Ultrafast Screening of a Novel, Moderately Hydrophilic Angiotensin

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Ultra-fast Screening of a Novel Moderate Hydrophilic Angiotensin Converting Enzyme Inhibitory Peptide RYL from Silkworm Pupa Using Fe-doped Silkworm Excrement Derived Biocarbon: Waste Conversion by Waste Long Liu, Yanan Wei, Qing Chang, Huaju Sun, Kungang Chai, Zuqiang Huang, Zhenxia Zhao, and Zhongxing Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04442 • Publication Date (Web): 07 Dec 2017 Downloaded from http://pubs.acs.org on December 12, 2017

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Journal of Agricultural and Food Chemistry

Ultra-fast Screening of a Novel Moderate Hydrophilic Angiotensin Converting Enzyme Inhibitory Peptide RYL from Silkworm Pupa Using Fe-doped Silkworm Excrement Derived Biocarbon: Waste Conversion by Waste Long Liu, Yanan Wei, Qing Chang, Huaju Sun, Kungang Chai, Zuqiang Huang, Zhenxia Zhao, and Zhongxing Zhao*1 Guangxi Colleges and Universities Key Laboratory of New Technology and Application in Resource Chemical Engineering, School of Chemistry and Chemical Engineering, Guangxi University, Nanning 530004, China

1*

Corresponding Authors Phone +86-771-3233718; fax +86-771-3233718; e-mail [email protected] (Zhongxing Zhao) 1

ACS Paragon Plus Environment


1

ABSTRACT: A novel moderate hydrophilic peptide (RYL) with high ACE inhibitory

2

activity was ultra-fast screened via a concept of waste conversion using waste. This

3

novel peptide was screened from silkworm pupa using Fe-doped porous biocarbon

4

(FL/Z-SE) derived from silkworm excrement. FL/Z-SE possessed magnetic property

5

and specific selection for peptides due to Fe’s dual functions. The selected RYL with

6

moderate hydrophilicity (LogP =-0.22) exhibited a comparatively high ACE

7

inhibitory activity (IC50=3.31±0.11 µM). Inhibitory kinetics and docking simulation

8

results show that, as a competitive ACE inhibitor, RYL formed 5 hydrogen bonds with

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ACE residues in S1 and S2 pockets. In this work, both of screening carbon material

10

and selected ACE inhibitory peptide were derived from agricultural wasters (silkworm

11

excrement and pupa), and it offers a new way of thinking for development of

12

advanced utilization of the silkworm by-product/waster.

13 14 15

KEYWORDS: silkworm pupa protein, silkworm excrement, moderate hydrophilic

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peptide, ACE inhibitory activity, ultra-fast screening, molecular docking

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2


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■ Introduction

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Hypertension has been one of the major cardiovascular diseases that seriously affects

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people’s health.1 One of the most commonly used anti-hypertensive therapies is an

21

inhibitor of angiotensin-converting enzyme (ACE). It can effectively control blood

22

vessels and decrease blood pressure.2 Currently, purification ACE inhibitor peptides

23

with high bioactivity from natural sources are attracting more attention than the

24

development of synthetic drugs due to their fewer side effects.3

25

Many researches have shown that many selected peptides having high ACE

26

inhibitory activity from new natural sources are known as be composed entirely or

27

partly of more hydrophobic amino acids. For instance, VVLTK and FQPS were

28

separated from plant proteins with IC50 values of 53.394 and 27.0 µM,5 respectively.

29

PAFG, MPFLKSPIVPF and AHLL purified from marine sources had the IC50 value of

30

35.9,6 1.717 and 13.5 µM.8 MPFLKSPIVPF derived from a food source possessed the

31

IC50 value of 27.0 µM.9 Many selected peptides having good ACE inhibitory function

32

exhibit hydrophobic property to our knowledge. However, hydrophobic peptides

33

usually have low solubility and high aggregation in water solution. It will strongly

34

decrease their physiological functions,10-12 drug absorption and bioavailability,13 and

35

the reaction efficiency of chemical catalysis.14, 15 Thus, exploration of some moderate

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hydrophilic peptides with high ACE inhibition activity has attracted increasing

37

scientific attention.

38

Adsorption and separation strategy using porous materials, including affinity

39

separation, magnetic separation and porous adsorption, facilitates rapid screening

40

peptides with specific activity from protein hydrolysates.16,17 Porous materials, as the

41

core of adsorption approach, can be efficiently captured peptides with specific

42

property by their surface and pore physical properties. Jiang and co-workers18 3



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prepared Fe3O4@SiO2@graphene microspheres to enrich bioactive peptides at low

44

concentrations with high efficiency. Hippauf et al.19 employed microporous activated

45

carbons to isolate ACE inhibitory peptides from lactalbumin hydrolysates, and

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obtained a sixfold-concentrated the selected ACE-inhibitors. So far, these researches

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are mainly focused on selection of peptides through molecular sieving effects.

48

However, few reports have been published on peptide selective adsorption based on

49

surface property of adsorbents.

