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Efficient display of active lipase LipB52 with a Pichia pastoris cell surface display system and comparison with the LipB52 displayed on Saccharomyces cerevisiae cell surface.

Jiang Z, Gao B, Ren R, Tao X, Ma Y, Wei D - BMC Biotechnol. (2008)

Bottom Line: The LipB52 displayed on the Pichia pastoris cell surface exhibited better stability than the lipase LipB52 displayed on Saccharomyces cerevisiae cell surface.The displayed lipases exhibited similar transesterification activity.But the Pichia pastoris dry cell weight per liter (DCW/L) ferment culture was about 5 times than Saccharomyces cerevisiae, the lipase displayed on Pichia pastoris are more suitable for whole-cell biocatalysts than that displayed on Saccharomyces cerevisiae cell surface.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, PR China. zhbjiang@hubu.edu.cn

ABSTRACT

Background: For industrial bioconversion processes, the utilization of surface-displayed lipase in the form of whole-cell biocatalysts is more advantageous, because the enzymes are displayed on the cell surface spontaneously, regarded as immobilized enzymes.

Results: Two Pichia pastoris cell surface display vectors based on the flocculation functional domain of FLO with its own secretion signal sequence or the alpha-factor secretion signal sequence were constructed respectively. The lipase gene lipB52 fused with the FLO gene was successfully transformed into Pichia pastoris KM71. The lipase LipB52 was expressed under the control of the AOX1 promoter and displayed on Pichia pastoris KM71 cell surface with the two Pichia pastoris cell surface display vectors. Localization of the displayed LipB52 on the cell surface was confirmed by the confocal laser scanning microscopy (CLSM). The LipB52 displayed on the Pichia pastoris cell surface exhibited activity toward p-nitrophenol ester with carbon chain length ranging from C10 to C18, and the optimum substrate was p-nitrophenol-caprate (C10), which was consistent with it displayed on the Saccharomyces cerevisiae EBY100 cell surface. The hydrolysis activity of lipase LipB52 displayed on Pichia pastoris KM71-pLHJ047 and KM71-pLHJ048 cell surface reached 94 and 91 U/g dry cell, respectively. The optimum temperature of the displayed lipases was 40 degrees C at pH8.0, they retained over 90% activity after incubation at 60 degrees C for 2 hours at pH 7.0, and still retained 85% activity after incubation for 3 hours.

Conclusion: The LipB52 displayed on the Pichia pastoris cell surface exhibited better stability than the lipase LipB52 displayed on Saccharomyces cerevisiae cell surface. The displayed lipases exhibited similar transesterification activity. But the Pichia pastoris dry cell weight per liter (DCW/L) ferment culture was about 5 times than Saccharomyces cerevisiae, the lipase displayed on Pichia pastoris are more suitable for whole-cell biocatalysts than that displayed on Saccharomyces cerevisiae cell surface.

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The effect of temperature on stability of the displayed lipases. The lipase LipB52 displayed on Pichia pastoris KM71-pLHJ047 and KM71-pLHJ048 cell surface retained over 90% activity after incubation at 60°C for 2 hours at pH 7.0, and they can still retained about 80% activity after incubation for 3 hours. The lipase LipB52 displayed on Saccharomyces cerevisiae EBY100-pLHJ026 retained near 90% activity after incubation at 60°C for 2 hours at pH 7.0 and it can only retained about 70% activity after incubation for 3 hours.
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Figure 5: The effect of temperature on stability of the displayed lipases. The lipase LipB52 displayed on Pichia pastoris KM71-pLHJ047 and KM71-pLHJ048 cell surface retained over 90% activity after incubation at 60°C for 2 hours at pH 7.0, and they can still retained about 80% activity after incubation for 3 hours. The lipase LipB52 displayed on Saccharomyces cerevisiae EBY100-pLHJ026 retained near 90% activity after incubation at 60°C for 2 hours at pH 7.0 and it can only retained about 70% activity after incubation for 3 hours.

Mentions: The p-nitrophenol ester with carbon chain length ranging from C10 to C18 (C10, C12, C14, C16, C18) was used as the substrate to characterize the displayed lipase LipB52 produced by KM71-pLHJ047 and KM71-pLHJ048. The lipase LipB52 displayed on Pichia pastoris KM71 cell surface exhibited evident hydrolysis activity towards the p-nitrophenol ester, but the optimum substrate was p-nitrophenol-caprate (C10). Lipase LipB52 displayed on Pichia pastoris and Saccharomyces cerevisiae exhibited similar substrate specificity (Fig. 4). The lipase LipB52 displayed on Pichia pastoris KM71-pLHJ047 and KM71-pLHJ048 cell surface have a temperature optimum of 40°C at pH8.0. The hydrolysis activity reached 94 and 91 U/g dry cell respectively (assayed under their optimum condition). The lipase LipB52 displayed on Saccharomyces cerevisiae EBY100-pLHJ026 cell surface had a temperature optimum of 37°C at pH8.0, its maximum activity was 92 U/g dry cell. The lipase LipB52 displayed on Pichia pastoris KM71-pLHJ047 and KM71-pLHJ048 cell surface retained over 90% activity after incubation at 60°C for 2 hours at pH 7.0, and they can still retained about 80% activity after incubation for 3 hours. They exhibited better stability than the lipase LipB52 displayed on Saccharomyces cerevisiae EBY100-pLHJ026 cell surface (Fig. 5). The transesterification activity of the displayed lipases had no evident difference (Fig. 6).


