Identification and molecular docking of novel antioxidant peptides from Candida utilis

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Food Chemistry 455 (2024) 139860

Available online 28 May 2024

Identication and molecular docking of novel antioxidant peptides from

Candida utilis

Yashu Wei

, Lingling Wang

, Yan He , Xia Ma

School of Perfume and Aroma Technology, Shanghai Institute of Technology, No. 100 Haiquan Road, Shanghai 201418, PR China.

ARTICLE INFO

Keywords:

Candida utilis

Antioxidant peptide

Identication

Molecular docking

Antioxidant mechanism

ABSTRACT

The current trend is the promotion of antioxidants that are benecial for both health and the environment.

Candida utilis have garnered considerable attention due to their commendable attributes such as non-toxicity and

the ability to thrive in waste. Therefore, Candida utilis was used as raw material to isolate and identify new

antioxidant peptides by employing methods such as ultraltration, DEAE Sepharose Fast Flow, and liquid

chromatography-tandem mass spectrometry. The antioxidant mechanism of peptides was investigated by mo-

lecular docking. The properties of antioxidant peptides were evaluated using a variety of computational tools.

This study resulted in the identication of two novel antioxidant peptides. According to the molecular docking

results, the antioxidant mechanism of Candida utilis peptides operates by obstructing the entry to the myelo-

peroxidase activity cavity. The (−) CDOCKER energy of antioxidant peptides was 6.2 and 6.1 kcal/mol,

respectively. Additionally, computer predictions indicated that antioxidant peptides exhibited non-toxicity and

poor solubility.

1. Introduction

The normal physiological functioning of organisms is invariably

accompanied by the production of free radicals (Wen et al., 2020).

Oxidative stress caused by excess free radicals is a major threatening

factor to non-communicable chronic diseases (chronic, non-self-healing,

almost incurable diseases such as diabetes, cardiovascular disease, and

obesity) (Gao, Li, Chen, Gu, & Mao, 2021; Wen et al., 2020; Xia et al.,

2022). The presence of antioxidants in the capture and neutralization of

free radicals, thereby mitigating their detrimental impact on the body

(Guo et al., 2023). Currently, the utilization of synthetic antioxidants in

food is subject to stringent regulations due to their inherent drawbacks

including effects on human health, exorbitant costs, and limited mech-

anisms of action (Guo et al., 2023; Zhang et al., 2019). In recent years,

numerous studies have demonstrated that certain bioactive peptides

possess the capacity to scavenge free radicals and inhibit peroxidation

within the body. Peptide antioxidants offer the benets of being safe and

well-tolerated (Chen et al., 2023).

In recent years, bioactive peptides have garnered signicant

attention due to their high potency, efcient absorption, and natural

safety (Lopez-Garcia, Dublan-Garcia, Arizmendi-Cotero, & Olivan,

2022). Moreover, they exhibit a diverse array of physiological activities.

For instance, the peptides from Pleurotus eryngii possess the ability to

impede the proliferation of tumor cells and exert an anti-tumor effect

(Sun, Hu, & Li, 2017). The antimicrobial peptide produced by Lacti-

plantibacillus plantarum FB-2 exhibits antibacterial activity (Yu et al.,

2024). Among bioactive peptides, they exhibit great potential as func-

tional substances for preventing and treating non-communicable

chronic diseases such as diabetes, cardiovascular disease and obesity

(Xia et al., 2022). To date, several peptides with robust antioxidant

activity have been isolated from various types of protein hydrolysates,

including selenium-rich yeast protein hydrolysate and brewer's spent

yeast (Guo, Guo, & Liu, 2020; Vieira et al., 2016).

Bioactive peptides are derived from three primary sources: endoge-

nous bioactive peptides of natural origin, microbial fermentation,

exogenous bioactive peptides obtained through protein hydrolysates

and synthetic peptides synthesized using chemical methods (Guo, Lin,

Guo, Zhang, & Zheng, 2017). However, industrial-scale production of

Abbreviations: VPPP, Val-Pro-Pro-Pro; MYP, Met-Tyr-Pro; ROS, reactive oxygen species; MPO, myeloperoxidase; CUH, Candida utilis hydrolysate; LC-MS/MS,

liquid chromatography-tandem mass spectrometry.

