Biofilm-Based Biocatalysis for Galactooligosaccharides Production by the Surface Display of β-Galactosidase in Pichia pastoris

Abstract

Galactooligosaccharides (GOS) are one of the most important functional oligosaccharide prebiotics. The surface display of enzymes was considered one of the most excellent strategies to obtain these products. However, a rough industrial environment would affect the biocatalytic process. The catalytic process could be efficiently improved using biofilm-based fermentation with high resistance and activity. Therefore, the combination of the surface display of β-galactosidase and biofilm formation in Pichia pastoris was constructed. The results showed that the catalytic conversion rate of GOS was up to 50.3% with the maximum enzyme activity of 5125 U/g by screening the anchorin, and the number of the continuous catalysis batches was up to 23 times. Thus, surface display based on biofilm-immobilized fermentation integrated catalysis and growth was a co-culture system, such that a dynamic equilibrium in the consolidated integrative process was achieved. This study provides the basis for developing biofilm-based surface display methods in P. pastoris during biochemical production processes.

Keywords: Pichia pastoris; biofilm; galactooligosaccharides; yeast surface display; β-galactosidase.

Figure 1

Biofilm-based biocatalysis for surface-displayed strains in dynamic growth and catalytic GOS production. (a) A schematic illustration of construction for the knockout of PAS_chr1-3_0226 and biofilm formation in P. pastoris. (b) The DNA components of the surface-displayed plasmids used in this study and a schematic diagram of the yeast surface display.

Figure 2

The impact of the knockout of PAS_chr1-3_0226 gene on biofilm formation and cell adhesion. (a) GS115 and GS115-Δ0226 strains were cultivated in 96-well plates for 72 h and tested for adhesion ability. Free cells were removed and washed twice with PBS buffer and stained with 0.1% crystal violet. The samples were washed repeatedly with water, lysed with glacial acetic acid, and then photographed. (b) Adhesion expressed as the optical density at 570 nm (OD₅₇₀) of solubilized crystal violet in acetic acid. Data are reported as the means and standard deviation of three independent experiments. The p-values were computed using Student’s t-test (*** p < 0.001). (c) Growth of GS115 and GS115-Δ0226 on YPD at an OD₆₀₀ = 1 and used to make 10⁻¹, 10⁻², and 10⁻³ gradient dilutions in sterile water. (d) Standard plate-wash assay of GS115 and GS115-Δ0226.

Figure 3

SEM (a) and TEM (b) images of cells after 48 h of fermentation. (a) The biofilm formation on the cotton fibers of the control, GS115 wild type, and GS115-Δ0226. The observation scale was 10 μm in I to III and 5 μm in IV to VI. The deletion of PAS_chr1-3_0226 could enhance biofilm formation. (b) The microstructure of cell morphology of GS115 wild type and GS115-Δ0226. WT was normal with smooth surface, while strain Δ0226 was rough and the specific surface area was increased. The observation scale was 2 μm in I to II and 500 nm in III to IV.

Figure 4

Expression and enzyme activity of surface-displayed β-galactosidase in P. pastoris. (a) β-galactosidase expression in (I) Δ0226-Aga2p-lacA, (II) Δ0226-Flo1p-lacA, (III) Δ0226-Pir1p-lacA, and (IV) the control. The darker the color of the sample, the higher the expression of β-galactosidase. (b) Enzyme activity was determined by adding 200–1200 μL oNPG to precipitate to determine the optimum substrate concentration. (c) The enzyme activities of precipitate by the strains Δ0226-Pir1p-lacA, Δ0226-Flo1p-lacA, and Δ0226-Aga2p-lacA. (d) The enzyme activity of supernatant by the free expression of WT-lacA and the three surface-displayed strains.

Figure 5

The characterization of β-galactosidase by (a) immunofluorescence microscopy and (b) flow cytometry in WT-lacA and Δ0226-Pir1p-lacA strains. (a) Panels Ⅰ and Ⅳ show representative immunofluorescence micrographs of the free expression WT-lacA and surface-displayed strain Δ0226-Pir1p-lacA using FITC excitation wavelengths; Ⅱ and Ⅴ show the visible light images, and Ⅲ and Ⅵ are merged composites of the visible light and FITC excitation images. All micrographs shown were taken at 1000× magnification and at the same angle. Scale bar = 10 μm. (b) Flow cytometry histograms reflect the fluorescence signal of the surface-displayed strain, with the x-axis indicating fluorescence intensity and the y-axis indicating the cell count. Red arrow: the peak shifted to the right indicated that the efficiency of surface display reached 80.63%.

Figure 6

The characterization of surface-displayed β-galactosidase enzyme activity. (a) The influence of temperature on the relative activity of recombinant strain. (b) Effect of pH on the relative activity of recombinant strain. (c) Effect of exogenous addition by different concentrations of metal ions at 5 mmol/L, 25 mmol/L, and 50 mmol/L on the relative activity of recombinant strain, calculated assuming 100% enzyme activity without the addition of metal ions. (d) The surface-displayed enzyme was maintained at 40–80 °C for 120 min and the relative enzyme activity was measured every 30 min. (e) The surface-displayed enzyme was maintained at pH 4.0–7.0 for 120 min and the relative enzyme activity was measured every 30 min. (f) The surface-displayed enzyme was held at pH 6.0 and 50 °C for 24 h and sampled at intervals to measure the enzyme activity. Calculations assumed 100% activity at 0 h.

Figure 7

Optimization of GOS production and cell reuse times. (a) Comparison of immobilized and free cell dry weights. Data are reported as the means and standard deviation of three independent experiments. The p-values were computed using Student’s t-test (*** p < 0.001). (b) Effect of lactose concentration and catalytic time on GOS production by the surface-displayed strains. GOS production was increased in a lactose concentration-dependent manner, with the highest production of 251 g/L GOS at 500 g/L lactose (50.3% yield), before a sudden drop in production at concentrations above 500 g/L lactose. The optimal production time for GOS was 12 h, after which the yield stabilizes. (c) Influence of different volumes of BMMY medium on GOS yield in multi-batch catalysis. A total of 4 catalysis batches were conducted, with the highest catalytic efficiency starting in the third batch with 20% BMMY. Adding more than 20% BMMY negatively impacted GOS yield. (d) Number of reuses in the free cell catalysis. (e) Number of reuses in the immobilized cell catalysis. When the GOS yield drops to a certain level, the immobilized cells were resuscitated for 48 h and before catalysis was resumed.