Preparation method and application of sinonovacula constricta enzymolysis polypeptide

A technology of enzymatic hydrolysis and razor clam, which is applied in the field of preparation of enzymatic hydrolyzed polypeptides of razor clam, achieving the effects of high sensitivity, good stability, and few side effects

Inactive Publication Date: 2014-05-21

ZHEJIANG OCEAN UNIV

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AI-Extracted Technical Summary

Problems solved by technology

For razor clam constriction, there are few studies on anti-tumor aspects, especially the anti-tumor ...

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Abstract

The invention discloses a preparation method and application of a sinonovacula constricta enzymolysis polypeptide. The method is characterized by comprising the following steps: shelling sinonovacula constricta, washing, mincing, adding water in the material-liquid mass ratio of 1:(3-5) for homogenizing, and adjusting the pH value to 5.5-6.5; adding protease for performing enzymolysis at the temperature of 60-70 DEG C for 1.5-2.5 hours, wherein the adding amount of the protease is 0.2-0.3 percent based on the total mass of sinonovacula constricta; after enzymolysis, performing enzyme deactivation, and centrifuging to obtain supernatant serving as enzymatic hydrolysate; performing ultrafiltration, G-25 gel chromatography and reversed phase high-performance liquid chromatography elution on the enzymatic hydrolysate to obtain the needed sinonovacula constricta enzymolysis polypeptide. A preparation process is simple, the obtained sinonovacula constricta enzymolysis polypeptide is high in sensitivity and stability and small in side effects, plays a remarkable role in inhibiting the proliferation of DU-145 and PC-3 cells through MTT detection, and can be applied to in-vitro resistance of prostatic cancer. The preparation method plays an important role in further researching and developing functional foods and medicaments based on the sinonovacula constricta enzymolysis polypeptide and increasing the economical value of the sinonovacula constricta.

Application Domain

Hydrolysed protein ingredientsPeptide preparation methods +2

Technology Topic

UltrafiltrationSinonovacula +12

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Examples

Experimental program(1)
Effect test(1)

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[0031] The present invention will be further described in detail below in conjunction with the embodiments of the drawings.

[0032] 1. Materials and Instruments

[0033] 1.1 Main materials and reagents

[0034] Sinonovacula constricta was purchased from Nanzhen Market in Zhoushan City, Zhejiang Province;

[0035] Fetal Bovine Serum (FBS), Hangzhou Sijiqing Biological Products Co., Ltd.;

[0036] HamF12 medium, Gibco;

[0037] Trypsin, SIGMA, USA;

[0038] Penicillin, Streptomycin, Shandong Lukang Pharmaceutical Co., Ltd.;

[0039] Sephadex G-25, Beijing Yatai Hengxin Biotechnology Co., Ltd.;

[0040] Pepsin, papain, alkaline protease, Asia Pacific Hengxin Biotechnology Co., Ltd.;

[0041] MTT was purchased from American Sigma Company;

[0042] Dimethyl sulfoxide (DMSO) was purchased from American AMRESCO;

[0043] The remaining reagents are of analytical grade.

[0044] 1.2 Main instruments

[0045] DS-1 high-speed tissue masher, Shanghai Specimen Model Factory;

[0046] BSA124S electronic balance, Sartorius AG, Germany;

[0047] SSW type microcomputer electric heating thermostat, Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory;

[0048] Ultrafiltration cup, ultrafiltration membrane, Shanghai Mosu Scientific Equipment Co., Ltd.;

[0049] BSW-100-ZCD automatic part collector, Shanghai Qite Analytical Instrument Co., Ltd.;

[0050] BT1-100 constant flow pump, Shanghai Qite Analytical Instrument Co., Ltd.;

[0051] PHS-250pH meter, Shanghai Lida Instrument Factory;

[0052] CF16RXII high-speed refrigerated centrifuge, Japan HITACHI company;

[0053] ZHJH-C1209C ultra-clean workbench, Shanghai Zhicheng Analytical Instrument Manufacturing Co., Ltd.;

[0054] Forma 3111 CO2 incubator, American Thermo Company;

[0055] Microplate reader, Bio-Rad, USA;

[0056] 752FC UV Spectrophotometer, Shanghai Spectrometer Co., Ltd.;

[0057] LGJ-18 freeze dryer, Beijing Songyuan Huaxing Technology Development Co., Ltd.;

[0058] High performance liquid chromatograph (P1201 Yilite), Dalian Yilite Analytical Instrument Co., Ltd.;

[0059] Micro oscillator, Shanghai Precision Instrument Co., Ltd.;

[0060] Electrophoresis instrument (BIO-RAD, 041BR), Hangzhou Baocheng Biotechnology Co., Ltd.;

[0061] FACS Calbur flow cytometer, BD company.