* Corresponding author at: School of Perfume and Aroma Technology, Shanghai Institute of Technology, No. 100 Haiquan Road, Shanghai 201418, PR China.

E-mail addresses: heyan@sit.edu.cn (Y. He), maxia@sit.edu.cn (X. Ma).

These authors contributed equally to this work.

Contents lists available at ScienceDirect

Food Chemistry

journal homepage: www.elsevier.com/locate/foodchem

https://doi.org/10.1016/j.foodchem.2024.139860

Received 19 October 2023; Received in revised form 23 May 2024; Accepted 26 May 2024

Food Chemistry 455 (2024) 139860

endogenous peptides content in living organisms is relatively low, which

hinders the achievement of commercialization (Wang, 2014). The pep-

tides can be prepared through microbial fermentation using Aspergillus

oryzae, Actinomycetes, Aspergillus niger, Bacillus subtilis and others (Wang,

Liu, Liu, Su, & Liu, 2022). The toxicity or harmfulness of certain enzyme-

producing strains to humans should be considered (Wei, Pan, & Ji,

2010). The activity and diversity of chemically synthesized peptides are

limited, and synthesis is only possible for peptides with known se-

quences (Wang, Liu, et al., 2022). The process of extracting peptides

from proteins through acid hydrolysis and alkaline hydrolysis raises

certain nutritional and toxicological safety concerns. The traditional

acid method was employed by Ma, Hu, and Yu (2010) for the hydrolysis

of protein from Corbicula uminea. Inadequate control over the con-

centration of hydrochloric acid could result in a chloropropanol content

in the hydrolysate that fails to meet the minimum standards set by most

countries, thereby posing signicant risks to human health and well-

being (Ma et al., 2010). The process of alkali hydrolysis of protein

leads to racemization, thereby reducing the effective absorption of the

dextrorotatory portion by the human body (Wang, Liu, et al., 2022). The

enzymatic hydrolysis products of proteins primarily consist of highly

pure and easily separable peptides. The enzymatic hydrolysis process is

characterized by its gentle conditions, short reaction time, and high

efciency. For instance, the optimal conditions for hydrolysis of protein

from Candida utilis were determined by Wang, He, Chen, and Ma (2022):

the enzyme amount was 3000 U/g, the hydrolysis temperature was set at

51 ◦C, and a pH value of 10.0 was maintained. These conditions led to a

degree of hydrolysis of 47.78%. Therefore, it is imperative to identify a

natural substance characterized by high protein content and low culti-

vation cost, capable of yielding bioactive peptides through enzymatic

hydrolysates that show safety and non-toxicity.

The studies on animal and plant bioactive peptides have been

extensively documented, whereas research on fungal bioactive peptides

remains relatively limited. Yeast offers a high utilization rate and quality

protein, making it a promising source of fungal active peptides (Guo

et al., 2020). Candida utilis is a high-yielding strain of single-cell protein,

which has been certied by the Food and Drug Administration of the

United States as a safe organism that can be used in food and pharma-

ceutical industry (Zhao, Liang, & Huang, 2002). Candida utilis has a high

fermentation density of 92 g/L (Kondo, Miura, Sone, Kobayashi, &

lijima, H., 1997) and can be grown in some industrial wastes (Rosma &

Cheong, 2007). Therefore, it could be a high-quality raw material for the

preparation of bioactive peptides.

The application of molecular docking has gained signicant promi-

nence in recent years, emerging as a pivotal technology within the realm

of computer-aided drug research (Chen et al., 2024). Moreover, it can be

effectively employed for studying antioxidant mechanisms (Ma et al.,

2018). Presently, molecular docking is employed to screen myeloper-

oxidase (MPO) inhibitors from protein hydrolysates (Gao et al., 2021;

Zhang, He, Bonneil, & Simpson, 2020). The molecular docking tech-

nique was employed to identify MPO inhibitors with antioxidant activity

from duck liver-derived antioxidant peptides and antioxidant peptides

from two sources of eggshell membrane hydrolysates, thereby demon-

strating the efcacy of this method (Fan et al., 2023; Zhu et al., 2022).

Therefore, molecular docking is a valuable tool for identifying antioxi-

dants and elucidating their underlying mechanisms.

