Sweet-tasting Protein Brazzein Research Articles

Study on the Thermal Stability of the Sweet-Tasting Protein Brazzein Based on Its Structure-Sweetness Relationship.

Brazzein (Brz) is a sweet-tasting protein composed of 54 amino acids and is considered as a potential sugar substitute. The current methods for obtaining brazzein are complicated, and limited information is available regarding its thermal stability. In this study, we successfully expressed recombinant brazzein, achieving a sweetness threshold of 15.2 μg/mL. Subsequently, we conducted heat treatments at temperatures of 80, 90, 95, and 100 °C for a duration of 2 h to investigate the structural changes in the protein. Furthermore, we employed hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) to analyze the effect of heating on the protein structure-sweetness relationships. Our results indicated that the thermal inactivation process primarily affects residues 6-14 and 36-45 of brazzein, especially key residues Tyr8, Tyr11, Ser14, Glu36, and Arg43, which are closely associated with its sweetness. These findings have significant implications for improving the thermal stability of brazzein.

Functional Characterization of the Venus Flytrap Domain of the Human TAS1R2 Sweet Taste Receptor.

The human sweet taste receptor is a heterodimeric receptor composed of two distinct G-protein-coupled receptors (GPCRs), TAS1R2 and TAS1R3. The TAS1R2 and TAS1R3 subunits are members of a small family of class C GPCRs whose members share the same architecture, comprising a Venus Flytrap (VFT) module linked to the seven transmembrane domains (TMDs) by a short cysteine-rich region (CRR). The VFT module of TAS1R2 contains the primary binding site for most of the sweet-tasting compounds, including natural sugars and artificial and natural sweeteners. However, cellular assays, molecular docking and site-directed mutagenesis studies have revealed that the VFT, CRR and TMD of TAS1R3 interact with some sweeteners, including the sweet-tasting protein brazzein. The aim of this study was to better understand the contribution of TAS1R2-VFT in the binding of sweet stimuli. To achieve this, we heterologously expressed human TAS1R2-VFT (hTAS1R2-VFT) in Escherichia coli. Circular dichroism spectroscopic studies revealed that hTAS1R2-VFT was properly folded with evidence of secondary structures. Using size-exclusion chromatography coupled with light scattering, we found that hTAS1R2-VFT behaves as a monomer. Ligand binding quantified by intrinsic tryptophan fluorescence showed that hTAS1R2-VFT is capable of binding sweet stimuli with Kd values, in agreement with physiological detection. Furthermore, we investigated whether the impact of point mutations, already shown to have deleterious effects on cellular assays, could impact the ability of hTAS1R2-VFT to bind sweet ligands. As expected, the ligand affinities of hTAS1R2-VFT were drastically reduced through the introduction of single amino acid substitutions (D278A and E382A) known to abolish the response of the full-length TAS1R2/TAS1R3 receptor. This study demonstrates the feasibility of producing milligram quantities of hTAS1R2-VFT to further characterize the mechanism of binding interaction and perform structural studies.

Functional expression of the sweet-tasting protein brazzein in transgenic tobacco

The sweet-tasting protein, brazzein, has potential as a low-calorie sugar substitute owing to its high sweetness, stability, and water solubility. In this study, the synthetic brazzein gene was expressed in the tobacco plant, Nicotiana tabacum. Three types of expression cassettes containing the brazzein gene were constructed to examine the expression and purification efficiency of the brazzein: pBI-BZ1 containing a signal sequence and His-tag, pBI-BZ2 containing a signal sequence, and pBI-BZ3 containing only the brazzein gene. Brazzein expression confirmed by ELISA was purified using ammonium sulfate precipitation, heat treatment, and CM-sepharose chromatography. The purity and conformational state of the brazzein were confirmed using SDS-PAGE, HPLC, and circular dichroism. The identity of the brazzein was confirmed by N-terminal amino acid analysis, ESI-MS/MS, and sweetness analysis. We successfully generated brazzein overexpression tobacco plants, suggesting that this method could be used as a brazzein production platform to provide an alternative to currently produced sweeteners.

Efficient brazzein production in yeast (Kluyveromyces lactis) using a chemically defined medium.

