Catalytic behavior of pancreatic lipase in crowded medium for hydrolysis of medium-chain and long-chain lipid: An isothermal titration calorimetry study

Accepted Manuscript Title: Catalytic behavior of pancreatic lipase in crowded medium for hydrolysis of medium-chain and long-chain lipid: an isothermal titration calorimetry study Authors: Yan Zhang, Xing-An Luo, Li-Juan Zhu, Shen-Zhi Wang, Ming-Qiang Jia, Zhong-Xiu Chen PII: DOI: Reference:

S0040-6031(18)30464-7 https://doi.org/10.1016/j.tca.2018.12.015 TCA 78171

To appear in:

Thermochimica Acta

Received date: Revised date: Accepted date:

1 July 2018 11 December 2018 15 December 2018

Please cite this article as: Zhang Y, Luo X-An, Zhu L-Juan, Wang S-Zhi, Jia MQiang, Chen Z-Xiu, Catalytic behavior of pancreatic lipase in crowded medium for hydrolysis of medium-chain and long-chain lipid: an isothermal titration calorimetry study, Thermochimica Acta (2018), https://doi.org/10.1016/j.tca.2018.12.015 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Catalytic behavior of pancreatic lipase in crowded medium for hydrolysis of medium-chain and long-chain lipid: an isothermal titration calorimetry study

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Yan Zhang, Xing-An Luo, Li-Juan Zhu, Shen-Zhi Wang, Ming-Qiang Jia, and

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Zhong-Xiu Chen*

Molecular Food Science Laboratory, College of Food & Biology Engineering,

Corresponding author (telephone +86 571 28008980; fax +86 571 28008900; e-mail

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*

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Zhejiang Gongshang University, Hangzhou, 310018, China

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[email protected]

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Graphical abstract

Highlights 

Macromolecular crowding effect on porcine pancreatic lipase activity was 1

disclosed. 

ITC was used to study the activity of lipase in hydrolyzing triglyceride.

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Modulation of the catalytic activity of lipase with crowded medium was achieved. Polysaccharide Ficoll400 can increase the activity of lipase by crowding effect.

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Polysaccharide Ficoll400 induced a more active conformation of the enzyme.

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

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Abstract

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Pancreatic lipase is an interfacial enzyme. The kinetic parameters of pancreatic

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lipase derived from heat release in crowded surroundings have not been reported. In

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this work, the effect of crowded medium on the catalytic activity of porcine pancreatic

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lipase (PPL) during lipid digestion was investigated by Isothermal Titration Calorimetry (ITC). The results showed that dietary polysaccharide Ficoll400 tended

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to increase the catalytic efficiency of PPL for both glyceryl trioleate and glyceryl trioctanoate substrates, whereas Dextran20 decreased the activity of the enzyme. The

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change in the fluorescence intensity and secondary structural content of PPL parallels the change of kcat/Km, of PPL, which was further confirmed in PEG2000. The findings

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of this research improve understanding of macromolecular crowding effects on interfacial catalysis by lipases. Moreover, it provides a useful method for research on enzyme kinetics in crowed medium by ITC.

Keywords: pancreatic lipase; macromolecular crowding; lipid; Isothermal Titration 2

Calorimetry; thermodynamics

1. Introduction Nonspecific interactions between macromolecules and the surroundings within a

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crowded medium can greatly influence the equilibrium and rates of reactions in which the macromolecules participate [1]. This “macromolecular crowding” alters the

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reaction rates depending on the nature of the reactions. For a diffusion-limited

reaction, the diffusion of substrates decreases due to the increased viscosity and reduced mobility of the reactants. However, for a transition-state-limited reaction, the

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rate is increased because crowding is expected to enhance the relative abundance of

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the transition state complex due to an increase in the effective concentration of

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substrates resulting from the excluded volume effect [2,3]. Most of the published

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work about crowding focuses on the influence of mutual volume exclusion on the

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energetics and transport properties of macromolecules such as proteins or nucleic acids [4-8]. Macromolecular crowding affects small-molecule catalytic reactions

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involving fatty acids. Hydrolysis of fatty acid anhydrides involves vesicle formation and interface catalysis. Fatty acids generated on the interfaces of vesicles display

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unusual kinetic behavior in crowded medium [9]. Pancreatic lipases are interfacial enzymes that can be activated at oil–water

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interfaces to access water insoluble substrates for hydrolysis, converting triglyceride substrates to monoglycerides and free fatty acids in human digestive system [10]. During lipid digestion, pancreatic lipases interact with other compounds, such as polysaccharides and proteins. These macromolecules create a highly crowded environment, which constitutes the native environment of the lipase. The question 3

then arises: Does the crowded medium affect fatty acid production from lipase-catalyzed lipid hydrolysis in a physiological environment? If so, can dietary polysaccharides be used to modulate the activity of lipase in a vivo-like medium? Is there any difference between different macromolecular surroundings in their influence on enzymolysis kinetics?

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For industrial bioprocessing applications, enzymes have been successfully immobilized by various means including chemically cross-linking, physical

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adsorption and entrapment to alter the conformation and enhance activity [11,12].

