Recombinant expressions of sweet plant protein mabinlin II in Escherichia coli and food-grade Lactococcus lactis

Original Paper

Abstract

Sweet plant proteins, which are safe, natural, low-calorie sweeteners, may be suitable replacements for sugars in the food and beverage industries. Mabinlin II, a sweet plant protein, shows the most pronounced heat stability and acid resistance of any of the six known types of plant sweet proteins. However, mabinlin II is difficult to extract from the Capparis masaikai plant, which is itself becoming increasingly scarce. This limits the use of naturally acquired mabinlin II. In this study, recombinant mabinlin II proteins were expressed and purified in Escherichia coli and in food-grade Lactococcus lactis. Recombinant mabinlin II proteins MBL-BH (containing the B-chains of mabinlinII downstream fused with His-tag) and MBL-ABH (containing the A- and B-chains of mabinlinII downstream fused with His-tag) were expressed in E. coli in the form of inclusion bodies. They were then purified and renatured. The refolded MBL-BH was found to be 100 times sweeter than sucrose by weight, but it was not heat-stable. Refolded MBL-ABH was neither sweet nor heat-stable. Recombinant mabinlin II proteins were secreted and expressed intracellularly in food-grade L. lactis, in which the concentrated cell samples and culture medium samples were detected using enzyme-linked immunosorbent assay and Western blotting analysis with anti-mabinlin II polyclonal antibody. This study demonstrated that the single B chain of mabinlin II has a sweet taste. The recombinant mabinlin II proteins have been successfully expressed in food-grade L. lactis, which is a crucial step in the production of mabinlin II through microorganism expression systems.

Keywords

Sweet protein Mabinlin II Recombinant expression Escherichia coli Lactococcus lactis 