The sweet-tasting protein brazzein offers considerable potential as a functional sweetener with antioxidant, anti-inflammatory, and anti-allergic properties. Here, we optimized a chemically defined medium to produce secretory recombinant brazzein in Kluyveromyces lactis, with applications in mass production. Compositions of defined media were investigated for two phases of fermentation: the first phase for cell growth, and the second for maximum brazzein secretory production. Secretory brazzein expressed in the optimized defined medium exhibited higher purity than in the complex medium; purification was by ultrafiltration using a molecular weight cutoff, yielding approximately 107mgL-1. Moreover, the total media cost in this defined medium system was approximately 11% of that in the optimized complex medium to generate equal amounts of brazzein. Therefore, the K. lactis expression system is useful for mass-producing recombinant brazzein with high purity and yield at low production cost and indicates a promising potential for applications in the food industry.

Removal of the N-terminal methionine improves the sweetness of the recombinant expressed sweet-tasting protein brazzein and its mutants in Escherichia coli.

The sweet-tasting protein brazzein is the smallest sweet protein with high sweet potency. Overexpression of this protein in a heterogenous host is an essential way for its production in food industry. In this study, the gene of minor form brazzein was cloned into the pET-SUMO vector with optimized codon usage and expressed in E. coli BL21-CodonPlus (DE3)-RIL. The recombinant protein in absence of the N-terminal methionine displayed a sweetness threshold about 1.5μg/ml, which is the sweetest brazzein protein reported up to now. The unexpected sweet potency of the protein was validated by a series of mutants (7Val→Arg, 9Glu→Lys and 9Glu→Asp), in which E9K exhibited about 50% enhancement of sweetness than the wild type. The superior sweetness of recombinant brazzein and its sweeter mutants suggest their potential applications in food and beverages. PRACTICAL APPLICATIONS: The sweet-tasting proteins are natural, low-, or non-caloric and nutritive, showing to be promising replacers of sugars and artificial sweeteners, and can be used as sweet additives in the fields of food, medicine, and biotechnology. In the present study, we report that the recombinant brazzein protein expressed in E. coli exhibits superior and improved sweetness than those previously reported. Furthermore, sweeter mutants were obtained with the expression procedure. The superior sweetness of recombinant brazzein and its sweeter mutants suggest their potential applications in food, beverages, and other fields.

Functional expression of recombinant sweet-tasting protein brazzein by Escherichia coli and Bacillus licheniformis

ABSTRACTBrazzein is an attractive sweetener candidate because of its sugar-like taste, high sweetness, and good stability at high temperature and wide pH range. This study was aimed to express and purify bioactive recombinant brazzein (rBrazzein). The rBrazzein gene was synthesized according to the preferred codons of Bacillus subtilis and successfully expressed in Escherichia coli and Bacillus licheniformis. In E. coli host, lower induction temperature of 30°C increased soluble rBrazzein (Ebrazzein) at high level. In B. licheniformis host, two signal peptides (Sec type and Tat type) were evaluated for the expression of rBarzzein in B. subtilis and B. licheniformis. However, only the Sec-type signal peptide guided the secretion expression of rBrazzein in B. licheniformis. The rBrazzein was expressed steadily and the highest yield reached about 57 mg/L at 36 h by small-scale fermentation. The purification procedure of rBrazzein by B. licheniformis (Bbrazzein) was thus established. Approximately 5 mg/L purified rBrazzein was obtained and the purity was 85%. The conformational state of rBrazzeins was confirmed by circular dichroism. The bioactivities of rBrazzeins were evaluated by sweet taste testing. The Bbrazzein and Ebrazzein were 266 times and 400 times sweeter than sucrose on a weight basis, respectively. The formation of disulfide bonds were both confirmed by LC/MS/MS and MALDI-TOF. The CD analysis indicated that Ebrazzein has a similar secondary structure with natural brazzein, which explained why Ebrazzein had a higher intensity of sweetness. This study demonstrated that B. licheniformis system is useful to produce active recombinant brazzein, and has potential food industry applications.

Optimized production and quantification of the tryptophan-deficient sweet-tasting protein brazzein in Kluyveromyces lactis

The sweet-tasting protein brazzein is a candidate sugar substitute owing to its sweet, sugar-like taste and good stability. To commercialize brazzein as a sweetener, optimization of fermentation and purification procedure is necessary. Here, we report the expression conditions of brazzein in the yeast Kluyveromices lactis and purification method for maximum yield. Transformed K. lactis was cultured in YPGlu (pH 7.0) at 25 °C and induced by adding glucose:galactose at a weight ratio of 1:2 (%/%) during the stationary phase, which increased brazzein expression 2.5 fold compared to the previous conditions. Cultures were subjected to heat treatment at 80 °C for 1 h, and brazzein containing supernatant was purified using carboxymethyl-sepharose cation exchange chromatography using 50 mM NaCl in 50 mM sodium acetate buffer (pH 4.0) as a wash buffer and 400 mM NaCl (pH 7.0) for elution. The yield of purified brazzein under these conditions was 2.0-fold higher than that from previous purification methods. We also determined that the NanoOrange assay was a suitable method for quantifying tryptophan-deficient brazzein. Thus, it is possible to obtain pure recombinant brazzein with high yield in K. lactis using our optimized expression, purification, and quantification protocols, which has potential applications in the food industry.