Currently, the primary focus of Candida utilis research lies in its

application as animal feed, with no existing reports on utilizing Candida

utilis for the preparation of bioactive peptides. This study postulates that

extracting, purifying, and identifying antioxidant peptides from Candida

utilis could enhance its value-added potential and application prospects.

Therefore, the objective of this study was to isolate, purify, and identify

novel antioxidant peptides derived from Candida utilis. The identied

peptides can be assessed for their potential to inactivate the enzyme

spontaneously via molecular docking, thereby elucidating the interac-

tion mechanism. The water solubility, allergenicity, toxicity, and

gastrointestinal stability of antioxidant peptides were assessed utilizing

a diverse range of computational methodologies. The isolation of pep-

tides from Candida utilis could open up new vistas in the development of

health-functional foods and medicinal formulations.

2. Materials and methods

2.1. Materials

The Candida utilis employed in this study was sourced from the lab-

oratory of the Shanghai Institute of Technology. The DEAE-Sepharose

Fast Flow was procured from General Electric Co. Ltd. (Boston, USA).

Alcalase (with an activity of 200,000 U/g) was obtained from Sigma-

Aldrich Co. Ltd. (Shanghai, China). Trypsin (featuring an activity of

50,000 U/g), hydrogen peroxide (H

), and salicylic acid were pur-

chased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

The 1,1-diphenyl-2-trinitrophenylhydrazine (DPPH) and 2,2

′

-Azinobis-

(3-ethylbenzthiazoline-6-sulphonate) (ABTS) were obtained from

Shanghai Baoman Biotechnology Co., Ltd. (Shanghai, China). Vitamin C

(Vc) and FeSO

were sourced from Shanghai Titan Scientic Co., Ltd.

(Shanghai, China). All reagents utilized in the study were of analytical

grade. The propagation of Candida utilis and the production of Candida

utilis hydrolysate (CUH) were conducted following the enzymatic solu-

tion method previously outlined by our team (Wang, He, et al., 2022).

2.2. Ultraltration separation of Candida utilis peptides

Candida utilis was prepared into 10% bacterial suspension, subjected

to ultrasonication and enzymatic treatment with alcalase and trypsin,

and the resulting supernatant from CUH was collected. The supernatant

was sequentially processed using ultraltration membranes

(UFSC20001, Millipore Amicon, Germany) with molecular weight cut-

off (MWCO) values of 10 and 3 kDa, respectively. In essence, the re-

sidual substance P3 (>10 kDa) retained in the concentration stage of the

10 kDa membrane was taken and subjected to freeze-drying, while the

the 3 kDa membrane then trapped the ltrate. From this, the retentate

P2 (3–10 kDa) and permeate P1 (<3 kDa) were collected. Both the

retentate and the nal permeate underwent freeze-drying (utilizing an

FDU-1200 instrument from Shanghai Eyela Co., Ltd., China) and were

subsequently stored at −20 ◦C. The yield of components with varying

molecular weights can be determined using the following formula.

Yield =Weight of each component (g)

Weight of enzymatic hydrolysate(g)×100% (1)

2.3. DEAE Sepharose fast ow separation of Candida utilis peptides

The research methods were modied based on previous ndings

(Zhang et al., 2019a). The component with a molecular weight <3 kDa

(P1) was prepared into a 20 mg/mL solution, and subsequently ltered

using a 0.22

m lter membrane. Following this, a pre-equilibrated

DEAE-Sepharose Fast Flow column (dimensions: 30 cm ×2.6 cm) was

loaded with 5 mL of the said solution. The ow rate was set at 1.5 mL/

min, with collections made every 5 min. After sample absorption by the

packing material, stepwise elution was performed using NaCl solutions

of increasing concentration (0.00, 0.10, 0.20, 0.40, 0.60, and 0.80 mol/

L, respectively). Each collected solution was subsequently examined at

wavelengths of 220 nm and 280 nm. This procedure enabled the sepa-

ration of four puried components of Candida utilis peptides (F1, F2, F3,

F4) using the DEAE-Sepharose Fast Flow column. Each peptide peak was

collected separately, leading to the acquisition of puried peptide

fractions following dialysis and lyophilization. Subsequently, the anti-

oxidant activities of the four components were assessed, and the struc-

tures of the components with higher activity were elucidated.

Y. Wei et al.

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