50

Silkworm industry is very developed in China's western region and other East

51

Asian countries. Herein, we proposed to synthesize a Fe-bifunctional porous

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biocarbon using a silkworm waste (silkworm excrement, SE) to high-throughput

53

screen peptide for ACE inhibition from another silkworm waste (silkworm pupa

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protein, SP). We used “waste” protein source via “waste” material to acquire a novel

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peptide with high ACE inhibitory activity. The high surface biocarbon was further

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modified by Fe elements doping (FL/Z-SE), which enables sample to have surface

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acidity and magnetic dual functions. The unique surface makes us realize a rapid

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capture and separation of moderate hydrophilic peptides from a complex mixed

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hydrolysis process. The novel ACE inhibitory peptide (RYL, LogP =-0.22) was

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quickly isolated and identified by using FL/Z-SE separation and matrix-assisted laser

61

desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-TOF),

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respectively. Its ACE inhibition pattern and ACE inhibition mechanism of RYL were

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then systematically studied by the classic Lineweaver-Burk model and molecular

64

docking. Herein, both of separation carbon material (Fe-C) and selected ACE

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inhibitory peptides were derived from silkworm wasters (silkworm excrement and

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pupa). It was then a promising alternative for replacement of the current high-cost and

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tedious peptides screening process and purification. Besides, it offers a new way of 4


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thinking for development of advanced utilization of the silkworm by-product/waster.

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■ Materials and Methods

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Reagents. Silkworm excrement purchased from Yi Zhou farmer (China). Neutral

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protease (AS1.398, 60 U/mg) was provided by Pangbo Biological Engineering Co.,

72

Ltd. (Nanning, China). ACE from rabbit lung, hippuryl-L-histidyl-L-leucine (HHL),

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bovine serum albumin, ovalbumin, cyyochrome, insulin and vitamin B12 were offered

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from Sigma-Aldrich Chemical Co., Ltd. (St. Louis, MO). Pepsin from porcine gastric

75

mucosa was supplied by Coolaber Science & Technology Co., Ltd. (Beijing, China),

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and pancreatin from porcine pancreas was offered by Shanghai Yuanye

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Bio-Technology Co., Ltd. (Shanghai, China). Methanol and acetonitrile for HPLC

78

analysis were supplied by Thermo Fisher Scientific Co., Ltd. FeCl2·4H2O (AR, 99.0%)

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and ZnCl2 (AR, 99.0%) were supplied by Aladdin Industrial Co. Ltd. (Shanghai,

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China). The ultrapure water purified via the Smart-S15UV (18.2 MegaOhm-cm,

81

Hitech Instruments Co., Ltd, Shanghai, China) was used throughout the experiments.

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All starting materials were commercially available reagents of analytical grade and

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used without further purification.

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Synthesis of Z-SE, FL/Z-SE, and FH/Z-SE Materials. Silkworm excrement (SE) was

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first washed with deionized water, filtered and dried at 110 °C overnight. Then, the SE

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was crushed to uniform particles in the range of 1-2 mm. 2.00 g of SE sample was

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first immersed a certain weight ratio of ZnCl2/FeCl2 solution for 8 h, followed by

88

freeze-drying process to contain the original three-dimensional morphology. After that,

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the frozen SE was heated to 900 °C at a rate of 5 °C/min and maintained at this

90

temperature for 2 hours under N2 atmosphere. Finally, activated samples were washed

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with 1.0 M HCl and 10% HF to remove the unconjugated iron, ZnO and SiO2. The

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corresponding FL/Z-SE and FH/Z-SE were designated as adjusting weight ratio of 5



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FeCl2/ZnCl2 equal to 0.5/1.0 and 1.0/1.0, respectively. For comparison, the porous

94

biocarbon (Z-SE) without loading irons was also prepared in this work. The synthesis

95

procedure of Z-SE was the same as that of FH/Z-SE expect for not adding FeCl2

96

during the activation process.

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Physical Characterization. The morphologies of the synthesized materials were

98

surveyed by a scanning electron microscope and a field-emission scanning electron

99

microscope equipped with an energy dispersive X-ray spectrometer (SEM-EDX,

100

Hitachi S-3400N). Powder X-ray diffraction (XRD) measurements were performed on

101

a X-ray spectrometer (Rigaku) with Cu Ka radiation (λ=1.5406 Å). Specific surface

102

area and pore size distribution were calculated on the basis of nitrogen physical

103

adsorption with a Micromeritics ASAP 2460. Zeta potential and dynamic light

104

scattering (DLS) were measured using a MALVERN Zetasizer Nano ZS90

105

(Brookhaven NanoBrook Omni). The magnetic properties were analyzed on a

106

Physical Property Measurement System PPMS-9 (Quantum Design).

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Assay of ACE Inhibition. ACE inhibitory activity assay was performed by

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measuring the concentration of hippuric acid liberated from hippuryl-histidyl-leucine

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(HHL) according to the method described by Cushman and Cheung20 with slight

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modifications. HHL was first dissolved in a 100 mM borate buffer (pH 8.3)

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containing 300 mM NaCl. Rabbit lung ACE was dissolved in the same buffer solution

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at a concentration of 10 mU·mL-1. After that, 40 µL of ACE solution and a certain

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concentration of peptide or their mixture solution (200 µL) were pre-incubated at

114

37 °C for 10 min and the mixture was subsequently incubated with 10 µL of HHL

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solution for another 30 min at the same temperature. The reaction was then terminated

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by adding 50 µL of 1.0 M HCl. Finally, 20 µL of the solution injected directly onto a

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Zorbax SB C18 column (4.6 mm×150 mm, particle size 5 µm; Agilent) to detect the 6


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product, hippuric acid (HA) from HHL. The column was eluted with 15% methanol

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(in water, v/v) containing 0.1% trifluoroacetic acid (TFA) with flow rate of 1.0

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mL/min and absorbance of the eluate measured at 228 nm. ACE Inhibition activity (I,

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percent) is calculated by using the following equation:

=

− −

(1)

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where Ae is the relative area of HA peak generated without ACE inhibitors, Af is the

123

relative area of HA peak generated in the presence of purified peptides, and Ab is the

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relative area of HA peak generated without ACE and purified peptides. The IC50 value

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defined as the concentration of inhibitor (millimolar) required to inhibit 50% of ACE

126

activity, which was determined by regression analysis of ACE inhibition versus

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peptide concentration.

