Preparation of Cyclic Lunasin by Heterologous Recombination Combined with Enzymatic Cyclization and Its Properties

SHI Fan; HOU Yi; ZHAO Jinsong; HU Songqing

doi:10.13386/j.issn1002-0306.2024040501

Volume 46 Issue 7

Mar. 2025

Turn off MathJax

Article Contents

Abstract

References

Supplements

Science and Technology of Food Industry > 2025 > 46(7): 133-139. > DOI: 10.13386/j.issn1002-0306.2024040501

SHI Fan, HOU Yi, ZHAO Jinsong, et al. Preparation of Cyclic Lunasin by Heterologous Recombination Combined with Enzymatic Cyclization and Its Properties[J]. Science and Technology of Food Industry, 2025, 46(7): 133−139. (in Chinese with English abstract). doi: 10.13386/j.issn1002-0306.2024040501.

Citation:

PDF (5476 KB)

Preparation of Cyclic Lunasin by Heterologous Recombination Combined with Enzymatic Cyclization and Its Properties

1.
School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China
2.
School of Light Industry Science and Engineering, South China University of Technology, Guangzhou 510640, China

More Information

Received Date: May 05, 2024
Available Online: February 07, 2025

Graphical Abstract

Abstract

Abstract

In order to prepare a cyclic polypeptide cLunasin by the method of total biosynthesis, the linear precursor aLunasin was designed for cLunasin synthesis, and a fusion expression vector with the double tags of MBP and SUMO was constructed. After the recombinant expression of aLunasin by Escherichia coli, the cLunasin was catalyzed to synthesize by the polypeptide ligase Butelase 1. The effects of cyclization on the thermal stability, structural properties and anticolorectal cancer activity of the peptide were further investigated by circular dichroism spectroscopy, endogenous fluorescence spectroscopy and cell experiments. The recombinant protein was successfully expressed after induction at 37 ℃, and aLunasin was successfully cyclized by Butelase 1, which was identified by the mass spectrometry identification. And 6.36 mg of cLunasin could be prepared form 1 liter of fermentation broth by the total biosynthesis. Cyclization could get the peptide structure more ordered and more compact, indicated by circular dichroism spectroscopy and endogenous fluorescence spectroscopy. Morover, the thermal stability of peptide was improved after cyclization. After incubation at 97 ℃ for 30 min, the peptide retention for cLunasin increased to 74.9%, while that of Lunasin was 62.3%. The half maximal inhibitory concentration of cLunasin on colorectal cancer HCT-116 cells was determined to 15.2 μmol/L, which was lower than that of for Lunasin 30.3 μmol/L, indicating that the anticolorectal cancer activity was enhanced significantly by cyclization. A green biosynthesis method for cLunasin was established, and an useful exploring was provided for the modification and recombinant expression of active peptides.
- soya bean,
- Lunasin,
- recombinant expression,
- cyclization,
- Butelase 1,
- stability,
- anti-colorectal cancer activity

FullText(HTML)

References (30)

