Dataset Information

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.

ABSTRACT: 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.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.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.

SUBMITTER: Jiang Z

PROVIDER: S-EPMC2267459 | biostudies-literature | 2008 Jan

REPOSITORIES: biostudies-literature

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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 Zhengbing Z Gao Bei B Ren Ren R Tao Xingyi X Ma Yushu Y Wei Dongzhi D

BMC biotechnology 20080128

<h4>Background</h4>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.<h4>Results</h4>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 ...[more]

PMID: 18221563

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Project description:Yeast cell-surface display technologies have been widely applied in the fields of food, medicine, and feed enzyme production, including lipase, ?-amylase, and endoglucanase. In this study, a treS gene was fused with the yeast cell-surface anchor protein gene Pir1p by overlap PCR, the Pir1p-treS fusion gene was ligated into pPICZ?A and pGAPZ?A and transformed into P. pastoris GS115 to obtain recombinant yeast strains that displays trehalose synthase(TreS) on its cell surface as an efficient and recyclable whole-cell biocatalyst. Firstly, the enhanced green fluorescence protein gene (egfp) was used as the reporter protein to fusion the Pir1p gene and treS gene to construct the recombinant plasmids containing treS-egfg-Pir1p fusion gene, and electrotransformed into P. pastoris GS115 to analyze the surface display characteristics of fusion gene by Western blot, fluorescence microscopy and flow cytometry. The analysis shown that the treS-egfg-Pir1p fusion protein can be successfully displayed on the surface of yeast cell, and the expression level increased with the extension of fermentation time. These results implied that the Pir1p-treS fusion gene can be well displayed on the cell surface. Secondly, in order to obtain surface active cells with high enzyme activity, the enzymatic properties of TreS displayed on the cell surface was analyzed, and the fermentation process of recombinant P. patoris GS115 containing pPICZ?A-Pir1p-treS and pGAPZ?A-Pir1p-treS was studied respectively. The cell surface display TreS was stable over a broad range of temperatures (10-45°C) and pH (6.0-8.5). The activity of TreS displayed on cell surface respectively reached 1,108 Ug-1 under PAOX1 control for 150 h, and 1,109 Ug-1 under PGAP control for 75h in a 5 L fermenter, respectively. Lastly, the cell-surface displayed TreS was used to product trehalose using high maltose syrup as substrate at pH 8.0 and 15°C. The surface display TreS cells can be recycled for three times and the weight conversion rate of trehalose was more than 60%. This paper revealed that the TreS can display on the P. pastoris cell surface and still had a higher catalytic activity after recycled three times, which was suitable for industrial application, especially the preparation of pharmaceutical grade trehalose products.

Dataset Information

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.

Publications

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.

Similar Datasets

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