Expression of Aspergillus niger glucose oxidase in Pichia pastoris and its antimicrobial activity against Agrobacterium and Escherichia coli

Strains and plasmids

A 1,749.0 bp GOD gene fragment was amplified from the genomic DNA of Aspergillus niger ZM-8 by the CTAB method (Porebski, Bailey & Baum, 1997). Primers for PCR were designed as Table S1 based on conserved sequences of glucose oxidase gene (No. JO5242) from GenBank Database, and then cloned into plasmid pUC19, The linearized vector pUC19-His-GOD by SmaI was inserted into the S. cerevisiae α-factor secretion signal molecule pPIC9k (Invitrogn, Carlsbad, CA, USA) to generate the expression vector pPIC9k-His-GOD under the action of the promoter AOX-1. The identified recombinant plasmid pPIC9K-His-GOD was linearized by Bgl-П and transformed into P. pastoris GS115 cells by electroporation. The electro-competent P. pastoris GS115 cells were prepared using standard methods (Manivasakam & Schiestl, 1993). The electroporation was performed using a Gene Pulser (Bio-Rad, Hercules, CA, USA) at 1.5 kV, 40.0 μF, and 150.0 Ω according to manufacturer’s instruction.

Screening of clones and determination of biomass

The recombinant yeast clones were screened on yeast extract peptone dextrose (YPD) (1% (w/v) yeast extract, 2% (w/v) tryptone, 2% (w/v) dextrose, 2% (w/v) agar) plus 1 M sorbitol (YPDS) plates containing 100.0 μg/mL G418 (Invitrogen, Carlsbad, CA, USA) for 2.0–4.0 days. Potential high-level secretion transformants were obtained from the YPDS agar plates containing a higher G418 concentration (300.0 μg/mL). All these potential high-level secretion clones were confirmed by PCR using genomic DNA as the templates.

One colony was picked among several high copy clones obtained from the plate containing P. pastoris GS115-pPIC9k-His-GOD. P. pastoris GS115-pPIC9k was used as a negative control for the experiment. Clones were inoculated in Buffered Glycerol-complex medium (BMGY) (1% (w/v) yeast extract, 2% (w/v) tryptone, 100 mM Potassium Phosphate (pH 6.0), 1.34% (w/v) YNB, 4 × 10⁻⁵ D-Biotin, 1% (w/v) glycerol), and cultured at 30 °C until OD₆₀₀ = 0.60. The culture was then transferred to Buffered methanol-complex medium (BMMY) and cultured at 30 °C. The absorbance of growing culture was measured every 3 h.

Expression of GOD in transgenic P. pastoris GS115

The P. Pastoris strains were cultivated in BMGY medium at 30 °C for 24 h. Biomass was generated after initial growth phase with glycerol as a carbon source. Finally, to induce AOX-1 dependent protein expression, the methanol fed-batch phase was started with methanol feed rate of 0.5 mL/12.0 h. Cell-free supernatant, the supernatant of cell lysate, and the precipitate of cell lysate was collected, and crushed by ultrasonic. The ultrasonication conditions used was 15 s, 25 s, 380 w, 99 times, and then stored at 4 °C (Cereghino & Cregg, 2000).

Analysis of glucose oxidase activity

Pichia pastoris GS115-His-GOD-01 and P. pastoris GS115-pPIC9k were cultured at 30 °C with supplement of 0.5% methanol per 12 h. Activities of glucose oxidase from cell-free supernatant, cell lysate supernatant and precipitation were carried out according to Gemba & Hara (1971) with a slight modification. In detail, the reaction system consisting of 4.0 mL of reaction mixture (0.20 M Acetic acid-Sodium acetate, pH 5.2; 0.2 M glucose and 1.0 mM Indigo Carmine) and 2.0 mL of the appropriately diluted enzyme solution was added into and incubated for 10 min at 37 °C. Then, the reaction was stopped by boiling water bath for 13 min. The absorbance was measured at a wavelength of 615.0 nm. One unit (U) of Glucose oxidase activity was defined as the amount of enzyme, that can oxidize 1.0 μM of β-D-glucose to D-gluconic acid and H₂O₂ per 3 min at pH 6.0 and 30 °C. All reactions were performed in triplicate. The formula of enzyme activity as follows: $$X_0=\left[\left(A-A_0\right)\times K+C_0\right]\times V\times F$$X₀: Enzyme activity; A: Absorbance value of trichloroacetic acid instead of glucose as the control; A₀: Absorbance value of the sample solution; K: Slope of the standard curve; C₀: Intercept of the standard curve. V: the volume of sample solution; F: the dilution factor.

The specific activity of GOD

Protein concentrations of cell-free liquid, cell lysate supernatant and precipitation from P. pastoris GS115-His-GOD-01 and P. pastoris GS115-pPIC9K were determined by the method of Bradford (Hammond & Kruger, 1988). Absorbance was measured at 615 nm wavelength and specific activity of GOD was the value of activity divided by the value of protein concentrations.