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Purification of ACE Inhibitory Peptides. The preparation of the SPP hydrolysate

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with neutral protease was already described elsewhere.21,22 The lyophilized SPP

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hydrolysate was dissolved in ultrapure water, and 10 mg of Z-SE, FL/Z-SE and

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FH/Z-SE was mixed with 1 mg/mL SPP hydrolysate at a ratio of 1:1 (w/v) and stirred

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at 30 °C for 15 min. Then, the FL/Z-SE was recovered through a magnet and washed

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three times with ultrapure water. After that, the adsorbed peptides were desorbed from

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the adsorbed FL/Z-SE by using absolute ethanol.

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The eluent with highest inhibitory activity was concentrated and freeze-dried and

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then was further separated on a Zorbax SB C18 column by RP-HPLC (Agilent 1260).

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The column was eluted by a linear gradient of acetonitrile (5-25%) containing 0.1%

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TFA at a flow rate of 0.50 mL/min within 60 min. The absorbance of the elution was

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monitored at 220 nm with diode array detector (DAD). The fractions collected from

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HPLC were lyophilized for further assay of ACE inhibitory activity. Subsequently, the

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lyophilized fraction with the highest ACE-inhibitory activity was purified by using the 7



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second step HPLC, eluting with 8% acetonitrile in water (v/v) containing 0.1% TFA at

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a flow rate of 0.50 mL/min. These fractions were collected and lyophilized to powder

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for further measurement of their ACE inhibitory activities and sequence identification.

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Characterization of Purified Peptide. Accurate relative molecular mass and amino

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acid sequence of the purified peptide (peptide A) with the highest ACE inhibitory

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activity were determined on a 4800 plus MALDI-TOF/TOF analyzer (Applied

148

Biosystems, Beverly, MA). Spectra were acquired on a matrix-assisted laser

149

desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer with a 337 nm

150

pulsed nitrogen laser (2 ns pulse duration, 3 Hz repetition rate). The purified peptides

151

mass spectra were acquired in linear positive ion mode at a mass range of m/z 50 to

152

500.

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Kinetics of ACE Inhibition. To determine the ACE inhibitory mechanism of

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inhibitory peptide A, kinetics of ACE inhibition was established by varying the

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concentration of the enzyme substrate HHL (1.044, 1.392, 1.740, and 2.088 mM) in

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the absence and presence of two different concentrations of inhibitory peptide (25.31

157

µM and 50.27 µM).23, 24 The kinetics of ACE in the presence of the inhibitory peptide

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was determined using Lineweaver-Burk plots, where the reciprocal of HHL

159

concentration is used as an independent variable (x-axis) and the reciprocal of

160

production rate of HA as a dependent variable (y-axis). The inhibitory constants ( )

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was determined as the intercept of the Lineweaver-Burk lines.

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Molecular Docking. The structure of peptide A was constructed using Sybyl

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X-2.1.1 (Tripos International, St. Louis, MO), and the structure was energy minimized

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using the Powell conjugate gradient optimization algorithm with the Tripos force

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field.25 The crystal structure of human ACE-lisinopril (1O86) complex was obtained

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from the Protein Data Bank (http://www.rcsb.org/pdb/home/home.do). Before the 8


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docking, water molecules and the inhibitor lisinopril were removed, whereas the

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cofactor zinc atom was retained in ACE model. Then the protein structure was

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preanalyzed and prepared for the docking runs using the biopolymer structure

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preparation tool with default settings and the protomol was created by automatic. The

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Surflex-Dock program was used for docking studies. During the docking process, the

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parameter of additional starting conformation per molecule is 5, considering ring

173

flexibility, and default settings for the rest. The binding affinity of the ligand is

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predicted by the software in terms of total score, which is expressed as LogKd, where

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Kd is the binding constant. A high value of total score indicates good protein-ligand

176

binding.

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Statistical Analysis. All assays of ACE inhibitory activity were conducted in

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triplicate. Data were presented as mean ± standard deviation. Statistical analysis was

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performed in MS Excel (Microsoft Windows 2003) by use of Student’s t-test.

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Significant difference in means between the samples were determined at a 5%

181

confidence level (p < 0.05).

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■ Results and Discussion

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Physical Characteristics. Figure 1 shows the SEM images and elemental

184

compositions of SE, Z-SE, FL/Z-SE, and FH/Z-SE. The original silkworm excrement

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(SE) had a very smooth surface with regular wrinkle structure (Figure 1A). After the

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SE was activated by ZnCl2 at 900 °C, its morphology of surface wrinkles disappeared

187

on Z-SE, and many holes with various sizes appeared on the sample surface (Figure

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1B). After being treated with FeCl2 and ZnCl2 at 900 °C, a honeycomb-like surface

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with large numbers of tiny holes appeared over the entire samples (FL/Z-SE and

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FH/Z-SE, Figure 1C-D). From element analysis in Figure 1, the original SE was

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mainly composed of carbon, nitrogen and oxygen elements, similar to other natural 9



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biomass materials. During activated process, large amount of oxygen groups was

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removed from original SE substrate, resulting in C element enrichment in three

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activated samples. Oxygen content presents an obvious decrease after being activated

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by ZnCl2 as activator. The ZnCl2 and FeCl2 activation process can generate Fe3C

196

bonds on the carbon surfaces, and the content of Fe element in FH/Z-SE apparently

197

more than that in FL/Z-SE accompany with the increase of adding FeCl2 amount.