However, how to control the catalytic activity of pancreatic lipase during digestion in

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vivo remains an open question. The interfacial activation of lipases depends on

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hydrophobic interfaces which might be related to conformational changes of lipases

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[10]. Previous research on lipase immobilization has indicated that the hydrophobic

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microenvironment created from nanopores of mesoporous silica greatly enhances the catalytic efficiency of lipases [13]. Additionally, high concentrations of lipase itself

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resulted in an enhanced turnover rate on surface-immobilized substrates [14]. These results indicate that the underlying principle of enzyme dynamics relating to the

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activity in crowded environments is expected to be generally applicable. Several macromolecules have been shown to directly or indirectly modulate the

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activity of lipases. Pretreatment with polyethylene glycol (PEG) stabilizes the chromobacterium viscosum lipase in ultrasound-assisted water-isooctane emulsion

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[15]. Dextran networks were used as covalent coupling reactants for agarose macroporous bead immobilization of bacterial thermoalkalophilic lipases [16]. These research efforts investigated the function of the macromolecules on the activity of the bioprocessed immobilized lipase, but the mechanistic basis of the macromolecular crowding was not elucidated. So far, only one paper has mentioned that crowding can 4

change lipoprotein lipase activity [17]; however, to our knowledge, the effect of macromolecular crowding on the catalytic kinetics of pancreatic lipase in crowded environment has not been reported. The most common methods to study enzyme kinetics are spectrophotometric and fluorometric assays. Isothermal titration calorimetry (ITC) overcomes several of the

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limitations of other techniques for quantitative measurement of classical or rapid

time-scale reaction kinetics such as enzyme catalytic kinetics [18-21]. Despite the

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widely available kinetic data on the enzyme catalysis of porcine pancreatic lipase

(PPL) [22], the dynamic heat release of pancreatic lipase reaction and enzyme kinetic

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parameter in crowded medium measured by ITC have not been elucidated. By using

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ITC, we obtained the thermodynamic parameters of structural transition of vesicles

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induced by sodium cholate in a vivo-like environment [23]. We also found a

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difference in binding of long-and medium-chain fatty acids with serum albumin in macromolecular crowding [24]. These results invoked an investigation into the

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catalytic kinetics of PPL under crowded conditions by ITC. In this work, different concentrations of putatively inert macromolecules, such as

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dietary polysaccharides Dextran20 and Ficoll400, and the synthetic macromolecule polyethylene glycol (PEG), were used as crowders. We first systematically

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characterized the enhanced or inactivated activity of PPL under different crowding conditions, and further, we elucidated the influencing factors behind the crowding

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effect. Using a combination of titration calorimetry and structure studies, we uncovered how crowding caused specific changes in the enzyme structure and thus modified the catalytic properties. The results improve our understanding of the effects of macromolecules on lipase activity. Additionally, it is helpful for food formulation for controlling digestion by modulating enzyme activity using dietary 5

macromolecules.

2. Materials and Methods 2.1 Materials. Porcine pancreatic lipase (PPL, porcine pancreatin extract (P7545, 8×USP specifications activity), triglyceride (TG) including glyceryl trioctanoate (TG-C8, ⩾99%), and glyceryl trioleate (TG-C18:1, ~65%) were purchased from

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Sigma-Aldrich. Polyethylene glycol (PEG), sodium phosphate monobasic dihydrate, sodium phosphate dibasic dihydrate, and sodium hydroxide were all obtained from

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Aladdin. Dextran20 (Dex20), Ficoll400 (Fic400), sodium cholate were purchased

from Tokyo Chemical Industry. All other chemicals were of analytical-grade. All the

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experiments were performed in a PPL assay buffer composed of 0.1 mol•L-1sodium

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phosphate at pH 7.4. All enzyme and substrate solutions were prepared in this buffer

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2.2 Preparation of PPL suspension

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to minimize the heat of dilution during injection.

An accurately weighed amount of PPL was suspended in 4 mL sodium cholate

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solution and 0.1 mol•L-1 phosphate buffer (pH=7.4) and vortex-mixed for 3 min at room temperature. The suspension was then centrifuged for 10 min at 5000 rpm at

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10oC and diluted 106 times before it was withdrawn and used in the experiment. The

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lipase suspension was freshly prepared (within 15 min) in each assay in order to avoid denaturation of the lipase. 2.3 Preparation of the emulsion

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Glyceryl trioctanoate (TG-C8), and glyceryl trioleate (TG-C18:1) served as

model lipid substrates with different chain lengths. Oil-in-water emulsions were prepared using sodium cholate as an emulsifier to investigate lipase catalytic efficiency under simulated intestinal conditions. Aqueous solutions of emulsifier were prepared by dispersing the emulsifier in distilled water and stirring gently for at least 6

2 h at 25oC for complete dissolution and hydration. Emulsions were prepared by a two-step process. In the first step, 20.5 mmol•L-1 of triglyceride (TG) and aqueous emulsifier solution were mixed by using vortex oscillator for 1 min. In the second step, the emulsion was prepared using a high-shear-dispersion emulsifier for 10 min at 10000 rpm at 25oC.

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2.4 Michaelis−Menten kinetic assays based on Isothermal Titration Calorimetry (ITC)

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All kinetic experiments were conducted using an ITC (VP-ITC, MicroCal) at

37oC. A single injection assay was used to determine the enthalpy change and multiple

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injection assays was used to determine kinetic parameters.