Antibacterial effects of glucose and glucose oxidase system on growth of Agrobacterium and E. coli in liquid medium

Glucose oxidase and glucose were used in three dilution-set combinations. The concentration of glucose used were 1.0, 2.5 and 5.0 mg/mL respectively. The GOD was from fermentation supernatant of transgenic P. pastoris GS115-His-GOD-01 that was induced by methanol and fermentation supernatant from P. pastoris GS115-pPIC9K as control. Concentrations of GOD used was 1.0, 5.0 and 10.0 U/mL. The GOD and glucose solutions were added in the medium of YEP or LB and arranged in a Latin-square design to study the effects of substrates and enzyme on growth of Agrobacterium LBA4404 and E. coli DH5α by measuring optical density in 600 nm.

GOD antibacterial activity to Agrobacterium and E. coli on agar plates

The antibacterial activity of GOD produced by P. pastoris GS115-His-GOD-01 against A. tumefaciens LBA4404 and E. coli DH5α (stored in Dr. Ma Jianzhong’s laboratory of Lanzhou University of Technology) was determined. Cultivate to bacterial liquid OD₆₀₀ = 1.0, spread on YPE (1% (w/v) yeast extract, 1% (w/v) tryptone, 0.5% (w/v) NaCl and 1.5% (w/v ) Agar) plates or LB (1% (w/v) yeast extract, 2% (w/v) tryptone, 2% (w/v) NaCl and 1.5% (w/v) agar) plates, on 51 h after adding methanol, add cell-free liquid, sonicate the supernatant by precipitating, collect the pellet and resuspend the pellet in an ice bath, immerse it in sterile filter paper, and inoculate with 0.20 M A. tumefaciens LBA4404 or E. coli DH5α on the surface of the glucose plate, observe its antibacterial effect.

Antibacterial activity of hydrogen peroxide solution to Agrobacterium and E. coli on agar plates

The inhibitory effects of A. tumefaciens LBA4404 and E. coli DH5α and the minimum hydrogen peroxide concentration to inhibit bacterial growth were detected. A. tumefaciens LBA4404 was cultured with shaking at 28 °C to OD₆₀₀ = 1.0, and 200 μL was coated on YPE medium, and then put into filter paper with different concentrations of hydrogen peroxide solution, and cultured at 28 °C for 14 h, and observed antibacterial effect. E. coli DH5α was shake-cultured at 37 °C to OD₆₀₀ = 1.5, 200 μL was applied to LB medium, and then filter paper of different concentrations of hydrogen peroxide solution was placed. The culture was allowed to stand at 37 °C for 14 h to observe the inhibitory effect.

Vector construction and screening of transgenic P. pastoris clones

Pichia pastoris strain GS115 was transformed using linearized pPIC9K-His-GOD as described in materials and methods to yield P. pastoris GS115-His-GOD (Fig. S1). Twelve clones were obtained and confirmed by PCR-testing for gene integration. These clones were then screened on YPDS plates with different concentrations of Geneticin (G418), that is, 100 mM, 200 mM, and 300 mM, respectively. A positive transgenic clone, designated as P. pastoris GS115-His-GOD 01 was grown on the YPDS plate with a high Geneticin concentration and was considered for subsequent experiments.

Expression of the GOD affecting the growth of the GOD-transgenic strain

Hydrogen peroxide, one of the products by GOD, injures living cells. In line with this statement, growth of the GOD-transgenic strain, P. pastoris GS115-His-GOD 01 was analyzed. Compared to P. pastoris GS115-pPIC9K, the growth of P. pastoris GS115-His-GOD 01 was slightly decreased during 51st h of incubation under GOD uninduced condition (Fig. 1A). Its optical density at 600 nm was 0.95-fold of that of P. pastoris GS115-pPIC9K at the time point of 51.0 h. However, the growth of P. pastoris GS115-His-GOD 01 was significantly lowered if the GOD was induced by methanol (Fig. 1B). During the growth of 51.0 h, the optical densities of P. pastoris GS115-His-GOD 01 were 0.54-fold of that of P. pastoris GS115-pPIC9K at 18.0h, 0.43-fold at 36 h, and 0.37-fold at 51.0 h, respectively. The inhibited growth of the GOD-transgenic P. pastoris could be attributed to the expression of the foreign GOD and, hereafter, accumulation of H₂O₂.

