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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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(a) SDS-PAGE analysis of the culture supernatant of recombinant P. pastoris strain egE. Lane 0: the samples of P. pastoris transformed with pPIC9K at 24 h of cultivation; Lanes 1–2: the samples of P. pastoris strain egE taken at 24 h, 12 h of cultivation, respectively. Lane M: the protein molecular marker; (b) CMC utilization by recombinant P. pastoris. The P. pastoris strain harboring pPICE (1) or empty plasmid pPIC9K (2) was inoculated in YSM medium to examine the CMC utilization. The CMC-containing plate was incubated at 30 °C for 60 h; (c) PCR verification of the celE gene integrated in the chromosome of P. pastoris egE. Lane 1–4: 1, 4, 6, 10 generations of the P. pastoris egE; Lane 0: P. pastoris transformed with pPIC9K. Lane M: DNA marker.
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f1-ijms-12-03366: (a) SDS-PAGE analysis of the culture supernatant of recombinant P. pastoris strain egE. Lane 0: the samples of P. pastoris transformed with pPIC9K at 24 h of cultivation; Lanes 1–2: the samples of P. pastoris strain egE taken at 24 h, 12 h of cultivation, respectively. Lane M: the protein molecular marker; (b) CMC utilization by recombinant P. pastoris. The P. pastoris strain harboring pPICE (1) or empty plasmid pPIC9K (2) was inoculated in YSM medium to examine the CMC utilization. The CMC-containing plate was incubated at 30 °C for 60 h; (c) PCR verification of the celE gene integrated in the chromosome of P. pastoris egE. Lane 1–4: 1, 4, 6, 10 generations of the P. pastoris egE; Lane 0: P. pastoris transformed with pPIC9K. Lane M: DNA marker.

Mentions: After P. pastoris transformation, thirty P. pastoris transformants with the highest resistance to G418 were selected and subjected to genomic DNA isolation. The PCR assay and nucleotides sequencing were performed, which confirmed that all the transfomants contained an integrated celE sequence in the genome DNA. Enzyme activity was then further determined, after induction in shaking flasks in BMGY and BMMY medium. With induction by 0.5% methanol for 48 h, all the recombinant strains expressed and secreted endo-β-1,4-glucanase into the culture broth. Among them, three strains exhibited relatively high production of the recombinant protein, and one of the strains, designed as P. pastoris egE, was selected for further study. The endo-β-1,4-glucanase activity reached 35.1 IU mL−1 within 48 h after 0.5% methanol induction in shaking flasks. The continuous production of CelE by transgenic P.pasoris due to the expression of the celE gene was also confirmed by SDS-PAGE analysis of culture supernatants of after growth for 12–24 h in BMMY broth. The total protein profile of transgenic P.pastoris on SDS-PAGE showed an apparent single band. It was estimated to be 52 kDa from its mobility relative to standard proteins by SDS-PAGE, similar to the predicted molecular mass (51.5 kDa) (Figure 1a).


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)

(a) SDS-PAGE analysis of the culture supernatant of recombinant P. pastoris strain egE. Lane 0: the samples of P. pastoris transformed with pPIC9K at 24 h of cultivation; Lanes 1–2: the samples of P. pastoris strain egE taken at 24 h, 12 h of cultivation, respectively. Lane M: the protein molecular marker; (b) CMC utilization by recombinant P. pastoris. The P. pastoris strain harboring pPICE (1) or empty plasmid pPIC9K (2) was inoculated in YSM medium to examine the CMC utilization. The CMC-containing plate was incubated at 30 °C for 60 h; (c) PCR verification of the celE gene integrated in the chromosome of P. pastoris egE. Lane 1–4: 1, 4, 6, 10 generations of the P. pastoris egE; Lane 0: P. pastoris transformed with pPIC9K. Lane M: DNA marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1-ijms-12-03366: (a) SDS-PAGE analysis of the culture supernatant of recombinant P. pastoris strain egE. Lane 0: the samples of P. pastoris transformed with pPIC9K at 24 h of cultivation; Lanes 1–2: the samples of P. pastoris strain egE taken at 24 h, 12 h of cultivation, respectively. Lane M: the protein molecular marker; (b) CMC utilization by recombinant P. pastoris. The P. pastoris strain harboring pPICE (1) or empty plasmid pPIC9K (2) was inoculated in YSM medium to examine the CMC utilization. The CMC-containing plate was incubated at 30 °C for 60 h; (c) PCR verification of the celE gene integrated in the chromosome of P. pastoris egE. Lane 1–4: 1, 4, 6, 10 generations of the P. pastoris egE; Lane 0: P. pastoris transformed with pPIC9K. Lane M: DNA marker.
Mentions: After P. pastoris transformation, thirty P. pastoris transformants with the highest resistance to G418 were selected and subjected to genomic DNA isolation. The PCR assay and nucleotides sequencing were performed, which confirmed that all the transfomants contained an integrated celE sequence in the genome DNA. Enzyme activity was then further determined, after induction in shaking flasks in BMGY and BMMY medium. With induction by 0.5% methanol for 48 h, all the recombinant strains expressed and secreted endo-β-1,4-glucanase into the culture broth. Among them, three strains exhibited relatively high production of the recombinant protein, and one of the strains, designed as P. pastoris egE, was selected for further study. The endo-β-1,4-glucanase activity reached 35.1 IU mL−1 within 48 h after 0.5% methanol induction in shaking flasks. The continuous production of CelE by transgenic P.pasoris due to the expression of the celE gene was also confirmed by SDS-PAGE analysis of culture supernatants of after growth for 12–24 h in BMMY broth. The total protein profile of transgenic P.pastoris on SDS-PAGE showed an apparent single band. It was estimated to be 52 kDa from its mobility relative to standard proteins by SDS-PAGE, similar to the predicted molecular mass (51.5 kDa) (Figure 1a).

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