References

[1]	HAO Y Q, FAN X, GUO H M, et al. Overexpression of the bioactive lunasin peptide in soybean and evaluation of its anti-inflammatory and anti-cancer activities in vitro[J]. Journal of Bioscience and Bioengineering,2020,129(4):395−404. doi: 10.1016/j.jbiosc.2019.11.001
[2]	HUANG P Y, CHIANG C C, HUANG C Y, et al. Lunasin ameliorates glucose utilization in C2C12 myotubes and metabolites profile in diet-induced obese mice benefiting metabolic disorders[J]. Life Sciences,2023,333:122180. doi: 10.1016/j.lfs.2023.122180
[3]	ELVIRA G D, ERICK D, WANG T, et al. Potential health benefits associated with lunasin concentration in dietary supplements and lunasin-enriched soy extract[J]. Nutrients,2021,13(5):1618. doi: 10.3390/nu13051618
[4]	SOUZA D, LEDESMA H. Lunasin as a promising plant-derived peptide for cancer therapy[J]. International Journal of Molecular Sciences,2022,23(17):9548. doi: 10.3390/ijms23179548
[5]	SZYMC A K, KACZMR W, GAJE K L, et al. Lunasin and its epigenetic impact in cancer chemoprevention[J]. International Journal of Molecular Sciences,2023,24(11):9187. doi: 10.3390/ijms24119187
[6]	MESSINA M, CASKILL W, LAMPE J W. Addressing the soy and breast cancer relationship:Review, commentary, and workshop proceedings[J]. Jnci-Journal of the National Cancer Institute,2006,98(18):1275−1284. doi: 10.1093/jnci/djj356
[7]	KERWIN S M. Soy saponins and the anticancer effects of soybeans and soy-based foods[J]. Current Medicinal Chemistry Anti-cancer Agents,2004,4(3):263−72. doi: 10.2174/1568011043352993
[8]	KHAN N, AFAQ F, MUKHTAR H. Cancer chemoprevention through dietary antioxidants:Progress and promise[J]. Antioxidants and Redox Signaling,2008,10(3):475−510. doi: 10.1089/ars.2007.1740
[9]	LOSSO J N. The biochemical and functional food properties of the bowman-birk inhibitor[J]. Critical Reviews in Food Science and Nutrition,2008,48(1):94−118. doi: 10.1080/10408390601177589
[10]	MEJIA E G, PRISECARU V I. Lectins as bioactive plant proteins:A potential in cancer treatment[J]. Critical Reviews in Food Science and Nutrition,2005,45(6):425−445. doi: 10.1080/10408390591034445
[11]	FAN X, QIN P Y, HAO Y Q, et al. Overexpression of soybean-derived lunasin in wheat and assessment of its anti-proliferative activity in colorectal cancer HT-29 cells[J]. International Journal of Molecular Sciences,2020,21(24):9594. doi: 10.3390/ijms21249594
[12]	SAMUEL F T, XU F, HAN Y H, et al. Inhibitory effects of peptide lunasin in colorectal cancer HCT-116 cells and their tumorsphere-derived subpopulation[J]. International Journal of Molecular Sciences,2020,21(2):537. doi: 10.3390/ijms21020537
[13]	REYES E D, MEJIA E, PEREA F, et al. Liposomes loaded with unsaponifiable matter from Amaranthus hypochondriacus as a source of squalene and carrying soybean lunasin inhibited melanoma cells[J]. Nanomaterials,2021,11(8):1960. doi: 10.3390/nano11081960
[14]	PATERSON S, TOME S, GALVEZ A, et al. Evaluation of the multifunctionality of soybean proteins and peptides in immune cell models[J]. Nutrients,2023,15(5):1220. doi: 10.3390/nu15051220
[15]	GULLA K, KARTH A, SHIVA N, et al. Quantification of bioactive peptide lunasin from soybean, wheat, and their commercial products by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry[J]. Journal of Food Measurement and Characterization,2023,17(5):4927−4937. doi: 10.1007/s11694-023-02008-1
[16]	ZHAO J S, GE G, HUANG Y B, et al. Butelase 1 mediated enzymatic cyclization of antimicrobial peptides:Improvements on stability and bioactivity[J]. Journal of Agricultural and Food Chemistry,2022,70(50):15869−15878. doi: 10.1021/acs.jafc.2c06588
[17]	LIU C F, PAN T M. Recombinant expression of bioactive peptide lunasin in Escherichia coli[J]. Applied Microbiology and Biotechnology,2010,88(1):177−186. doi: 10.1007/s00253-010-2754-5
[18]	何佳彧, 梁菊, 宣茂松, 等. 提高多肽体内稳定性的有效策略[J]. 药学学报,2020,55(1):25−32. [HE J Y, LIANG J, XUAN M S, et al. Effective strategies to improve the stability of polypeptides in vivo[J]. Acta Pharmacologica Sinica,2020,55(1):25−32.] HE J Y, LIANG J, XUAN M S, et al. Effective strategies to improve the stability of polypeptides in vivo[J]. Acta Pharmacologica Sinica, 2020, 55（1）: 25−32.
[19]	NI Z F, LI N, XU P, et al. Enhancement of thermostability and catalytic properties of ammonia lyase through disulfide bond construction and backbone cyclization[J]. International Journal of Biological Macromolecules,2022,219:804−811. doi: 10.1016/j.ijbiomac.2022.07.213
[20]	NGUYEN G, WANG S, QIU Y, et al. Butelase 1 is an asx-specific ligase enabling peptide macrocyclization and synthesis[J]. Nature Chemical Biology,2014,10(9):732−738. doi: 10.1038/nchembio.1586
[21]	SOUZA S, KALUME D, LIMA L, et al. Physicochemical and structural properties of lunasin revealed by spectroscopic, chromatographic and molecular dynamics approaches[J]. BBA Proteins and Proteomics,2020,1868(8):140−154.
[22]	SOUZA S, FERRETTI G, KALUME D, et al. Novel method for the production, purification, and characterization of recombinant lunasin:Identification of disulfide cross-linked dimers[J]. International Journal of Peptide Research and Therapeutics,2022,28:159−162. doi: 10.1007/s10989-022-10466-2
[23]	GILL H S. Evaluating the efficacy of tryptophan fluorescence and absorbance as a selection tool for identifying protein crystals[J]. Acta Crystallographica Section F-Structural Biology Communications,2010,66(3):364−372. doi: 10.1107/S1744309110002022
[24]	BAHAR A A, REN D. Antimicrobial peptides[J]. Pharmaceuticals,2013,6(12):1543−1575. doi: 10.3390/ph6121543
[25]	STEWART E J, ASLUND F, BECK J. Disulfide bond formation in the Escherichia coli cytoplasm:An in vivo role reversal for the thioredoxins[J]. Embo Journal,1998,17(19):5543−5550. doi: 10.1093/emboj/17.19.5543
[26]	LOBSTEIN J, EMRICH C A, JEANS C, et al. SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm[J]. Microbial Cell Factories,2012,11(56):506−510.
[27]	VASSIL A A, KOZLOV S A, GRISHIN E V. Antimicrobial peptide precursor structures suggest effective production strategies[J]. Recent Patents on Inflammation & Allergy Drug Discovery,2008,2(1):58−63.
[28]	NGUYEN G K, QIU Y, CAO Y, et al. Butelase-mediated cyclization and ligation of peptides and proteins[J]. Nature Protocols,2016,11(10):1977−1988. doi: 10.1038/nprot.2016.118
[29]	MARTIN L A, GONG X, DUSZYK M, et al. The three-dimensional structure of carnocyclin A reveals that many circular bacteriocins share a common structural motif[J]. Journal of Biological Chemistry,2009,284(42):28674−28681. doi: 10.1074/jbc.M109.036459
[30]	HUANG L R, ZHANG W X, YAN D D, et al. Solubility and aggregation of soy protein isolate induced by different ionic liquids with the assistance of ultrasound[J]. International Journal of Biological Macromolecules,2020,164:2277−2283. doi: 10.1016/j.ijbiomac.2020.08.031