Efficient display of active lipase LipB52 with a Pichia pastoris cell surface display system and comparison with the LipB52 displayed on Saccharomyces cerevisiae cell surface.

Jiang Z, Gao B, Ren R, Tao X, Ma Y, Wei D - BMC Biotechnol. (2008)

The effect of temperature on stability of the displayed lipases. The lipase LipB52 displayed on Pichia pastoris KM71-pLHJ047 and KM71-pLHJ048 cell surface retained over 90% activity after incubation at 60°C for 2 hours at pH 7.0, and they can still retained about 80% activity after incubation for 3 hours. The lipase LipB52 displayed on Saccharomyces cerevisiae EBY100-pLHJ026 retained near 90% activity after incubation at 60°C for 2 hours at pH 7.0 and it can only retained about 70% activity after incubation for 3 hours.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2267459&req=5

Figure 5: The effect of temperature on stability of the displayed lipases. The lipase LipB52 displayed on Pichia pastoris KM71-pLHJ047 and KM71-pLHJ048 cell surface retained over 90% activity after incubation at 60°C for 2 hours at pH 7.0, and they can still retained about 80% activity after incubation for 3 hours. The lipase LipB52 displayed on Saccharomyces cerevisiae EBY100-pLHJ026 retained near 90% activity after incubation at 60°C for 2 hours at pH 7.0 and it can only retained about 70% activity after incubation for 3 hours.
Mentions: The p-nitrophenol ester with carbon chain length ranging from C10 to C18 (C10, C12, C14, C16, C18) was used as the substrate to characterize the displayed lipase LipB52 produced by KM71-pLHJ047 and KM71-pLHJ048. The lipase LipB52 displayed on Pichia pastoris KM71 cell surface exhibited evident hydrolysis activity towards the p-nitrophenol ester, but the optimum substrate was p-nitrophenol-caprate (C10). Lipase LipB52 displayed on Pichia pastoris and Saccharomyces cerevisiae exhibited similar substrate specificity (Fig. 4). The lipase LipB52 displayed on Pichia pastoris KM71-pLHJ047 and KM71-pLHJ048 cell surface have a temperature optimum of 40°C at pH8.0. The hydrolysis activity reached 94 and 91 U/g dry cell respectively (assayed under their optimum condition). The lipase LipB52 displayed on Saccharomyces cerevisiae EBY100-pLHJ026 cell surface had a temperature optimum of 37°C at pH8.0, its maximum activity was 92 U/g dry cell. The lipase LipB52 displayed on Pichia pastoris KM71-pLHJ047 and KM71-pLHJ048 cell surface retained over 90% activity after incubation at 60°C for 2 hours at pH 7.0, and they can still retained about 80% activity after incubation for 3 hours. They exhibited better stability than the lipase LipB52 displayed on Saccharomyces cerevisiae EBY100-pLHJ026 cell surface (Fig. 5). The transesterification activity of the displayed lipases had no evident difference (Fig. 6).

Bottom Line: The LipB52 displayed on the Pichia pastoris cell surface exhibited better stability than the lipase LipB52 displayed on Saccharomyces cerevisiae cell surface.The displayed lipases exhibited similar transesterification activity.But the Pichia pastoris dry cell weight per liter (DCW/L) ferment culture was about 5 times than Saccharomyces cerevisiae, the lipase displayed on Pichia pastoris are more suitable for whole-cell biocatalysts than that displayed on Saccharomyces cerevisiae cell surface.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Bioreactor Engineering, Newworld Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237, PR China. zhbjiang@hubu.edu.cn

ABSTRACT

Background: For industrial bioconversion processes, the utilization of surface-displayed lipase in the form of whole-cell biocatalysts is more advantageous, because the enzymes are displayed on the cell surface spontaneously, regarded as immobilized enzymes.

Results: Two Pichia pastoris cell surface display vectors based on the flocculation functional domain of FLO with its own secretion signal sequence or the alpha-factor secretion signal sequence were constructed respectively. The lipase gene lipB52 fused with the FLO gene was successfully transformed into Pichia pastoris KM71. The lipase LipB52 was expressed under the control of the AOX1 promoter and displayed on Pichia pastoris KM71 cell surface with the two Pichia pastoris cell surface display vectors. Localization of the displayed LipB52 on the cell surface was confirmed by the confocal laser scanning microscopy (CLSM). The LipB52 displayed on the Pichia pastoris cell surface exhibited activity toward p-nitrophenol ester with carbon chain length ranging from C10 to C18, and the optimum substrate was p-nitrophenol-caprate (C10), which was consistent with it displayed on the Saccharomyces cerevisiae EBY100 cell surface. The hydrolysis activity of lipase LipB52 displayed on Pichia pastoris KM71-pLHJ047 and KM71-pLHJ048 cell surface reached 94 and 91 U/g dry cell, respectively. The optimum temperature of the displayed lipases was 40 degrees C at pH8.0, they retained over 90% activity after incubation at 60 degrees C for 2 hours at pH 7.0, and still retained 85% activity after incubation for 3 hours.

Conclusion: The LipB52 displayed on the Pichia pastoris cell surface exhibited better stability than the lipase LipB52 displayed on Saccharomyces cerevisiae cell surface. The displayed lipases exhibited similar transesterification activity. But the Pichia pastoris dry cell weight per liter (DCW/L) ferment culture was about 5 times than Saccharomyces cerevisiae, the lipase displayed on Pichia pastoris are more suitable for whole-cell biocatalysts than that displayed on Saccharomyces cerevisiae cell surface.

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Related in: MedlinePlus