In the single injection assay, the enzymatic reaction was initiated by injection of

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40 μL of TG-C8 (12.2 mmol•L-1) into the calorimeter cell (1.4592 mL) containing

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0.75 μmol•L-1 PPL in the reaction buffer after equilibration for 180 s. And 20 μL of

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TG-C18:1 (15 mmol•L-1) into the 35 nmol•L-1 PPL in the reaction buffer after

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equilibration for 120 s. Data points were collected at 1200-3000 s intervals ones. 𝑡=∞

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[𝑆]Total ∗ 𝑉

∫ 𝑡=0

𝑑𝑄 𝑑𝑡 𝑑𝑡

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Δ𝐻app =

[S]Total is the concentration of the limiting substrate, V is the volume of the reaction

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cell (1.4592 mL) and dQ is the heat change measured at time t. After ΔHapp was obtained, ITC experiments for enzyme kinetics reactions were

then conducted in the multiple titration injection mode, with successive injections of

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the substrate solution into an enzyme solution. These experiments involve multiple injections of a highly concentrated solution of TG (20.5 mmol•L-1) into the cell (5 μL/injection) containing the enzyme. The concentration of the substrate in the reaction mixture increases stepwise until saturation of the enzyme and maximum velocity are reached. The lipase concentration in the reaction cell is generally lower than in the 7

single injection assay, so the amount of the product produced is negligible, and the effect of product inhibition is minimized. The TG solution is injected multiple times, allowing for baseline equilibration after each injection. ITC enables the determination of enzymatic parameters of the reaction under study because the rate of reaction is proportional to the measured heat flow with ΔHapp

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as the proportionality constant. The thermal power at time t is equal to the heat rate (dQ/dt) and thereby to the change in concentration of the product over time: 𝑑𝑄 𝑑[𝑃]𝑡 = ∗ 𝑉cell ∗ Δ𝐻app 𝑑𝑡 𝑑𝑡

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Power =

The rate of reaction as a function of the kinetic parameters is then:

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𝑑[𝑃]𝑡 1 𝑑𝑄 𝑣𝑚𝑎𝑥 ∗ [𝑆]𝑡 = = 𝑑𝑡 𝑉cell ∗ Δ𝐻app 𝑑𝑡 𝐾𝑚 + [𝑆]𝑡

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𝑅𝑡 =

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In the multiple injection assays, the TG concentration was gradually increased by

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a total of 20 injections with an interval of 120-180 s; each injection containing TG-C8 or TG-C18:1 (5 μL, 20.5 mmol•L-1). The enzyme concentration in the multiple

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injection assay was 0.35 nmol•L-1. Manufacturers make approximations in correcting for displaced volume effects which occur with each injection (see descriptions in

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supplementary data). Cholate was used as colipase. To avoid the effect of cholate

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concentration on the activity of PPL in the ITC cell, we use cholate at the same concentration as the emulsifier for emulsion preparation. All experiments were performed in the buffer containing Dextran20, Ficoll400,

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PEG2000 at varying concentrations at a stirring speed of 416 rpm at 37oC. A minimum of three titrations were done for each sample. Control experiments were performed in the absence of PPL (Figure S1 in supplementary data). Kinetic data were analyzed from ITC thermogram using Origin software without subtracting the control due to negligible dilution heat. Also, the data points for the first few minutes were 8

removed to avoid the subtraction the heat of dilution from the thermogram according to literature [18]. 2.5 Steady-State fluorescence spectral measurements All steady-state fluorescence experiments were performed at 37°C using a Hitachi F-7000 fluorescence spectrophotometer with excitation and emission slits set

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at 5 nm. PPL solution (0.35 μmol•L-1) was titrated with Dextran20, Ficoll400, PEG or buffer, respectively. An excitation wavelength of 278 nm was selected to monitor the

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fluorescence of tryptophan. The emission spectra were recorded from 280 to 400 nm

with the scan rate 240 nm/min. A minimum of three scans were taken for each sample.

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Inherent fluorescence of Dextran20, Ficoll400, PEGs was also measured.

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2.6 Circular Dichroism (CD) assay

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CD measurements were made in a Jasco J-815 spectropolarimeter equipped with a Peltier-type temperature controller with six accumulations. The concentration of

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PPL used for the CD measurements was 8.5 μmol•L-1. Cells of 0.1 cm path lengths

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were used for the measurements of the far and near-UV spectra. The scan wavelength range was of 190-260 nm and the scanning speed was 200 nm/min. Three scans were

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accumulated and averaged for each spectrum after the background of diluted blank buffer was subtracted. All readings for native state structural analysis were obtained at

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37oC, and all data were analyzed using Yang's equations provided by CD Spectra Measurement.

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3. Results and discussion 3.1 Influence of dietary polysaccharides, Ficoll400 and Dextran20, on the kinetic parameters of PPL Figure 1 shows a typical calorimetric trace of one injection of TG-C18:1 or TG-C8 into the cell containing PPL. The reaction goes to completion, allowing ΔHapp 9

to be determined by integrating the total peak area in the calorimeter trace (Table 1).

Figure 2 shows the raw data for thermal power plotted against time from the experiment involving multiple injections of a highly concentrated solution of TGs (C8

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or C18:1) into PPL in the absence or presence of dietary polysaccharide (Dextran20 or Ficoll400 at varying concentrations). In the presence of Ficoll400, the fitted lines

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(Figure 2b, 2d) are located above the control line, suggesting enhancement of lipase activity by crowding. In contrast, in the presence of Dextran20 (Figure 2f, 2h), the reaction rate was lower.

