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Constitutive expression of Thermobifida fusca thermostable Acetylxylan Esterase gene in Pichia pastoris.

Yang CH, Lin KI, Chen GH, Chen YF, Chen CY, Chen WL, Huang YC - Int J Mol Sci (2010)

Bottom Line: About 70% of the original activity remained after heat treatment at 60 °C for three hours.The optimal pH and temperature of the purified enzyme were 8.0 and 60 °C, respectively.The properties of the purified AXE from the P. pastoris transformant are similar to those of the AXE from an E. coli transformant.

View Article: PubMed Central - PubMed

Affiliation: Department of Cosmetic Science, Providence University, 200, Chung-Chi Rd., Taichung, 43301, Taiwan; E-Mails chyang@pu.edu.tw (C.-H.Y.); ghchen2@pu.edu.tw (G.-H.C); yfchen@pu.edu.tw (Y.-F.C.); zychan1268@gmail.com (C.-Y.C); wei.lin46@gmail.com (W.-L.C).

ABSTRACT
A gene encoding the thermostable acetylxylan esterase (AXE) in Thermobifida fusca NTU22 was amplified by PCR, sequenced and cloned into the Pichia pastoris X-33 host strain using the vector pGAPZαA, allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular AXE production, as high as 526 U/mL in the Hinton flask culture broth. The purified enzyme showed a single band at about 28 kDa by SDS-polyacrylamide gel electrophoresis after being treated with endo-β-N-acetylglycosaminidase H; this agrees with the predicted size based on the nucleotide sequence. About 70% of the original activity remained after heat treatment at 60 °C for three hours. The optimal pH and temperature of the purified enzyme were 8.0 and 60 °C, respectively. The properties of the purified AXE from the P. pastoris transformant are similar to those of the AXE from an E. coli transformant.

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Related in: MedlinePlus

Time course for the expression of AXE activity by P. pastoris transformant (pGAPZ-axe). Cells were grown aerobically in a 500 mL Hinton flask loaded with 50 mL of medium consisting of YPD broth, and were incubated at 28 °C, 150 rpm for 96 hours. (•), esterase activity; (□), OD600.
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f1-ijms-11-05143: Time course for the expression of AXE activity by P. pastoris transformant (pGAPZ-axe). Cells were grown aerobically in a 500 mL Hinton flask loaded with 50 mL of medium consisting of YPD broth, and were incubated at 28 °C, 150 rpm for 96 hours. (•), esterase activity; (□), OD600.

Mentions: The fermentation conditions for constitutive expression of the AXE were investigated in a 500 mL Hinton flask loaded with 50 mL YPD broth at 28 °C. Transformant (pGAPZ-axe) grew logarithmically from 24 to 48 hours and then entered a stationary phase (Figure 1). The biomass reached approximately 22 of the OD600 value after 48-hours incubation. The enzyme production pattern in this transformant indicated that AXE synthesis began in the middle logarithmic phase and continued to be produced during the stationary phase where the maximum activity (526 U/mL) was reached in the culture broth. The AXE activity was about 500-times higher than that observed when expressed from an E. coli transformant [18]. The control strain, P. pastoris (pGAPZαA), could also produce esterase activity under the same culture conditions (data not shown). In the GAP promoter expression system, the cloned heterologous protein will be expressed along with cell growth if the protein is not toxic for the cell [23,24]. Such a result was observed in this study. As shown in Figure 1, the rapid production of the extracellular AXE occurred in parallel with an increase in biomass accumulation of the P. pastoris transformant.


Constitutive expression of Thermobifida fusca thermostable Acetylxylan Esterase gene in Pichia pastoris.

Yang CH, Lin KI, Chen GH, Chen YF, Chen CY, Chen WL, Huang YC - Int J Mol Sci (2010)

Time course for the expression of AXE activity by P. pastoris transformant (pGAPZ-axe). Cells were grown aerobically in a 500 mL Hinton flask loaded with 50 mL of medium consisting of YPD broth, and were incubated at 28 °C, 150 rpm for 96 hours. (•), esterase activity; (□), OD600.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3100850&req=5

f1-ijms-11-05143: Time course for the expression of AXE activity by P. pastoris transformant (pGAPZ-axe). Cells were grown aerobically in a 500 mL Hinton flask loaded with 50 mL of medium consisting of YPD broth, and were incubated at 28 °C, 150 rpm for 96 hours. (•), esterase activity; (□), OD600.
Mentions: The fermentation conditions for constitutive expression of the AXE were investigated in a 500 mL Hinton flask loaded with 50 mL YPD broth at 28 °C. Transformant (pGAPZ-axe) grew logarithmically from 24 to 48 hours and then entered a stationary phase (Figure 1). The biomass reached approximately 22 of the OD600 value after 48-hours incubation. The enzyme production pattern in this transformant indicated that AXE synthesis began in the middle logarithmic phase and continued to be produced during the stationary phase where the maximum activity (526 U/mL) was reached in the culture broth. The AXE activity was about 500-times higher than that observed when expressed from an E. coli transformant [18]. The control strain, P. pastoris (pGAPZαA), could also produce esterase activity under the same culture conditions (data not shown). In the GAP promoter expression system, the cloned heterologous protein will be expressed along with cell growth if the protein is not toxic for the cell [23,24]. Such a result was observed in this study. As shown in Figure 1, the rapid production of the extracellular AXE occurred in parallel with an increase in biomass accumulation of the P. pastoris transformant.

Bottom Line: About 70% of the original activity remained after heat treatment at 60 °C for three hours.The optimal pH and temperature of the purified enzyme were 8.0 and 60 °C, respectively.The properties of the purified AXE from the P. pastoris transformant are similar to those of the AXE from an E. coli transformant.

View Article: PubMed Central - PubMed

Affiliation: Department of Cosmetic Science, Providence University, 200, Chung-Chi Rd., Taichung, 43301, Taiwan; E-Mails chyang@pu.edu.tw (C.-H.Y.); ghchen2@pu.edu.tw (G.-H.C); yfchen@pu.edu.tw (Y.-F.C.); zychan1268@gmail.com (C.-Y.C); wei.lin46@gmail.com (W.-L.C).

ABSTRACT
A gene encoding the thermostable acetylxylan esterase (AXE) in Thermobifida fusca NTU22 was amplified by PCR, sequenced and cloned into the Pichia pastoris X-33 host strain using the vector pGAPZαA, allowing constitutive expression and secretion of the protein. Recombinant expression resulted in high levels of extracellular AXE production, as high as 526 U/mL in the Hinton flask culture broth. The purified enzyme showed a single band at about 28 kDa by SDS-polyacrylamide gel electrophoresis after being treated with endo-β-N-acetylglycosaminidase H; this agrees with the predicted size based on the nucleotide sequence. About 70% of the original activity remained after heat treatment at 60 °C for three hours. The optimal pH and temperature of the purified enzyme were 8.0 and 60 °C, respectively. The properties of the purified AXE from the P. pastoris transformant are similar to those of the AXE from an E. coli transformant.

Show MeSH
Related in: MedlinePlus