Activities of the glucose oxidase

After 51 h-induced incubation, the cultures were processed into three parts of which were the cell-free supernatant, the supernatant and the precipitation of the cell lysates. The activities of the GOD preparations from P. pastoris GS115-His-GOD 01 were 14.27 U/mL in the cell-free supernatant, 18.2 U/mL in the supernatant of the cell lysate, and 0.48 U/mL in the precipitation (Fig. 2A). As a control, the activities of the three GOD preparations from P. pastoris GS115-pPIC9K were 3.22 U/mL, 1.76 U/mL and 0.41 U/mL, respectively (Fig. 2A). The specific activities of the three GOD preparations from P. pastoris GS115-His-GOD 01 were 8.30 U/mg in the cell-free supernatant, 6.52 U/mg in the supernatant of the cell lysate, and 0.73 U/mg in the precipitation, respectively (Fig. 2B). The specific activities of the three preparations from P. pastoris GS115-pPIC9K were 0.859 U/mg, 1.483 U/mg, and 0.529 U/mg, respectively (Fig. 2B). According to the specific activities, the cell-free supernatant of P. pastoris GS115-His-GOD 01 had the highest value, but the supernatant of the cell lysate of P. pastoris GS115-pPIC9K gave the highest specific activity. These results suggested that the native GOD of P. pastoris GS115 was mainly an intracellular enzyme. In the GOD-transgenic P. pastoris GS115, the enzyme was mainly secreted. This is in accordance with that the recombinant GOD was directed to an extra-cellular fraction by a signal peptide, α-mating factor.

The concentration of hydrogen peroxide from GOD catalyzed glucose

The concentration of hydrogen peroxide produced by GOD from P. pastoris GS115-His-GOD-01 and P. pastoris GS115-pPIC9K catalytic glucose was showed in Fig. 3. According to the results in Fig. 3, the concentration of hydrogen peroxide was 14.3 μg/mL and 3.05 μg/mL in cell-free supernatant. The concentration of H₂O₂ from the supernatant of cell lysate was 18.2 μg/mL and 1.86 μg/mL, however, that of the precipitate of cell lysate was 0.48 μg/mL and 0.46 μg/mL. These results indicated that the GOD could be secreted out of the cells with the form of soluble protein.

Inhibition of the GOD preparations on the growth of A. tumefaciens LBA4404 and E. coli in liquid medium

The GOD was prepared in fermentation supernatant of P. pastoris GS115-His-GOD 01. When the concentration of GOD was set as 1.0 U/mL, the growth curve of A. tumefaciens LBA4404 and E. coli DH5α under the gradually increasing of Glu were shown in Figs. 4A and 4B. From these two figures, almost no inhibition to the growth of A. tumefaciens LBA4404 compared with the control that was added with equal volume of P. pastoris GS115-pPIC9K fermentation supernatant. However, the marked inhibition to the growth of E. coli DH5α were observed from 4th h to 14th h with substrate concentration increasing (Fig. 4B). When the concentration of GOD was set as 5.0 U/mL, a slight inhibition on the growth of A. tumefaciens LBA4404 after 14th h with substrate concentration increasing (Fig. 4C), and the same change trends with Fig. 4B can be observed for the inhibition on E. coli DH5α from Fig. 4D. With the increasing of Glu concentration, the delay of growth of A. tumefaciens LBA4404 also be observed by the given GOD concentration of 10 U/mL (Fig. 4E), and the minor effect on E. coli DH5α also could be found in Fig. 4F. Conclusions were drawn from Fig. 4, it showed these combinations did not completely inhibit growth of A. tumefaciens and LBA4404 E. coli DH5α, but influenced the time at which growth was initiated. Delay of growth initiation was greatest with the enzyme concentration, 5.0 U/mL, and the impact increased also with substrate concentration.

Antibacterial effects of glucose and glucose oxidase on growth of A. tumefaciens and E. coli on agar plates

Analysis of the antibacterial activity of hydrogen peroxide (H₂O₂) produced by GOD catalyzed substrates glucose. A. tumefaciens LBA4404 (Fig. 5A) and E. coli DH5α (Fig. 5B) were plated on YPE or LB which were contained 0.2 M glucose. Filter papers were soaked by cell-free supernatant, the supernatant of cell lysate, and the precipitate of cell lysate from P. pastoris GS115-His-GOD 01, cell-free supernatant of P. pastoris GS115-pPIC9K as the negative control. The results showed that H₂O₂ derived from glucose which catalyzed by GOD had no effect on the growth of A. tumefaciens LBA4404 and E. coli DH5α.

Antibacterial activity of hydrogen peroxide solution to A. tumefaciens LBA4404 and E. coli DH5α

To detect the minimum concentration of hydrogen peroxide solution inhibit the growth of A. tumefaciens LBA4404 and E. coli DH5α, the sterile filter articles were soaked with a volume of 10 μL hydrogen peroxide that was diluted to different concentrations. Different concentrations of hydrogen peroxide solution effect on A. tumefaciens LBA4404 and E. coli DH5α were determined. As shown in Figs. 6A–O and 6P–AA, the inhibition effect of H₂O₂ on the two strains gradually decreased with the concentration reducing. The diameters of inhibition zone for these strains also were displayed in Figs. 6BB and 6CC, implying that the minimum concentration of hydrogen peroxide solution inhibits the growth of A. tumefaciens LBA4404 and E. coli DH5α was 5.6 × 10³ μg/mL and 6.0 × 10³ μg/mL.