Enzymatic Degradation of Aromatic and Aliphatic Polyesters by P. pastoris Expressed Cutinase 1 from Thermobifida cellulosilytica

Abstract

To study hydrolysis of aromatic and aliphatic polyesters cutinase 1 from Thermobifida cellulosilytica (Thc_Cut1) was expressed in P. pastoris. No significant differences between the expression of native Thc_Cut1 and of two glycosylation site knock out mutants (Thc_Cut1_koAsn and Thc_Cut1_koST) concerning the total extracellular protein concentration and volumetric activity were observed. Hydrolysis of poly(ethylene terephthalate) (PET) was shown for all three enzymes based on quantification of released products by HPLC and similar concentrations of released terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET) were detected for all enzymes. Both tested aliphatic polyesters poly(butylene succinate) (PBS) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) were hydrolyzed by Thc_Cut1 and Thc_Cut1_koST, although PBS was hydrolyzed to significantly higher extent than PHBV. These findings were also confirmed via quartz crystal microbalance (QCM) analysis; for PHBV only a small mass change was observed while the mass of PBS thin films decreased by 93% upon enzymatic hydrolysis with Thc_Cut1. Although both enzymes led to similar concentrations of released products upon hydrolysis of PET and PHBV, Thc_Cut1_koST was found to be significantly more active on PBS than the native Thc_Cut1. Hydrolysis of PBS films by Thc_Cut1 and Thc_Cut1_koST was followed by weight loss and scanning electron microscopy (SEM). Within 96 h of hydrolysis up to 92 and 41% of weight loss were detected with Thc_Cut1_koST and Thc_Cut1, respectively. Furthermore, SEM characterization of PBS films clearly showed that enzyme tretment resulted in morphological changes of the film surface.

Keywords: Pichia pastoris; Thermobifida cellulosilytica; aliphatic polyesters; cutinase; enzymatic hydrolysis; poly(3-hydroxybutyrate-co-3-hydroxyvalerate); poly(butylene succinate); poly(ethylene terephthalate).

Figure 1

Representation of Thc_Cut1 glycosylation site knockout mutants. (A) Thc_Cut1_koAsn, (B) Thc_Cut1_koST; mutated AA are in pink (A) and blue (B), active site residues are in red, ALE linker sequence for 6× His-Tag is shown in yellow.

Figure 2

SDS-PAGE (12%) of P. pastoris fermentation supernatant samples withdrawn at different time points after induction. (A) Thc_Cut1, (B) Thc_Cut1_koAsn, (C) Thc_Cut1_ko_ST; M = peqGold protein marker IV, #= time after methanol induction in h, each sample set was once loaded after treatment with Endo Hf (left) and once without Endo Hf treatment (right).

Figure 3

Glycostain gel analysis of P. pastoris and E. coli expressed Thc_Cut1. Left, SDS PAGE gel; Right, Glycostained gel; same samples were applied on both gels; C, Candy cane protein marker; M, peqGold protein marker IV; Pp, P. pastoris expressed Thc_Cut1; Ec, E. coli expressed Thc_Cut1.

Figure 4

Total protein concentration (A) and esterase activity (B) of fermentation supernatant samples of different time points after methanol induction. Thc_Cut1 (black line), Thc_Cut1_ko_Asn (blue line), and Thc_Cut1_ko_ST (red line). Time point 0 h indicates the start of methanol feed. Error bars show standard deviations of triplicate measurements.

Figure 5

Concentrations of soluble released products terephthalic acid (TPA) and mono(2-hydroxyethyl) terephthalate (MHET) upon enzymatic hydrolysis of PET powder by Thc_Cut1 (dark gray bars), Thc_Cut1_ko_Asn (light gray bars) and Thc_Cut1_ko_ST (middle gray bars). Time scan for 24, 48, 72, or 96 h was performed at 65 °C with 5 μM enzyme in 1 M KPi pH 8.0 with 50 mg/mL substrate at 100 rpm.

Figure 6

Concentrations of soluble released 3-hydroxybutyric acid (3-HBA) upon enzymatic hydrolysis of PHBV powder by Thc_Cut1 (dark gray bars) and Thc_Cut1_ko_ST (middle gray bars). Time scan for 24, 48, 72, or 96 h was performed at 65 °C with 5 μM enzyme in 1 M KPi pH 8.0 with 5 mg/mL substrate at 100 rpm.

Figure 7

Concentrations of soluble released products succinic acid (SA) and 1,4-butanediol (BDO) upon enzymatic hydrolysis of PBS powder by Thc_Cut1 (dark gray bars) and Thc_Cut1_ko_ST (middle gray bars). Time scan for 24, 48, 72 or 96 h was performed at 65°C with 5 μM enzyme in 1 M KPi pH 8.0 with 5 mg/mL substrate at 100 rpm.

Figure 8

Quartz Crystal Microbalance (QCM) measurements of hydrolysis of spin-coated PBS and PHBV films by Thc_cut1. (A,B). Progress curves of the polyester adlayer mass coated onto the QCM sensors during the hydrolysis of PBS (A) and PHBV (B). At time t = 0 h we added Thc_cut1 to the buffer running over the films, as indicated by the vertical dashed lines in panels (A,B). Black and gray lines represent duplicate experiments (C). Dry masses of the sensor adlayers before and after the hydrolysis experiments. Hydrolysis experiments were performed at 40°C and pH 7.0 (3 mM Tris buffer).

Figure 9

Weight loss of PBS films upon enzymatic hydrolysis by Thc_Cut1 (dark gray bars) and Thc_Cut1_ko_ST (middle gray bars). Time scan for 24, 48, 72, or 96 h was performed at 65 °C with 5 μM enzyme in 1 M KPi pH 8.0 with 0.5 × 1.0 cm PBS films at 100 rpm

Figure 10

SEM surface characterization of PBS films. (A,B) control reactions (polymer film + buffer only); (C,D) Thc_Cut1-catalyzed hydrolysis (polymer film + Thc_Cut1 in buffer); (E,F) Thc_Cut1_ko_ST-catalyzed hydrolysis (polymer film + Thc_Cut1_ko_ST in buffer). All samples were measured after 24 (left column) and 96 h (right column) of treatment. It's clearly visible the increase of the hydrolysis reaction from 24 to 96 h (C–F) and the higher damage to the film by Thc_Cut1_ko_ST that yield, after 96 h to holes that go throughout the film (E).