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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 curve of displayed lipase production. The lipase activity produced by pichia pastoris KM71-pLHJ047 reached its maximum 92 U/g dry cell after induced for 96 h; The lipase activity produced by pichia pastoris KM71-pLHJ048 reached its maximum 89 U/g dry cell after induced for 96 h; The lipase activity produced by Saccharomyces cerevisiae EBY100-pLHJ026 reached its maximum 92 U/g dry cell after induced for 48 h. Assayed under the same condition: p-nitrophenol-caprate used as substrate, assayed at 37°C, pH8.0.
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Figure 2: The curve of displayed lipase production. The lipase activity produced by pichia pastoris KM71-pLHJ047 reached its maximum 92 U/g dry cell after induced for 96 h; The lipase activity produced by pichia pastoris KM71-pLHJ048 reached its maximum 89 U/g dry cell after induced for 96 h; The lipase activity produced by Saccharomyces cerevisiae EBY100-pLHJ026 reached its maximum 92 U/g dry cell after induced for 48 h. Assayed under the same condition: p-nitrophenol-caprate used as substrate, assayed at 37°C, pH8.0.

Mentions: The recombinant plasmids linearized with Sal I were electroporated into Pichia pastoris KM71 as described in Methods. The lipase gene was expressed under the control of the AOX1 promoter and the displayed lipase activity were detected by BMMY medium plates supplemented with 1% olive oil and 0.002% rhodamine B, the expression pattern can be determined by the fluorescent halo around them. The single transformant colony with lipase activity was inoculated and named as KM71-pLHJ047 and KM71-pLHJ048, respectively. By using the total DNA of KM71-pLHJ047 and KM71-pLHJ048 as the template, a DNA fragment with the same size as lipase gene lipB52 was obtained with primers LipB52Pf-EcoR I and LipB52Pr-Not I by PCR amplification (total DNA of Pichia pastoris KM71 transfected with the plasmid pPIC9K was used as the negative control template), which convinced that the KM71-pLHJ047 and KM71-pLHJ048 were recombinant Pichia pastoris with lipase gene lipB52. The lipase activity reached their maximum (92 and 89 U/g dry cell, p-nitrophenol-caprate used as substrate, assayed at 37°C, pH8.0) after induced for 96 h, while the lipase LipB52 displayed on Saccharomyces cerevisiae EBY100-pLHJ026 reached its maximum (92 U/g dry cell, assayed under the same condition) after induced for 48 h (Fig. 2) and the cells were harvested for further analysis.


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 curve of displayed lipase production. The lipase activity produced by pichia pastoris KM71-pLHJ047 reached its maximum 92 U/g dry cell after induced for 96 h; The lipase activity produced by pichia pastoris KM71-pLHJ048 reached its maximum 89 U/g dry cell after induced for 96 h; The lipase activity produced by Saccharomyces cerevisiae EBY100-pLHJ026 reached its maximum 92 U/g dry cell after induced for 48 h. Assayed under the same condition: p-nitrophenol-caprate used as substrate, assayed at 37°C, pH8.0.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: The curve of displayed lipase production. The lipase activity produced by pichia pastoris KM71-pLHJ047 reached its maximum 92 U/g dry cell after induced for 96 h; The lipase activity produced by pichia pastoris KM71-pLHJ048 reached its maximum 89 U/g dry cell after induced for 96 h; The lipase activity produced by Saccharomyces cerevisiae EBY100-pLHJ026 reached its maximum 92 U/g dry cell after induced for 48 h. Assayed under the same condition: p-nitrophenol-caprate used as substrate, assayed at 37°C, pH8.0.
Mentions: The recombinant plasmids linearized with Sal I were electroporated into Pichia pastoris KM71 as described in Methods. The lipase gene was expressed under the control of the AOX1 promoter and the displayed lipase activity were detected by BMMY medium plates supplemented with 1% olive oil and 0.002% rhodamine B, the expression pattern can be determined by the fluorescent halo around them. The single transformant colony with lipase activity was inoculated and named as KM71-pLHJ047 and KM71-pLHJ048, respectively. By using the total DNA of KM71-pLHJ047 and KM71-pLHJ048 as the template, a DNA fragment with the same size as lipase gene lipB52 was obtained with primers LipB52Pf-EcoR I and LipB52Pr-Not I by PCR amplification (total DNA of Pichia pastoris KM71 transfected with the plasmid pPIC9K was used as the negative control template), which convinced that the KM71-pLHJ047 and KM71-pLHJ048 were recombinant Pichia pastoris with lipase gene lipB52. The lipase activity reached their maximum (92 and 89 U/g dry cell, p-nitrophenol-caprate used as substrate, assayed at 37°C, pH8.0) after induced for 96 h, while the lipase LipB52 displayed on Saccharomyces cerevisiae EBY100-pLHJ026 reached its maximum (92 U/g dry cell, assayed under the same condition) after induced for 48 h (Fig. 2) and the cells were harvested for further analysis.

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.

Show MeSH
Related in: MedlinePlus