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Raw data were obtained from the multiple injection assays at 37oC at a stirring speed

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of 416 rpm. The concentration of TGs in the calorimetric cell was increased by

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injections of 20×5 μL of TG-C18:1 (20.5 mmol•L-1) with 150 s spacing time or TG-C8 with 120 s spacing time between each injection. The concentration of enzyme

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was 0.35 nmol•L-1. The enzyme concentration decreases by 0.34% during each injection. This was corrected for by VP-ITC manufacturers. (The detailed descriptions

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can be found in supplementary data).

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The dependence of the reaction rate on TG concentration followed typical

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Michaelis–Menten kinetics in Figure 2, and the parameters kcat (rate constant) and Km (Michaelis constant) were obtained by fitting the curve using non-linear regression. To account for day-to-day variability, kcat/Km values for crowding were normalized to

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those obtained concurrently in buffer and the relative catalytic efficiency (RCE) was defined. As shown in Table 2, the catalytic parameters of PPL were determined in a concentrated solution of Ficoll400, a globular-shaped neutral polysaccharide recommended for mimicking intracellular macromolecular crowding. For TG-C18:1, the crowding agent reduced kcat to a small extent but significantly decreased the Km, 10

resulting in a steady enhancement in catalytic efficiency (kcat/Km), and the value of RCE (relative catalytic efficiency) is more than 1. For TG-C8, the crowder at low concentration reduced Km to a small extent but significantly decreased the kcat, resulting in decrease in kcat/Km; the value of RCE is less than 1. In a solution of Dextran20, another polysaccharide crowding agent with an elongated shape, a lower

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kcat/Km was observed. The RCEs of both TG-C18:1 and TG-C8 in Dextran20 are less

than 1. These results suggest that Ficoll400 and Dextran20 displayed different effects

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on the activity of PPL. The reduction in Km may be ascribable to an increase in the chemical activity of the small molecule substrates in highly non-ideal crowded

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solutions [25].

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Previous studies have shown that the effects of Dextran20 or Ficoll400 on the

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intrinsic catalytic efficiency (kcat/Km) of enzymes are varied. For example, Jiang and

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Guo[25] investigated the effects of crowding on the activity of enterobactin-specific isochorismate synthase and found an increase in catalytic efficiency (kcat/Km) with

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increased Dextran and Ficoll concentrations. In contrast to an enhancement of the catalytic efficiency, rate reduction can occur due to a combination of increased

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viscosity and a minor offsetting increase in enzyme activity due to crowding. For

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example, Homchaudhuri et al [26] used Ficolls and Dextrans to study the kinetics of alkaline phosphatase-catalyzed hydrolysis of p-nitrophenyl phosphate. A reduction in the rate was observed in all cases when the crowding agent was added. Pastor et al [27]

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studied the crowding effect of large macromolecules on the reaction rates of the hydrolysis of N-succinyl-L-phenyl-Ala-p-nitroanilide catalyzed by R-chymo-trypsin by adding Dextrans of various molecular weights to the reaction solutions. The decrease in maximum velocity was explained by considering the effect of product inhibition, which was enhanced in the crowded medium. In addition, several studies 11

have observed a non-linear response of enzyme activity to macromolecular crowding in vitro. Pozdnyakova et al [28] studied the effects of macromolecular crowding on the enzymatic activity of multi-copper oxidase and found that the increase in kcat outweighed the increase in Km at low concentrations of macromolecular crowding agents (within the 1–5% range), resulting in an initial increase in catalytic efficiency,

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whereas at higher crowding agent concentrations, the increase in Km was higher than the effect on kcat, and kcat/Km was lower compared to the initial ratio in buffer. Taken

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together, these results imply that the effects of crowding by the dietary

polysaccharides Ficoll400 and Dextran20 on PPL activity appear to depend on

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numerous, sometimes competing factors.

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3.2 Investigation of the factors affecting the catalytic efficiency of PPL

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The effect of polarity change of crowded medium on the catalytic efficiency of PPL To investigate the effect of polarity change induced by the crowders on the

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intrinsic catalytic efficiency of PPL, control experiments with sucrose at the same concentration as polysaccharide crowding were done by ITC. The results in Table 3

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revealed that no enhancement of kcat/Km was observed in sucrose solution, suggesting that enhanced or decreased rate of PPL is not due to crowding-induced polarity

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change [25].

The effect of structural changes of PPL induced by the crowded medium on its

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intrinsic catalytic efficiency Addition of macromolecules changes the lipase microenvironment and increases

the quantum yield and the fluorescence intensity, resulting in fluorescence sensitization [30]. The microscopic conformation of the enzyme molecule is changed by adding macromolecules so that the extent of exposure of Phe, Tyr or Trp residues, 12

which were initially surrounded by other amino acid residues, is increased [31]. To uncover the factors involved in the drastic difference in PPL kinetic parameters in the presence or absence of Dextran20 and Ficoll400 polysaccharide crowding, conformational changes of PPL were examined. As shown in Figure 3a, with increasing Ficoll400 concentration, the combined intensity increases, and a blueshift

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is observed. In contrast, the fluorescence intensity decreases along with a redshift

when the concentration of Dextran20 is increased (Figure 3b), suggesting a change in

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the microenvironment of tryptophan residues in PPL [29]. Enzymes in solution are in a constant state of motion, with a rapid balance between the various conformational

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states.