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Figure 2 shows the PXRD patterns of the original SE, Z-SE, FL/Z-SE and FH/Z-SE

199

samples. Four sharp diffraction peaks at 15.23/24.65º and 26.79/29.32º appeared in

200

the original SE sample, which represented the species of CaCO3 and SiO2 in SE

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sample. These species might derive from silkworms’ food. Thus, we used water and

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hydrofluoric acid (HF) to remove these useless impurities afterwards. After being

203

treated with Fe/Zn chlorides, some new and sharp peaks appeared in the patterns of

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FL/Z-SE and FH/Z-SE samples. It can be attributed to characteristic diffraction peaks

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of iron carbide (Fe3C/Fe5C2) and α-Fe26 respectively, indicating the formation of Fe-C

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structure. Besides, there were two broad peaks locating at 23º and 43º in three

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obtained samples, which corresponded to the (002) and (100) crystal planes of a

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typical graphitic structure.27

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Nitrogen adsorption/desorption isotherms and the physical properties of Z-SE,

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FL/Z-SE and FH/Z-SE are displayed in Figure 3 and Table 1, respectively. As shown,

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all of the Z-SE, FL/Z-SE and FH/Z-SE showed a type IV isotherm with hysteresis

212

loop (H4) in Figure 3A. It indicated a typical hierarchical structure with micropores

213

and mesopores. Z-SE showed a remarkable hysteresis loop from the N2 isotherm as

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evidence of more mesopores. Its BET surface areas and total volume were

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respectively calculated to be 1415.5 m2/g and 1.03 cm3/g. Its micropore area and

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micropore volume were only 350.8 m2/g and 0.14 cm3/g. After adding FeCl2 specie 10


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for activation, the surface area was decreased to a variable extent in FL/Z-SE and

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FH/Z-SE. It was probable that these species tend to aggregate into large particles and

219

led to a possibility of pore blocking resulting in reduced surface area. Clearly, low

220

content of FeCl2 did not much affect the surface area. However, the surface area

221

suffered a sharp decline when the ratio of Fe/Zn increasing to 1:1 in FH/Z-SE. Figure

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3B shows the pore size distribution of these three samples. All of the samples

223

displayed very similar profile of pore size distribution. According to the DFT

224

calculation, the pore sizes were mainly concentrated at 5.0, 6.5-7.8, 11.6 and 27.4 Å,

225

possessing micropores and mesopores. From Table 1, although the pore size

226

distribution was almost similar in these three samples, both of the micropore surface

227

area and pore volume were significantly increased with adding amount of FeCl2.

228

These phenomena were possibly ascribed to the two functions of Fe species during

229

carbonization process. Ferrous chloride could react with microcrystalline carbon on

230

the edge, and generate the micropore structure in iron-containing SE samples.

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Moreover, Fe species were reduced to Fe3C and Fe5C2 and 0-valent iron in the process

232

of high-temperature carbonization. Another possibility might be that these new

233

micropores came from the partly-blocked mesopores by reduced Fe species.

234

Figure 4 shows the zeta potential and particle size of Z-SE, FL/Z-SE and FH/Z-SE.

235

The surface charges (ζ potential) of the Z-SE, FL/Z-SE and FH/Z-SE samples were

236

-24.66, -6.77 and 5.93 mV in water solution, respectively. The surface charge shows

237

an apparent rise with increasing Fe species in the sample. This indicated that iron

238

species in FH/Z-SE and FL/Z-SE enhanced the surface positive charge density.

239

Subsequently, the particle size and aqueous stability of Z-SE, FH/Z-SE and FL/Z-SE

240

were further tested, and the result data are also shown in Figure 4. As seen, FH/Z-SE

241

and FL/Z-SE (~550 nm) had larger particle size than Z-SE (490 nm). Their particle 11



242

sizes did not show the notable difference among these three samples. However, their

243

soluble stability in water was significantly different. Z-SE suspension had almost

244

precipitated off and the water solution with Z-SE sample became clear quickly. On the

245

contrary, iron-containing Z-SE particles (FH/Z-SE and FL/Z-SE) were found to be

246

more stable under similar condition. Considering the similar particle size of these

247

samples, FH/Z-SE and FL/Z-SE exhibited much higher compatibility with water than

248

Z-SE sample (inset photographs of Figure 4), which may be ascribed to their

249

enhanced hydrophilic property of carbon surface. The decoration of Fe specie

250

increased ‘Lewis acidity’ sites of carbon surface,28 and thus increased surface local

251

polarity to a certain extent compared to Z-SE. Therefore, the Fe-containing Z-SE

252

sample possesses two main adsorption sites: (1) hydrophobic (non-polar) sites on the

253

graphitic carbon surface and (2) acid/hydrophilic (polar) metal sites. These active

254

adsorption sites will tend to adsorb some moderate hydrophilic peptides containing

255

basic and phenyl groups.29

256

The magnetic properties of FL/Z-SE and FH/Z-SE were determined by a vibrating

257

sample magnetometer (VSM) and the results are shown in Figure 5. Both of these

258

samples could not be observed obvious remanence and coercivity, signifying a

259

superpara-magnetism property of these Fe-C materials. Saturation magnetization

260

values of FL/Z-SE and FH/Z-SE were 21.49 and 35.64 emu g/L at room temperature,

261

respectively. Apparently, FH/Z-SE exhibited a much higher saturation magnetization

262

than FL/Z-SE mainly due to the higher content of Fe in carbon materials. As shown in

263

inset photographs of Figure 5, with an external magnet, the homogeneous dispersion

264

of FH/Z-SE (sample 1) and FL/Z-SE (sample 2) microparticles could be separated

265

quickly from the solution and formed aggregates in only 10 s. The aggregate was

266

easily redispersed into the solution quickly after removing the magnet. Thus, similar 12


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to magnetic Fe3O4 nanoparticles, Fe-C (Fe3C/Fe5C2) carbons also possessed excellent

268

magnetic responsivity and redispersibility, which is beneficial for peptides fast

269

separation and screening.

