Abstract
Yeast cell-surface display technologies have been widely applied in the fields of food, medicine, and feed enzyme production, including lipase, α-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 pPICZαA and pGAPZαA 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 pPICZαA-Pir1p-treS and pGAPZαA-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.
Highlights
Since 2000, trehalose has been accepted as a novel food ingredient under the “generally recognized as safe” designation in the United States and the European Union
The fusion proteins in the extracts from the cell of P. pastorisGS115/pPICZαA-Trehalose synthase (TreS)-enhanced green fluorescence protein (EGFP)-Pir1p and P. pastoris GS115 were detected in Figure 1 (Lanes 2 and 5), indicating that they may attach to the cell wall as previously reported
No specific polypeptides could be detected from the concentrated growth and the supernatant of cell wall removed from P. pastoris GS115/pPICZαA-TreS-EGFP-Pir1p and P. pastoris GS115 (Figure 1, Lanes 1, 3, 4, 6), indicating that the fusion was displayed mainly on the cell wall
Summary
Since 2000, trehalose has been accepted as a novel food ingredient under the “generally recognized as safe” designation in the United States and the European Union It is a white, odorless powder and is a non-reducing sugar formed from two glucose units joined by an α-1,1-glycosidic bond. Odorless powder and is a non-reducing sugar formed from two glucose units joined by an α-1,1-glycosidic bond This special bonding can prevent Maillard reactions with proteins or amino acids (Higashiyama, 2002) and makes the compound stable at high temperatures or very acidic conditions. These heterologous expression systems are successful, lack of processing mechanism after protein translation and produced endotoxin limit their application to food and medicine
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