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The fluorescence intensity increases and the maximum emission wavelength is gradually blueshifted with an increase in Ficoll400 concentrations. In Ficoll400, the

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micro-environment polarity of the PPL surface is reduced, or the amino acid residues are allowed to remain in the hydrophobic core of the molecules, which is

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advantageous for maintaining the stability of the natural conformation of PPL. However, with the increase in the Dextran20 concentration, the redshift to longer

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wavelengths indicates that Trp is more exposed to the polar solvent and hence suggests that the active conformation of PPL is less stable.

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Taken together, for Ficoll400, which enhances the lipase activity, the

fluorescence intensity at 350 nm increases along with a blueshift; for Dextran20, which reduces the catalytic efficiency of the enzyme, the fluorescence intensity decreases, suggesting that a hydrophobic area is critical for enzyme activity. These phenomena were also found in research on the effect of Dextran on alcohol 13

dehydrogenase activity, in which the addition of dextran resulted in a redshift and a decrease in fluorescence intensity, suggesting a change in the tryptophan microenvironment [29]. The crowding-induced Trp fluorescence change that was observed for the allosteric ADP-glucose pyrophosphorylase was an intensity increase and a more pronounced blueshift, suggesting that crowding can induce structural

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changes in proteins in different ways. This structural change is most likely the

additional entropy pressure imposed by crowding [25].

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system’s way of reducing excluded volume effects and compensating for the

Because change in Km was usually correlated with the observed conformational

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change, we further examined possible alteration to the secondary structure of PPL

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induced by Dextran20 and Ficoll400 at the same conditions used for fluorescence measurements using a far-UV CD technique. The results are shown in Figure 4.

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For Ficoll400, the α-helix content of PPL increases from 5.7% to 26.1% with

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increasing concentrations of Ficoll400. The loop (the sum of β-turn and random

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contents) content decreases as Ficoll400 concentration is increased from 0 to 10wt%. However, for Dextran20, the α-helix content of PPL decreases from 5.7% to 0.2% as

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the concentration of Dextran20 increases, and the loop content increases instead.

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Among the secondary structural components of the enzyme, the decrease in

α-helices was associated with the reduction in PPL activity. This has also been found

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in research on the hypolipidemic mechanism of gypenosides via inhibition of pancreatic lipase [31]. Around the catalytic site of PPL, there are several α-helices that stabilize the enzyme and maintain its activity. Among the secondary structural components of the enzyme, loops represent a highly flexible conformation. Less loop content means less opportunity for the loop to move to the active center or binding 14

site, thereby reducing the possibility to cover the substrate binding sites, resulting in an increased possibility of substrate and enzyme combination, so that the enzyme activity is increased [32].

Figure 5 shows the correlation of the change in enzyme structure with

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enhancement or inhibition of catalytic efficacy. With Ficoll400 crowding, the

enhancement of fluorescence intensity and reduced loop content results in a steady

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increase in the catalytic efficiency (kcat/Km) of PPL. The decrease in fluorescence

intensity and greater loop content led to decreased catalytic efficiency of PPL (kcat/Km),

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which was the case with Dextran20.

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Kinetic parameters of PPL in PEG2000 confirmed the relationship between the

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intrinsic catalytic efficiency and the structural changes in the hydrolysis of TGs in

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crowded medium

To further investigate the universality of the correlation between structural

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change and kinetic parameters of PPL induced by crowded medium, we extended our research to include synthetic macromolecules. PEG2000 was selected because it is a

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neutral molecule, and there is no specific interaction among the molecules involved. The ITC raw data and fitting graph are shown in Figure S2 in the supplementary data.

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The kinetic parameters obtained from ITC are listed in Table 4. Addition of PEG2000 results in an increase in the kcat/Km of PPL for both TG-C18:1 and TG-C8, which are

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similar to but more pronounced than that observed in the Ficoll solution. We hypothesize that the differences in PPL activity with different PEGs are also

related to structural changes in the enzyme. As shown in Figure S3, when TG-C18:1 was used as the PPL substrate, the fluorescence intensity increased with increasing PEG concentration. Additionally, the α-helix content of PPL increased, and the loop 15

content decreased. Intrinsic fluorescence emission spectra of crowders at different concentrations in buffer (pH=7.4) at 37oC were also tested as the control (Figure S4). The change in the fluorescence intensity and secondary structural content of PPL with the substrate TG-C8 showed a similar pattern. This conformational change parallels the change of kcat/Km, which was higher with increasing PEG2000 concentrations. The

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the hydrolysis of TGs in the presence of PEG are shown in Figure 6.