270

Rapid Screening of ACE Inhibitory Peptides with Fe-C Porous Carbon. The

271

influence of adsorption time of three synthesized carbon samples on their fractional

272

residual amount of peptides was shown in Figure 6A. The fractional residual amount

273

of peptides in three solutions with different carbon adsorbents remained nearly

274

constant after 10 min. That means all peptides showed very fast adsorption kinetics,

275

which enabled samples to achieve equilibrium within very short time (10 min). The

276

fast equilibrium may be attributed to two reasons: (1) the high adsorption affinity

277

between carbon surface and the adsorbed peptides with similar property,30 and (2)

278

hierarchical pore size distribution with small diffusion resistance for peptides.29

279

Besides, Fe-doped samples (FL/Z-SE and FH/Z-SE) had slightly lower fractional

280

adsorbed peptides in comparison to Z-SE samples, and their adsorbed amount showed

281

a similar trend with sample’s specific surface area (Table 1). Selective adsorption of

282

peptides with bioactivity or specific function is considered as a more crucial factor

283

except for the sample’s adsorption capacity. ACE inhibitory activity and relative

284

adsorbed amount within 10 min of Z-SE, FL/Z-SE and FH/Z-SE were shown in

285

Figure 6B. Surprisingly, the FL/Z-SE sample with lower Fe content exhibited the

286

highest ACE inhibitory activity, whose value increased to 2-fold of the original SPP

287

hydrolysate under similar tested conditions. In this regard, pore structure and surface

288

property are critical to determine to screen peptides with high bioactivity.

289

Pore size distribution and surface functional groups were also investigated to

290

understand the selection strategy of the FL/Z-SE sample. From Figure 7A, it can be

291

seen that FL/Z-SE was mainly composed of ultra-micropores (5.5, 6.6 and 8.3 Å), 13



292

micropores (11.3 and 15.3 Å) and a wide mesopores distribution from 17-39 Å. These

293

peaks almost disappeared except for one at 12.7 Å in the peptide-adsorbed sample.

294

Obviously, only small chain peptides not exceeding 39 Å could be selected and

295

adsorbed on FL/Z-SE sample. The specific surface area of FL/Z-SE dramatically

296

dropped from 1396.1 to 150 m2/g after adsorbing peptides (Figure S1 and Table S1).

297

Ultramicropores of FL/Z-SE (< 10 Å) were completely filled with these small

298

peptides. Moreover, size-exclusion chromatography (SEC) was used to analyze these

299

adsorbed peptides from FL/Z-SE. For comparison, the SEC spectrum of SPP

300

hydrolysates before and after being adsorbed using FL/Z-SE were also present in

301

Figure 7B. The eluate SPP hydrolysate showed an obvious enhancement of peak

302

intensity between molecular weight of 73-799 Da and a weakness of peak intensity in

303

the region of 1390-4200 Da compared with original counterpart. It is suggested that

304

FL/Z-SE was preferably adsorbed some peptide fractions with low molecular weights

305

due to its pore size screening, and thus those peptides with high molecular weights

306

were excluded.31 As expected, the peptides with small molecule from FL/Z-SE sample

307

have high ACE inhibitory activity, and it is in accordance with previous reports.32,33

308

Besides their length of peptide chain, the hydrophobic/hydrophilic character of

309

peptides is also very crucial for their ACE inhibitory activity as well as their

310

biocompatibility.34 Moderate hydrophilic peptides with a certain ratio of hydrophobic

311

amino acid chains are considered to be ideal ACE inhibitors. Therefore, we prepared

312

hydrophobic porous carbon containing hydrophilic adsorption sites (Lewis acid Fe

313

ions) to screen moderate hydrophilic peptides from SPP hydrolysate.

314

To better understand the mechanism underlying the selective adsorption of the

315

obtained carbons, we next employed X-ray photoelectron spectroscopy (XPS)

316

analysis to investigate carbon component and surface properties of the carbon Z-SE 14


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and FL/Z-SE samples (Figure 8). Both of these two carbons (Z-SE and FL/Z-SE)

318

showed a high proportion of Sp2 C, indicating a relative higher graphitized carbon

319

structure than common activated carbon.35 After Fe species being doped in Z-SE,

320

some notable changes had taken place in their XPS spectra. First, the ratio of Sp2

321

C/Sp3 C was further increased due to the graphitization of amorphous carbon

322

catalyzed by Fe metal (Figure 8B).26 Second, the peaks of the FeC-like structures

323

(Fe3C) at 282.3 eV appeared in the FL/Z-SE (Figure 8B). Third, the concentration of

324

oxygen groups was significantly decreased in Fe-doped Z-SE. Thus, it can be deduced

325

that FL/Z-SE surface properties represented more heterogeneous surface containing

326

hydrophobic graphitic carbon36 and hydrophilic “Lewis acid” Fe37 in comparison to

327

Z-SE surface property. Based on the principle of high affinity between adsorbate with

328

adsorbent having similar property, the FL/Z-SE is hypothesized to prefer adsorbing

329

peptides with short and moderate hydrophilic chains. This was consistent with the

330

screening result showing from the above size-exclusion chromatography (Figure 7B).

