed in P. pastoris was 45 °C (Figure 2b). The activity at 40–55 °C was more than 80% of that at 45 °C, but decreased rapidly outside this temperature range. In addition, the pH and temperature optima of the enzyme activity were consistent with those of enzymes expressed from E. coli. Depending on these results, the endo-β-1,4-glucanase expressed in P. pastoris GS115 can be classified as a mesophilic neutral endoglucanase, which can be used as a good source for industrial applications, especially for denim biofinishing/washing.


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)

Effects of pH (a) and temperature (b) on EG activity for P. pastoris transformant egE. Each value is means of three replicates. Error bars indicate standard error.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2-ijms-12-03366: Effects of pH (a) and temperature (b) on EG activity for P. pastoris transformant egE. Each value is means of three replicates. Error bars indicate standard error.
Mentions: At 40 °C, the effect of pH on EG activity for the hydrolysis of CMC was determined. The result showed that the optimal pH of EG was at pH 6.0 (Figure 2a) and the activity at pH 5.5 and pH 7.5 was more than 80% of that at pH 6.0. At pH 6.0, EG activities were measured at various temperatures to determine the optimal temperature. The result showed that the optimal temperature for the enzyme of interest expressed in P. pastoris was 45 °C (Figure 2b). The activity at 40–55 °C was more than 80% of that at 45 °C, but decreased rapidly outside this temperature range. In addition, the pH and temperature optima of the enzyme activity were consistent with those of enzymes expressed from E. coli. Depending on these results, the endo-β-1,4-glucanase expressed in P. pastoris GS115 can be classified as a mesophilic neutral endoglucanase, which can be used as a good source for industrial applications, especially for denim biofinishing/washing.

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