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relationship between conformational changes and the intrinsic catalytic efficiency in

4. Conclusion

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In summary, the effect of crowded medium on the catalytic activity of porcine

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pancreatic lipase (PPL) was thoroughly investigated by ITC. The results showed that

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two dietary polysaccharides display opposite effects on the catalytic activity of PPL:

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Ficoll400 enhanced the catalytic efficiency (kcat/Km) of the enzyme, whereas

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Dextran20 decreased the activity regardless of the triglyceride chain length. With Ficoll400 crowding, the enhancement of fluorescence intensity and reduced loop

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content results in a steady increase in the catalytic efficiency (kcat/Km) of PPL. The decrease in fluorescence intensity and greater loop content led to decreased catalytic

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efficiency of PPL (kcat/Km), which was the case with Dextran20. The correlation between the kinetics parameter of PPL with fluorescence site and secondary structure

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change in the presence of macromolecules was further confirmed by PEG2000. Macromolecular crowding increases the intrinsic activity of an enzyme through inducing conformational and possibly other structural changes in the enzyme. All crowding-induced conformational changes appear to directly affect the active sites. Control experiments in sucrose solution excluded crowding-induced polarity change, 16

suggesting a specific macromolecular crowding effect. The results improve our understanding of the thermodynamics of macromolecular effects on lipase activity and shed light on the physiochemistry involved in the digestion process. It will also contribute to food formulation for regulating dietary fat digestion in the prevention and treatment of obesity and related disorders.

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Acknowledgment

The authors are grateful to the National Nature Science Foundation of China (NSFC,

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No.21673207), Nature Science Foundation of Zhejiang Province (Y19B030002) and the Zhejiang Provincial Top Key Discipline of Food Science and Biotechnology

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(JYTSP20141012) for financial support.

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[11] H. Zaak, E. Siar, J.F. Kornecki, L. Fernandez-Lopez, S.G. Pedrero, J.J.

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Virgen-Ortíz, R. Fernandez-Lafuente, Effect of immobilization rate and enzyme

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crowding on enzyme stability under different conditions. The case of lipase from thermomyces lanuginosus immobilized on octyl agarose beads, Process Biochem. 56

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(2017) 117-123.

[12] J.N. Talbert, L. Wang, B. Duncan, Y. Jeong, S.M. Andler, V.M. Rotello, J.M.

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Goddard, Immobilization and Stabilization of Lipase (CaLB) through Hierarchical Interfacial Assembly, Biomacromolecules, 15 (2014) 3915-3922.

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[13] J. Liu, J. Peng, S. Shen, Q. Jin, C. Li, Q. Yang, Enzyme entrapped in polymer‐ modified nanopores: The effects of macromolecular crowding and surface hydrophobicity, Chem.-Eur. J. 19 (2013) 2711-2719. [14] Z. Balevicius, D. Ignatjeva, G. Niaura, I. Ignatjev, V. Vaicikauskas, G.J. Babonas, G. Valincius, Crowding enhances lipase turnover rate on surface-immobilized substrates, Colloids Surf. B Biointerfaces. 131(2015) 115-121. 18

[15] M.M.R. Talukder, S.C.S. Shiong, Stabilization of chromobacterium viscosum lipase (CVL) against ultrasound inactivation by the pretreatment with polyethylene glycol (PEG), Appl. Biochem .Biotech. 177 ( 2015) 1742-1752. [16] S. Herranz, M. Marciello, D. Olea, M. Hernández, C. Domingo, M. Vélez, L.A. Gheber, J.M. Guisán, M.C. Moreno-Bondi, Dextran–Lipase Conjugates as Tools for

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Low Molecular Weight Ligand Immobilization in Microarray Development, Anal. Chem. 85 (2013) 7060-7068.

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Res. 58 (2017) 279-288.

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[18] S.N. Olsen, Applications of isothermal titration calorimetry to measure enzyme

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kinetics and activity in complex solutions, Thermochim Acta. 448 (2006)12-18.

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[19] K. Maximova, J. Trylska, Kinetics of trypsin-catalyzed hydrolysis determined by isothermal titration calorimetry, Anal. biochem. 486(2015) 24-34.

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[20] N. Rohatgi, S. Guðmundsson, Ó. Rolfsson, Kinetic analysis of gluconate phosphorylation by human gluconokinase using isothermal titration

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calorimetry, FEBS letters. 589 (2015) 3548-3555. [21] Q. Luo, D.Chen, R. M. Boom, A. E. Janssen, Revisiting the enzymatic kinetics of

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pepsin using isothermal titration calorimetry. Food Chemistry, 268 (2018) 94-100. [22] Z.D. Knezevic, S.S. Siler-Marinkovic, L.V. Mojovic, Kinetics of lipase-catalyzed

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hydrolysis of palm oil in lecithin/izooctane reversed micelles, Appl. Microbial .biot., 49 (1998) 267-271. [23] J.N. Tian, B.Q. Ge, Y.F. Shen, Y.X. He, Z.X. Chen, Thermodynamics and structural evolution during a reversible vesicle-micelle transition of a vitamin-derived bolaamphiphile induced by sodium cholate, J. Agric. Food Chem. 64 (2016) 19

1977-1988. [24] T.T. Zhu, Y. Zhang, X.A. Luo, S.Z. Wang, M.Q. Jia, Z. X. Chen,

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Binding of Long- and Medium-Chain Fatty Acids with Serum Albumin: The Role of Macromolecular Crowding Effect, J. Agric. Food Chem. 66 (2018) 1242-1250. [25] M. Jiang, Z. Guo, Effects of macromolecular crowding on the intrinsic catalytic

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efficiency and structure of enterobactin-specific isochorismate synthase, J. Am. Chem. Soc. 129 (2007) 730-731.

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[26] L. Homchaudhuri, N. Sarma, R. Swaminathan, Effect of crowding by dextrans and Ficolls on the rate of alkaline phosphatase–catalyzed hydrolysis: A size‐

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dependent investigation, Biopolymers. 83 (2006) 477-486.