331

HPLC Purification of ACE Inhibitory Peptides. The adsorbed peptides on FL/Z-SE

332

were eluted and then purified fraction with SB C18 column with gradient elution.

333

Figure 9A shows the reverse-phase gradient HPLC chromatogram of the SPP

334

hydrolysate elutes from FL/Z-SE, which was divided into 10 fractions named from

335

FSP-1 to FSP-10 according to retention time. Their ACE inhibitory activities were

336

tested at each fraction concentration of 80 mg/L and shown in Figure 9B. From Figure

337

9B, it can be observed that FSP-5 fraction exhibited the highest ACE inhibitory

338

activity with a value of 81.23 ± 3.15%, and thus was chosen for further purification

339

using the second RP-HPLC.

340

Figure 10 shows the chromatogram of FSP-5 fraction and their ACE inhibitory

341

activities. Another 4 fractions corresponding to four peaks were separated and named 15



342

as FSP-5x (x from 1 to 4) in Figure 10A. Among these four fractions (each fraction

343

concentration of 60 mg/L), FSP-53 possessed the highest ACE inhibitory activity with

344

a value of 78.44 ± 2.40% (Figure 10B). RP-HPLC technique is an important way to

345

separate peptide compounds with different polarities through non-polar stationary

346

phase.38 Based on this theory, the selected fractions of FSP-5 and FSP-53 with highest

347

ACE inhibitory activities were both obtained at the middle position of the whole

348

HPLC. From this, it can be deduced that some peptides with potential ACE inhibitory

349

activity come from some polar fractions containing greater amounts of hydrophilic

350

amino acids. Most amino acids are made of two parts: (1) typical hydrophilic

351

amino/carboxyl groups and (2) some other hydrophobic (benzene or alkyl) groups or

352

hydrophilic (N, O-containing) groups. Thus, Fe doping was proposed to modulate

353

hydrophilic surface and polarity, so that it can screen some moderate polar peptides

354

rather than high hydrophobic peptides from SPP hydrolysate source.

355

Identification of ACE Inhibitory Peptide by MALDI-TOF/TOF MS. The molecular

356

mass and amino acid sequence of fraction FSP-53 were identified by

357

MALDI-TOF/TOF MS and shown in Figure 11. The molecular mass of fraction

358

FSP-53 was determined to be of 452.55 Da. On the basis of this molecular mass and

359

tandem MS, the amino acid sequence was identified as Arg-Tyr-Leu (RYL), which is

360

a novel ACE-inhibitory peptide from silkworm pupa protein with the IC50 value of

361

3.31 ± 0.11 µM. The IC50 value was comparatively similar to some other peptides

362

GNPWM (IC50 = 21.70 µM) and GAMVVH (IC50 = 19.39 µM) obtained from the

363

same source (silkworm pupa protein).32

364

Comparison Adsorption Capacities of Different Tripeptides on FL/Z-SE.

365

LogP-value can be used to represent the hydrophobic/hydrophilic property of some

366

small molecules, whose definition is described in Eq. 2.39,40 As seen in this Eq. 2, 16


Page 16 of 45

Page 17 of 45


367

negative value of LogP means the molecule having hydrophilic behavior, while

368

positive value means the molecule having hydrophobic behavior. Thus, we calculated

369

the LogP value of the identified RYL using ChemDraw Software V15.0 (Cambridge

370

Soft), and its value was about -0.22 presenting a moderate hydrophilic peptide. LogP = Log

Cn − octanol C water

(2)

371

where Cn−octanol and C water mean the concentration of peptide in n-octanol and water

372

solvent, respectively.

373

To further investigate influence of hydrophilic property of peptide on selective

374

adsorption on the synthesized FL/Z-SE. One designed experiment was carried out to

375

explore the relationship between LogP-value of tripeptides with similar molecular

376

weights and their corresponding adsorption capacity on FL/Z-SE. Figure 12 shows the

377

adsorption capacities of five selected tripeptides (FYL, RYF, RYL, RYM and RYN)

378

with different LogP-values on FL/Z-SE. As shown, the adsorption capacities of these

379

peptides display a volcano trend with the increase of LogP value. It indicated that the

380

FL/Z-SE is inappropriate to adsorb peptides having either strong hydrophilicity or

381

hydrophobicity. As expected, the moderate hydrophilic peptides RYL (LogP = -0.22)

382

possessed the highest adsorption capacity (1.21 mmol/g) among these selected

383

peptides on FL/Z-SE. The strong hydrophilic and hydrophobic peptides had

384

significantly lower adsorbed capacity (0.64 and 0.77 mmol/g). This consequence can

385

be attributed to the synergistic effect of hydrophobic graphitic carbon and hydrophilic

386

“Lewis acid” Fe on FL/Z-SE surface. The amphiphilic property of FL/Z-SE could

387

efficiently enhance the selective enrichment for moderate hydrophilic peptides from

388

hydrolysates, which was verified the XPS results in Figure 8.

389

Inhibition Pattern of the Inhibitory Peptide. To elucidate the mechanism of ACE 17



390

inhibition, Lineweaver-Burk plot was used to determine for the inhibitory peptides

391

(RYL). As shown in Figure 13, the plot with coinciding intercept on the 1/S axis

392

indicate that RYL was competitive type of inhibition, which means that the bioactive

393

peptide (RYL) binding to the same catalytic site of ACE-HHL on the ACE molecule

394

to produce an inactive complex (enzyme-substrate-inhibitor).41 In general, lower

395

values indicate higher affinity and more potent inhibitory activity. The calculated

396

inhibition constant ( ) value of RYL was 1.4×10-5 M. In this case, the value of

397

RYL was clearly lower than some reported values of hemp protein hydrolysates

398

(6.0-8.9×10-4 M)42 and terminalia chebula retz protein hydrolysate (2.81×10-5 M).43

399

Thus, RYL has a potential ACE inhibitory capacity.