References

  1. Assadi-Porter FM, Aceti DJ, Cheng H, Markley JL (2000) Efficient production of recombinant brazzein, a small, heat-stable, sweet-tasting protein of plant origin. Arch Biochem Biophys 376(2):252–258CrossRefGoogle Scholar
  2. Berlec A, Jevnikar Z, Majhenic AC, Rogelj I, Strukelj B (2006) Expression of the sweet-tasting plant protein brazzein in Escherichia coli and Lactococcus lactis: a path toward sweet lactic acid bacteria. Appl Microbiol Biotechnol 73(1):158–163CrossRefGoogle Scholar
  3. Berlec A, Tompa G, Slapar N, Fonovic UP, Rogelj I, Strukelj B (2008) Optimization of fermentation conditions for the expression of sweet-tasting protein brazzein in Lactococcus lactis. Lett Appl Microbiol 46(2):227–231CrossRefGoogle Scholar
  4. Bogovic-Matijasic B, Rogelj I, Batic M, Raspor P (2001) Influence of pH on bacteriocin production by Lactobacillus K7 during batch fermentation. Period Biol 103(2):163–167Google Scholar
  5. Cheigh CI, Choi HJ, Park H, Kim SB, Kook MC, Kim TS, Hwang JK, Pyun YR (2002) Influence of growth conditions on the production of a nisin-like bacteriocin by Lactococcus lactis subsp. lactis A164 isolated from kimchi. J Biotechnol 95(3):225–235CrossRefGoogle Scholar
  6. Chen Z, Cai H, Lu F, Du L (2005) High-level expression of a synthetic gene encoding a sweet protein, monellin, in Escherichia coli. Biotechnol Lett 27(22):1745–1749CrossRefGoogle Scholar
  7. Chen Z, Heng C, Li Z, Liang X, Xinchen S (2007) Expression and secretion of a single-chain sweet protein monellin in Bacillus subtilis by sacB promoter and signal peptide. Appl Microbiol Biotechnol 73:1377–1381CrossRefGoogle Scholar
  8. Daniell S, Mellits KH, Faus I, Connerton I (2000) Refolding the sweet-tasting protein thaumatin II from insoluble inclusion bodies synthesised in Escherichia coli. Food Chem 71(1):105–110CrossRefGoogle Scholar
  9. de Ruyter PG, Kuipers OP, de Vos WM (1996) Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl Environ Microbiol 62(10):3662–3667Google Scholar
  10. Ding M, Hu Z (1986) Study on mabinlin, a sweet protein from the seeds of C. masaikai Levl. IV: stability and denaturation. Acta Bot Yunanica 8(2):181–192Google Scholar
  11. Elliker PR, Anderson AW, Hannesson G (1956) An agar medium for lactic acid streptococci and lactobacilli. J Dairy Sci 39(11):1611–1612CrossRefGoogle Scholar
  12. Faus I (2000) Recent developments in the characterization and biotechnological production of sweet-tasting proteins. Appl Microbiol Biotechnol 53(2):145–151CrossRefGoogle Scholar
  13. Holo H, Nes IF (1995) Transformation of Lactococcus by electroporation. In: Nickoloff JA (ed) Methods in molecular biology, vol 47., Electroporation protocols for microorganismsHumana Press, Totowa, pp 195–199Google Scholar
  14. Hu Z, He M (1983) Study on mabinlin, a sweet protein from the seeds of C. masaikai Levl. I: extraction, purification and certain characteristics. Acta Bot Yunanica 5(2):207–212Google Scholar
  15. Hu Z, Liang HX, Liu XZ (1985a) Study on mabinlin, a sweet protein from the seeds of C. masaikai Levl. III: storage site and state. Acta Bot Yunanica 7(2):187–194Google Scholar
  16. Hu Z, Peng LP, He M (1985b) Study on mabinlin, a sweet protein from the seeds of C. masaikai Levl. II: comparison of mabinlin I to mabinlin II. Acta Bot Yunanica 7(1):1–10Google Scholar
  17. Kondo K, Miura Y, Sone H, Kobayashi K, Iijima H (1997) High-level expression of a sweet protein, monellin, in the food yeast Candida utilis. Nat Biotechnol 15:453–457CrossRefGoogle Scholar
  18. Li C, Bai JH, Cai ZL, Fan OY (2002a) Optimization of a culture medium for bacteriocin production by Lactococcus lactis using response surface methodology. J Biotechnol 93(1):27–34CrossRefGoogle Scholar
  19. Li XD, Staszewski L, Xu H, Durick K, Zoller M, Adler E (2002b) Human receptors for sweet and umami taste. PNAS 99(7):4692–4696CrossRefGoogle Scholar
  20. Li DF, Jiang PH, Zhu DY, Hu YL, Max M, Wang DC (2008) Crystal structure of mabinlin II: a novel structural type of sweet proteins and the main structural basis for its sweetness. J Struct Biol 162(1):50–62CrossRefGoogle Scholar
  21. Liu XZ, Maeda S, Hu Z, Aiuchi T, Nakaya K, Kurihara Y (1993) Purification, complete amino acid sequence and structural characterization of the heat-stable sweet protein, mabinlin II. Eur J Biochem 211(1–2):281–287CrossRefGoogle Scholar
  22. Masuda T, Tamaki S, Kaneko R, Wada R, Fujita Y, Mehta A, Kitabatake N (2004) Cloning, expression, and characterization of recombinant sweet-protein thaumatin II using the methylotrophic yeast Pichia pastoris. Biotechnol Bioeng 85(7):761–769CrossRefGoogle Scholar
  23. Mierau I, Olieman K, Mond J, Smid EJ (2005) Optimization of the Lactococcus lactis nisin-controlled gene expression system NICE for industrial applications. Microb Cell Fact 4:16CrossRefGoogle Scholar
  24. Nirasawa S, Nishino T, Katahira M, Uesugi S, Hu Z, Kurihara Y (1994) Structures of heat-stable and unstable homologues of the sweet protein mabinlin. The difference in the heat stability is due to replacement of a single amino acid residue. Eur J Biochem 223(3):989–995CrossRefGoogle Scholar
  25. Nirasawa S, Masuda Y, Nakaya K, Kurihara Y (1996) Cloning and sequencing of a cDNA encoding a heat-stable sweet protein, mabinlin II. Gene 181(1–2):225–227CrossRefGoogle Scholar
  26. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  27. Schägger H (2006) Tricine–SDS-PAGE. Nat Protoc 1(1):16–22CrossRefGoogle Scholar
  28. Zhang XJ, Duan GC, Zhang RG (2009) Optimized expression of Helicobacter pylori ureB gene in the Lactococcus lactis nisin-controlled gene expression (NICE) system and experimental study of its immunoreactivity. Curr Microbiol 58(4):308–314CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  1. 1.Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and BiotechnologyChinese Academy of Tropical Agricultural SciencesHaikouChina
  2. 2.College of AgricultureHainan UniversityHaikouChina
  3. 3.Spice and Beverage Research InstituteChinese Academy of Tropical Agricultural SciencesWanningChina

Personalised recommendations