[0062] 2. experimental method

[0063] 2.1 Selection of the best enzyme

[0064] Use alkaline protease, trypsin, papain, and pepsin to carry out enzymolysis under optimal enzymolysis conditions, and then use MTT method to screen the protease with the strongest anti-tumor activity according to the proliferation inhibition rate of DU-145 cells to determine the best Enzyme species. The hydrolysis process is as follows: Sinonovacula constricta → shelling → washing and mincing → 50g homogenized with water → pH adjustment → enzymatic hydrolysis → boiling water bath to inactivate enzyme (90℃, heating 10min) → 5000r/min centrifuge for 10min → take centrifuge Clear liquid→freeze-drying→enzymatic hydrolysis→DU-145 cell proliferation inhibition rate (MTT)→determine the best enzyme species.

[0065] 2.2 Optimization of enzymatic hydrolysis conditions

[0066] Choose papain, use 5 factors of A (temperature), B (pH), C (enzyme addition), D (material-to-liquid ratio) and E (time), and choose 4 levels for L 16 (4 5 ) Orthogonal experiment to screen the enzymolysis conditions with the highest inhibition rate of proliferation of prostate cancer cells in the enzymolysis solution under different enzymolysis conditions, so as to determine the best hydrolysis conditions and carry out a large number of enzymolysis.

[0067] 2.3 Cell culture

[0068] Select human prostate cancer cells DU-145 and PC-3 hormone-independent cells (originally purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, passaged and preserved by our laboratory). DU-145 and PC-3 use F12 medium containing 10% fetal bovine serum at 37℃, 5% CO 2 Cultivate in the incubator to the logarithmic growth phase.

[0069] 2.4 Ultrafiltration and Sephadex G-25 gel chromatography

[0070] Ultrafiltration: Add the sample to the ultrafiltration cup, use a 10KD ultrafiltration membrane for ultrafiltration, collect the enzymatic hydrolysate with a molecular weight of 10KD or more, and freeze-dry it. The Sephadex G-25 gel particles were packed into the column by wet method, the column height was 80cm, the diameter was 2.6cm, and the column was equilibrated with deionized water. The concentration of the sample is 200mg/mL, the sample size is 3mL each time, the eluent is deionized water, the elution speed is 2.3mL/min, and the protein detector is used for detection at 280nm. Each elution peak is collected and concentrated. After freeze-drying. The anti-tumor activity experiment of each peak component obtained after drying was carried out by the MTT method, and the elution peak where the target peptide was located was further determined, and a large amount of the peak was collected, concentrated and freeze-dried.

[0071] 2.5 Reversed-phase high performance liquid chromatography (RT-HPLC)

[0072] RT-HPLC for purification. Chromatographic conditions: C18 reversed-phase column; column type: 10×250mm; acetonitrile/water as mobile phase, flow rate of 0.8mL/min, sample volume of 20μL, and detection wavelength of 220nm. Elution conditions: acetonitrile concentration is 15%, elution is 20min, and sample loading is repeated.

[0073] 2.6 LC-MS/MS identification of anti-tumor peptide structure

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PUM

Property	Measurement	Unit
Molecular weight	66149.0
tensile		MPa
Particle size		Pa
strength	10

Description & Claims & Application Information

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Classification and recommendation of technical efficacy words

High sensitivity
Simple preparation process

Preparation method and application of sinonovacula constricta enzymolysis polypeptide

A technology of enzymatic hydrolysis and razor clam, which is applied in the field of preparation of enzymatic hydrolyzed polypeptides of razor clam, achieving the effects of high sensitivity, good stability, and few side effects

AI-Extracted Technical Summary

Problems solved by technology

Abstract

Image

Examples

Example Embodiment

PUM

Description & Claims & Application Information

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