400

Molecular Docking. To further investigate the inhibition mechanism of inhibitor

401

(RYL) towards ACE, docking simulation with Sybyl X-2.1.1 was used to carry out in

402

this system. Figure 14A showed the interaction between RYL and ACE. As shown, the

403

RYL formed 5 hydrogen bonds with ACE residues, including Gln281 (2.0 Å), Lys511

404

(1.8 Å), Tyr520 (1.9 Å), Glu384 (2.1 Å) and Ala354 (2.1 Å), respectively. These

405

positions were mainly belonged to the active sites of S1 and S2 pockets from ACE.

406

We also used the same way to investigate the molecular docking between ACE and

407

HHL (Figure 14B). Docking result showed that HHL was found to form hydrogen

408

bonds with ACE residues on similar positions, which were Gln281 (1.8 Å), Ala354

409

(2.1 Å), His513 (2.1 Å) and Tyr520 (1.8 Å), respectively.44 Clearly, the selected RYL

410

would efficiently occupy some high active sites of ACE, and inhibited its bound with

411

HHL. This was consistent with RYL’s inhibition pattern from the inhibition kinetics

412

result.

413 414

■ Supporting Information 18


Page 18 of 45

Page 19 of 45


415

Isotherms of nitrogen adsorption for FL/Z-SE before and after addition of protein

416

hydrolysate; physical properties for FL/Z-SE before and after adsorbed protein

417

hydrolysate.

418 419

Funding

420

This work was financially supported by National Natural Science Foundation of

421

China (No. 31401629, 21666004, 21676059 and 21606054), Guangxi Distinguished

422

Experts Special Foundation of China, Natural Science Foundation of Guangxi Zhuang

423

Autonomous

424

2017GXNSFFA198009), Scientific Research Foundation of Guangxi University (No.

425

XGZ130963) and Innovation and Entrepreneurship Training Program of Guangxi

426

Zhuang Autonomous Region (No. 201710593185).

Region,

China

(No.

2016GXNSFAA380229

and

427 428

Notes

429

The authors declare no competing financial interest.

430 431

■ Acknowledgments

432

We appreciate helpful suggestions from Dr. Wei Hu of state Key Laboratory for

433

Conservation and Utilization of Subtropical Agro-bioresources.

434 435

■ References

436

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26


Page 26 of 45

Page 27 of 45


Figure captions

Figure 1

SEM images and element contents of (A) SE, (B) Z-SE, (C) FL/Z-SE, and (D) FH/Z-SE.

Figure 2

PXRD patterns of (A) SE and (B) Z-SE, FL/Z-SE and FH/Z-SE.

Figure 3

(A) Isotherms of nitrogen adsorption/desorption and (B) DFT pore size distributions for Z-SE, FL/Z-SE and FH/Z-SE composites.

Figure 4

Zeta potentials and particle sizes of Z-SE, FL/Z-SE and FH/Z-SE at the concentration of about 0.1 mg/mL.

Figure 5

Magnetic hysteresis curves of FL/Z-SE and FH/Z-SE.

Figure 6

(A) Effects of adsorption time on residual fraction of peptides fractions and (B) fractional adsorbed amounts and the corresponding ACE inhibitory activity of peptides from protein hydrolysates eluted from Z-SE,

FL/Z-SE

and

FH/Z-SE

samples

(concentration

of

hydrolysate/elute: 1.00 g/L) Figure 7

(A) DFT pore size distributions for FL/Z-SE before and after addition of protein hydrolysate and (B) SEC of untreated silkworm pupea protein hydrolysate (dashed line) and the enriched eluate fraction from FL/Z-SE after elution (straight line).

Figure 8

High-resolution C1s XPS spectra of (A) Z-SE and (B) FL/Z-SE samples.

Figure 9

(A) Chromatogram of SPP hydrolysate elute from the FL/Z-SE and (B) their ACE inhibitory activities of the separated ten peptide fractions (concentration of each FSP fraction was ~80 mg/L).

Figure 10

(A) Chromatogram of FSP-5 fraction and (B) their ACE inhibitory 27



activities of the separated four peptide fractions (concentration of each FSP-5x fraction was ~5 mg/L). Figure 11

Identification of molecular mass and amino acid sequence of the purified peptide. MS/MS spectrum of molecular ion m/z 452.55 Da.

Figure 12

Comparison between capacities of FL/Z-SE towards different tripeptides, starting concentration C0 = 1 mM, adsorbent mass mFL/Z-SE = 5 mg, volume of the liquid phase V0 = 25 mL, Temperature T = 23 °C.

Figure 13

Inhibition kinetic curves of ACE by inhibitory RYL.

Figure 14

Predicted binding modes between ACE and inhibitor RYL (A) or substrate HHL (B) after being docked at the ACE active site. The residues of ACE were shown as line, the inhibitor and substrate were shown as stick, and the hydrogen bond was shown as yellow dashed lines.