Supplements (1)

Supplements
Other Related Supplements

Cited By

Cited by

Periodical cited type(9)

1.	张彪，杨晓宽. 纳豆菌发酵板栗渣工艺优化及其多糖的抗氧化性. 食品研究与开发. 2025(03): 160-166 .
2.	屈雅宁，李慧，向大松，郭瑞，曾长立，王红波. 两种细菌发酵豆粉粗多糖体外降血糖、降血脂活性比较. 食品工业科技. 2024(12): 140-150 . 本站查看
3.	许梦粤，余金毅，李慧，刘琴，曾长立，王红波. 不同品种纳豆的多种功能活性成分比较. 食品工业科技. 2024(13): 140-149 . 本站查看
4.	屈雅宁，吴子龙，陈禅友，万何平，王亚珍，曾长立，王红波. 食用豆类多糖提取纯化、结构特征与生物活性研究进展. 中国调味品. 2024(07): 200-207 .
5.	王芳，刘红，李燕红，龚田. 大黄多糖的提取及其抗氧化和降血糖活性研究. 海峡药学. 2024(07): 8-12 .
6.	屈雅宁，许梦粤，李慧，王璐，郭瑞，王红波. 干酪乳杆菌发酵豆粉粗多糖结构特征与抗氧化活性. 中国食品学报. 2024(08): 112-121 .
7.	许梦粤，田鑫，王君可，武傲雪，邹芷怡，吴佳辉，王红波. 菜豆多糖与香菇多糖结构特征以及体外重要生物活性综合对比评价. 食品科学. 2024(23): 2318-2327 .
8.	李雪艳，罗钰颖，杨华杰，张雄，金浩鑫，杨双玲，饶毅，魏惠珍. 六神曲微生物菌群及功能性成分研究进展. 江西中医药大学学报. 2024(06): 116-120+128 .
9.	刘凯青，张涵，殷澳，张会佳，侯相竹，冯晨曦，高阳，徐多多. 苦碟子注射液中多糖的提取分离、结构表征及其抗氧化活性的研究. 中国医院药学杂志. 2023(23): 2618-2623 .