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[27] I. Pastor, E. Vilaseca, S. Madurga, J. L. Garcés, M. Cascante, F. Mas, Effect of Crowding by Dextrans on the Hydrolysis of N-Succinyl-l-phenyl-Ala-p-nitroanilide

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Catalyzed by α-Chymotrypsin, J. Phys. Chem. B.115 (2010) 1115-1121. [28] I. Pozdnyakova, P. Wittung-Stafshede, Non-linear effects of macromolecular

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crowding on enzymatic activity of multi-copper oxidase, BBA-Proteins and Proteom., 1804 (2010) 740-744.

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[29] J. Skjold-Jørgensen, J. Vind, A. Svendsen, M. J. Bjerrum, Altering the activation mechanism in Thermomyces lanuginosus lipase, Biochemistry. 53(2014) 4152-4160.

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[30] A.E. Wilcox, M.A. LoConte, K.M. Slade, Effects of Macromolecular Crowding on Alcohol Dehydrogenase Activity Are Substrate-Dependent, Biochemistry.

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55(2016)3550-3558. [30] J.D. Schrag, Y.Li, Ser-His-Glu triad forms the catalytic site of the lipase from Geotrichum candidum, Nature. 351 (1991) 761. [32] Jr,J.P. Hennessey, Jr,W.C. Johnson, Information content in the circular dichroism of proteins, Biochemistry. 20 (1981) 1085-1094. 20

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Figure 1. PPL-catalyzed hydrolysis of triglyceride (TG) in the absence or presence of crowded medium from a single injection assay performed on VP-ITC. (P: Thermal power) (a) TG-C18:1 (15 mmol•L-1) (b) TG-C8 (12.2 mmol•L-1) Assays containing PPL (35 nmol•L-1 for TG-C18:1 and 0.75 μmol•L-1for TG-C8), 40 mmol•L-1 sodium

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cholate and phosphate buffer (0.1 mol•L-1, pH=7.4). All titrations were performed at a

A

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A

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stirring speed of 416 rpm. Integration baselines are shown as dash lines.

22

IP T SC R U N A M ED PT CC E A Figure 2. PPL-catalyzed hydrolysis of TG from the multiple injection assays performed on VP-ITC (P: thermal power). (a), (c): hydrolysis of TG-C18:1 or TG-C8 in the presence of Ficoll400 at varying concentration ( 0wt%, black line; 0.1wt%, red 23

line; 0.5wt%, cyan line; 1wt%, olive line; 5wt%, blue line, 10wt%, green line). (e), (g): hydrolysis of TG-C18:1 or TG-C8 in the presence of Dextran20 at varying concentration (0wt%, black line; 0.1wt%, red line; 0.5wt%, cyan line; 1wt%, olive line). (b), (d), (f), (h): the steady state heat rates after each injection transformed into

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reaction rates and plotted against the concentrations of TG.

24

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Figure 3. Effect of Ficoll400 (a) and Dextran20 (b) on the fluorescence emission

spectra of PPL. Assays containing PPL (0.35 μmol•L-1) and 0.1 mol•L-1 of phosphate buffer (pH=7.4) were performed at 37oC in the presence of varying concentrations of Dextran20 or Ficoll400. The red arrow indicates that the fluorescence intensity

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increases or decreases as the concentration increases of Dextran20 and Ficoll400.

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Figure 4. CD spectra of PPL in the presence of (a) Ficoll400 and (b) Dextran20 at varying concentrations, and changes of α-helices and β-sheets content (c), and loop content (d) in the presence of polysaccharides. Assays containing PPL (8.5 μmol•L-1)

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and 0.1 mol•L-1 phosphate buffer (pH=7.4) were performed at 37oC in the presence of Dextran 20 and Ficoll400. Loop is the sum of β-turn and Random contents of the

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secondary structure of the enzyme protein.

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Figure 5. The relationship between conformational changes and the intrinsic catalytic efficiency in the hydrolysis of TGs in the presence of (a) and (c) Ficoll400, (b) and (d) Dextran20. RCE# , the relative catalytic efficiency of PPL, which was defined as the

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ratio of the kcat/Km in crowding to that in buffer only. F#, the ratio of fluorescence intensity in the presence of dietary polysaccharides to that in buffer. Loop#, the ratio

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of loop content in the presence of dietary polysaccharides to that in buffer.

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Figure 6. The relationship between conformational changes and the intrinsic catalytic

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efficiency in the hydrolysis of TGs in the presence of PEG2000. RCE#, the relative

catalytic efficiency of PPL, was defined as the ratio of the kcat/Km in crowding to that in buffer only. F# is the ratio of fluorescence intensity in the presence of PEGs to that in buffer only. Loop# is the ratio of loop content in the presence of PEGs to that in

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buffer.