28


Page 28 of 45

Page 29 of 45


Table Table 1 Physical properties of Z-SE, FL/Z-SE and FH/Z-SE samples Sample

BET

Langmuir

surface area

SMeso/SMicro

Pore

Micropore

surface area

volume

volume

(m2/g)

(m2/g)

(mL/g)

(mL/g)

Z-SE

1415.5

1671.1

3.04

1.03

0.14

FL/Z-SE

1396.1

1651.3

2.36

0.85

0.16

FH/Z-SE

1246.8

1477.8

1.41

0.69

0.21

29



Figure graphics

Figure 1

30


Page 30 of 45

Page 31 of 45


180 160

•

♣ CaCO3 ♠ SiO2

♣ ♣

140

♦ •

♠

120 Intensity (a.u.)

∗ --- Fe • --- Fe3C ♥ --- Fe5C2 ♦ --- Graphitized Carbon ♥♦ ♥ ∗ ♥♥ FH/Z-SE

(B)

(A)

100

•

♠

•

80 60

♦ •

•

40 20

♥♦ ♥ ♥

∗

FL/Z-SE

♦ ♦

0

Z-SE

-20 10

20

30

40

50

60

70

10

80

20

2θ (Degrees)

Figure 2

31


30

40 50 2θ (Degrees)

60

70

80


Quantity adsorbed (mmol/g)

30

(B)

(A)

Page 32 of 45

FH/Z-SE FL/Z-SE Z-SE

25 20 15

Adsorption Z-SE Desorption Z-SE Adsorption FL/Z-SE Desorption FL/Z-SE Adsorption FH/Z-SE Desorption FH/Z-SE

10 5 0 0.0

0.2

0.4 0.6 0.8 Relative pressure (P/P0)

1.0

5

10 15 20 25 30 35 40 45 50 55 60 Pore diameter (Å)

Figure 3

32



700

60

Zeta potential (mv)

40

Zeta potential Paricle size

650 600

20 5.93 mv 557.2 nm 553.9 nm 0

550

-6.77 mv

492.7 nm -20 -24.66 mv

500 450

-40 Z-SM

FL/Z-SM

FH/Z-SM

Figure 4

33


Particle size (nm)

Page 33 of 45


40 FH/Z-SE (1)

Ms (emu/g)

20

FL/Z-SE (2)

0

-20

-40 -20000

-10000

0 Field (G)

10000

Figure 5

34


20000

Page 34 of 45


(A)

Z-SE FL/Z-SE FH/Z-SE

80 60 40 20 0 0

100

Relative bound amount (%)

Relative unbound amount (%)

100

20 10 Equilibrium time / min

60

Relative adsorbed amount ACE inhibitory activity

100.0%

(B)

76.6% 80

80

67.8% 56.4%

60

53.7%

100

49.6%

51.8% 60

39.4% 40

40

20 Protein hydrolysate Z-SE

Figure. 6

35


20 FL/Z-SE

FH/Z-SE

ACE inhibitory activity (%)

Page 35 of 45


Page 36 of 45

2.1 0.6

1.8 1.5

0.2

0.6

0.0

AU

1.2

Original hydrolysate Eluate hydrolysate Residue hydrolysate

1390Da

0.4 4200 Da

19.5

21.0 Time (min)

0.9

22.5

AU

FL/Z-SM (before adsorption) FL/Z-SM (after adsorption)

AU

(A)

0.4

799 Da

0.2

73 Da

0.0 24

0.6 0.3

26 28 Time (min)

30

(B)

0.0 10

20

30 40 Pore diameter (Å)

50

60

0

10

Figure 7

36


20 30 Time (min)

40

50

Page 37 of 45


(A)

Raw Simulated

2

Sp C

(B)

Raw Simulated

2

Intensity (a.u.)

Intensity (a.u.)

Sp C

3

Sp C C-O

3

Sp C Fe3C

O-C=O

282

284

286

288

290

282

Binding energy (eV)

C-O 284 286 Binding Energy (eV)

Figure 8

37


O-C=O 288

290


FSP-1~4 FSP-5

100

FSP-6 ~ FSP-10

Relative inhibitory activity (%)

800

mAU

Intensity (mAU)

120

600

400

Page 38 of 45

110 100 90 14

15

Time (min)

16

200

(B)

81.23 74.83 67.44 66.12

80 60

50.74

47.86 43.54

37.48

40

28.10 20

18.74

(A)

0 0

10

20

30 40 Time (min)

50

0

60

1

Figure 9

38


2

3

4

7 5 6 FSP - Series

8

9

10

Page 39 of 45


(A)

100

FSP-52

(B) ACE inhibitory activity (%)

500

Intensity (mAU)

400 300

FSP-53 FSP-51

200

FSP-54 100 0

78.44

80 60 40

30.59 19.41

20

10.73

-100 0

5

10 Time (min)

15

20

0

FSP-51

Figure 10

39


FSP-52

FSP-53

FSP-54

338.46


b1 b2

100

R Y L

b1 157.34

40 392.49

20

451.72

434.43

320.45

60

60.23

Intensity (%)

80

b2

0 0

100

200 300 Mass (m/z)

400

Figure 11

40


500

Page 40 of 45


1.4 Adsorption capacity (mmol/g)

Page 41 of 45

RYL Mw: 450.6

1.2

RYF Mw: 484.6 RYM Mw: 468.6

1.0

0.8

FYL Mw: 450.6

RYN Mw: 451.5

0.6 -3

-2

-1

LogP

0

1

Figure 12

41


2


1.4 1/V0 (L⋅min⋅mM)

1.2

0 µM 25.31 µΜ 50.27 µΜ

1.0 0.8 0.6 0.4 0.2 0.0 0.0

0.2

0.4

0.6

0.8

1.0 -1

1/[S] (mM )

Figure 13

42


Page 42 of 45

Page 43 of 45


Figure 14

43



Table of Contents Graphic

44


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Ultrafast Screening of a Novel, Moderately Hydrophilic Angiotensin

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