N-terminal domain of turkey pancreatic lipase is active on long chain triacylglycerols and stabilized by colipase

Abstract

The gene encoding the TPL N-terminal domain (N-TPL), fused with a His6-tag, was cloned and expressed in Pichia pastoris, under the control of the glyceraldehyde-3-phosphate dehydrogenase (GAP) constitutive promoter. The recombinant protein was successfully expressed and secreted with an expression level of 5 mg/l of culture medium after 2 days of culture. The N-TPL was purified through a one-step Ni-NTA affinity column with a purification factor of approximately 23-fold. The purified N-TPL, with a molecular mass of 35 kDa, had a specific activity of 70 U/mg on tributyrin. Surprisingly, this domain was able to hydrolyse long chain TG with a specific activity of 11 U/mg using olive oil as substrate. This result was confirmed by TLC analysis showing that the N-TPL was able to hydrolyse insoluble substrates as olive oil. N-TPL was unstable at temperatures over 37°C and lost 70% of its activity at acid pH, after 5 min of incubation. The N-TPL exhibited non linear kinetics, indicating its rapid denaturation at the tributyrin-water interface. Colipase increased the N-TPL stability at the lipid-water interface, so the TPL N-terminal domain probably formed functional interactions with colipase despite the absence of the C-terminal domain.

Figure 1. Time-course of the expression of His6-tagged N-TPL by four isolated clones of recombinant Pichia pastoris.

The culture was carried out in 250 ml Erlen-meyer’s falsks with shaking at 150 rpm and 30°C.

Figure 2. Q-PCR analysis of the N-TPL cDNA copies number in the genomic DNA of N3, N4, N5 and N6 clones.

Correlation coefficient: 0.999; Slope: −3.484; PCR Efficiency: 93.6%.

Figure 3. SDS-PAGE of the purified His6-tagged N-TPL performed on 13% acrylamide gel.

Lane1: Low molecular weight marker; Lane 2∶30 µg of purified His6-tagged N-TPL.

Figure 4. Stability of His6-tagged N-TPL and TPL.

Effect of temperature (A) and pH (B). His6-tagged N-TPL was incubated at different temperatures and pH for 5 min and TPL was incubated at different temperatures and pH for 30 min. The activity is measured under optimal conditions (without colipase) and using TC4 as substrate. 100% activity corresponds to AS = 70 U/mg and AS = 9500 U/mg for His6-tagged N-TPL and TPL, respectively. Bars represent means ± SD. *p<0.05, **p<0.01, ***p<0.001 versus TPL.

Figure 5. Hydrolysis kinetics of tributyrin by His6-tagged N-TPL and TPL.

(A) His6-tagged N-TPL hydrolysis kinetic. (B) TPL hydrolysis kinetic. Lipolytic activity was followed at pH 8.5 and at 37°C in the presence of 0.5 mM NaTDC and 0.1 mM CaCl₂ (without colipase).

Figure 6. Hydrolysis rate of TC3 by His6-tagged N-TPL and TPL as a function of substrate concentration.

The TC3 solutions were systematically prepared in 30 mL of 0.33% GA and 0.15 M NaCl. The CMC of TC3 (12 mM) is indicated by vertical dotted line. Bars represent means ± SD. *p<0.05, **p<0.01, ***p<0.001 versus TPL.

Figure 7. Effect of E600 on the activity of His6-tagged N-TPL.

The protein was incubated with 4 mM E600, in the presence and absence of NADC (2 mM), using TC4 as substrate. Activity is measured at 37°C and pH 8.5.

Figure 8. Hydrolysis kinetic of tributyrin by His6-tagged N-TPL in the presence of colipase.

Lipolytic activity was followed at pH 8.5 and at 37°C.

Figure 9. TLC analysis of hydrolysis products of triolein by His6-tagged N-TPL.

Lane1: reaction products after 15 min incubation at 37°C of triolein in the absence of enzyme. Lane2: reaction products after 15 min incubation at 37°C of Triolein in the presence of His6-tagged N-TPL (14 µg). Reaction products were extracted with Chloroform/methanol mixture (2∶1, v/v).