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Table 1. ΔHapp (kJ•mol-1) of PPL titrated with glyceryl trioleate (TG-C18:1) or glyceryl trioctanoate (TG-C8) in various concentrations of Ficoll400 and Dextran20. (Uncertainties are given as standard deviations of 3 determinations.) 0

0.1

0.5

1.0

5.0

ΔHapp (kJ•mol-1) -6.82±0.13

-5.36±0.10

-7.74±0.26

-9.19±0.08

-10.20±0.09

TG-C8

-1.11±0.02

-1.73±0.06

-1.93±0.17

-1.97±0.04

-2.11±0.16

TG-C18:1

-6.82±0.13

-4.46±0.07

-4.77±0.05

-7.07±0.03

TG-C8

-1.11±0.02

-2.55±0.16

-2.26±0.03

-2.15±0.12

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Dextran20

TG-C18:1

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-10.21±0.13 -2.20±0.18

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Ficoll400

10.0

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Concentration (wt%)

Table 2. Kinetic parameters of PPL for glyceryl trioleate (TG-C18:1) and glyceryl trioctanoate (TG-C8) in Ficoll400 and in Dextran20 crowding. (Uncertainties are given as standard deviations of 3 determinations.) TG-C18:1

TG-C8 kcat (s-1)

Km

kcat/ Km

RCE#

kcat (s-1)

(mmol•L-1)

-

824±86

1.39±0.30

593±56

1.00±0.06

605±106

3.98±0.02

152±59

1.00±0.05

0.1wt%

617±206

0.61±0.07

1012±213

1.71±0.25*

381±188

2.90±0.87

131±48

0.86±0.06*

0.5wt%

290±93

0.45±0.15

645±84

1.09±0.12*

120±52

1.53±0.59

78±32

0.51±0.17*

1.0wt%

209±63

0.30±0.17

697±56

1.18±0.15*

166±53

0.59±0.28

279±45

1.82±0.01*

5.0wt%

437±91

0.56±0.06

780±78

1.31±0.08*

498±116

1.84±0.74

270±53

1.78±0.04*

10.0wt%

491±105

0.73±0.13

670±95

1.13±0.15*

344±36

1.06±0.25

324±56

2.13±0.14*

0.1wt%

403±29

0.89±0.10

453±30

0.77±0.04*

7.10±1.64

107±32

0.70±0.02*

0.5wt%

516±64

1.17±0.12

440±56

0.74±0.02*

409±32

3.22±0.39

127±32

0.84±0.03*

1.0wt%

764±66

2.04±0.42

374±66

331±8

3.05±0.14

108±9

0.71±0.01*

#

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763±288

0.63±0.05*

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Dextran20

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buffer

Ficoll400

RCE#

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Diluted

kcat/ Km

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(mmol•L-1)

Km

Relative catalytic efficiency (RCE) was defined as the ratio of kcat/Km in crowding to

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that in diluted buffer. “±” represent standard deviations (n = 3). *indicate a significant difference between diluted buffer and macromolecular

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crowding at various concentrations, *P<0.05.

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Table 3. Kinetic parameters of PPL for glyceryl trioleate (TG-C18:1) and glyceryl trioctanoate (TG-C8) in sucrose solution. TG-C18:1 kcat (s-1)

TG-C8 Km

kcat/ Km

RCE*

kcat (s-1)

(mmol•L-1) Diluted

Km

kcat/ Km

RCE*

(mmol•L-1)

-

824±86

1.39±0.30

593±56

1.00±0.06

605±106

3.98±0.02

152±59

1.00±0.08

0.1 wt%

703±102

1.19±0.12

591±99

1.00±0.05

117±37

0.74±0.04

158±37

1.04±0.08

0.5 wt%

612±93

1.03±0.09

594±85

1.00±0.13

250±56

1.63±0.06

1.0 wt%

320±54

0.55±0.03

581±66

0.99±0.25

105±25

0.70±0.13

5.0 wt%

419±56

0.71±0.07

590±54

1.00±0.12

119±16

0.80±0.12

10.0 wt%

264±23

0.45±0.05

587±20

1.00±0.19

650±50

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“±” represents standard deviations (n = 3).

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153±55

1.00±0.12

150±45

0.99±0.16

149±30

0.98±0.06

152±42

1.00±0.08

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Sucrose

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buffer

4.29±0.51

Table 4. Kinetic parameters of PPL for glyceryl trioleate (TG-C18:1) and glyceryl trioctanoate (TG-C8) in PEG2000 at varying concentrations. TG-C18:1

TG-C8 kcat (s-1)

Km(mmol •L-1)

kcat/ Km

RCE#

kcat(s-1)

Km(mmol• L-1)

kcat/ Km

RCE#

824±86

1.39±0.30

593±56

1.00±0.06

605±106

3.98±0.02

152±59

1.00±0.05

0.1wt%

649±47

0.80±0.01

812±54

1.37±0.15*

703±268

1.93±0.83

364±99

2.40±0.21*

0.5wt%

548±22

0.79±0.15

694±32

1.17±0.11*

393±56

1.23±0.15

320±45

2.11±0.36*

5.0wt%

618±9

0.83±0.21

747±15

1.26±0.17*

1092±77

2.54±0.18

430±62

2.83±0.30*

10.0wt%

496±65

0.68±0.18

729±52

1.23±0.20*

519±35

Diluted

#

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PEG2000

IP T

buffer

1.93±0.42

269±55

Relative catalytic efficiency (RCE) was defined as the ratio of kcat/Km in crowding to

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that in diluted buffer. “±” represent standard deviations (n = 3).

* indicate a significant difference between diluted buffer and macromolecular

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crowding at various concentrations: ** P<0.01 very significantly; 0.01<*P<0.05

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significant difference.

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1.77±0.04**