Antioxidant, anti-inflammatory, and anti-allergic activities of the sweet-tasting protein brazzein

Sweet-tasting proteins may be useful as low-calorie sugar substitutes in foods, beverages, and medicines. Brazzein is an attractive sweetener because of its high sweetness, sugar-like taste, and good stability at high temperature and wide pH ranges. To investigate the bioactivities of brazzein, the antibacterial, antifungal, antioxidant, anti-inflammatory, and anti-allergic activities were determined in vitro. Brazzein showed no antibacterial and antifungal activities, although it showed approximately 45% or greater similarity to defensin, which has antimicrobial effects, and drosomycin, which is used as an antifungal agent. However, brazzein exhibited strong antioxidant effects, showing ABTS radical scavenging activity (IC50=12.55μM) and DPPH activity (IC50>30μM). Brazzein also showed anti-inflammatory activity and anti-allergic activity in a β-hexosaminidase assay (IC50>15μM) and cyclooxygenase-2 inhibition assay (IC50=12.62μM), respectively. These results suggest that brazzein has antioxidant, anti-inflammatory, and anti-allergic activities and considerable potential as a functional sweetener.

Role of Lys5 Residue in β-Strand I of the Sweet-Tasting Protein Brazzein

To identify critical residues responsible for sweetness in brazzein and elucidate the interaction mechanisms of brazzein with the sweet taste receptor, three mutants of Lys5 residue in N-terminal β-strand I of brazzein were constructed by site-directed mutagenesis. Mutations of Lys to Asp or Glu at position 5 of brazzein significantly decreased its sweetness, while mutation of Lys5 to Arg resulted in a molecule with slightly decreased sweetness to des-pE1M-brazzein. From these results, it is suggested that the positive charge of Lys5 in β-strand I of brazzein is essential for its function and necessary for structural integrity. Practical Applications Brazzein is a sweet-tasting protein which has been isolated from the fruit of the West African plant Pentadiplandra brazzeana Baillon and is 500 to 2,000 times sweeter than sucrose. Brazzein is attractive as a candidate sweetener for control of obesity, because of its potential sweetness, sugar-like taste and good stability. The present study offers information on the precise interaction mechanism of brazzein with human sweet taste receptor responsible for the sweetness of brazzein, and can be of great value in future design of sweeter brazzein variants.

Design and Evaluation of Synthetic Peptides Corresponding to the Sweetness Loop of the Sweet-Tasting Protein Brazzein

E-mail: khkong@cau.ac.krReceived May 14, 2014, Accepted July 19, 2014Key Words : Brazzein, Model building, Peptide, Sweetness loop, Sweet-tasting proteinHumans recognize five tastes, namely, sweet, bitter, sour,salty, and umami, through taste buds on the tongue. Thesefive tastes give organism essential information with which toevaluate the nutritional components of food, as well asprevent the intake of toxic substances. The desirable tastesof umami and sweetness promote consumption of nutritivefood. On the other hand, bitterness and sourness alertorganisms to toxins and promote rejection of harmful foods.Identifying sweet-tasting foods is particularly important as itprovides organisms with a means to seek out carbohydrateswith high nutritive value.

Efficient secretory expression of the sweet-tasting protein brazzein in the yeast Kluyveromyces lactis

Brazzein is an intensely sweet-tasting protein with high water solubility, heat stability, and taste properties resembling those of carbohydrate sweeteners. In the present study, we describe the expression of the synthetic gene encoding brazzein, a sweet protein in the yeast Kluyveromyces lactis. The synthetic brazzein gene was designed based on the biased codons of the yeast, so as to optimize its expression, as well as on the extracellular secretion for expression in an active, soluble form. The synthesized brazzein gene was cloned into the secretion vector pKLAC2, which contains the yeast prepropeptide signal from the Saccharomycescerevisiae α-mating factor. The constructed plasmid pKLAC2-des-pE1M-brazzein was introduced into the yeast K. lactis GG799. The yeast transformants were cultured for high-yield secretion of the recombinant des-pE1M-brazzein in YPGal medium for 96h at 30°C. The expressed recombinant des-pE1M-brazzein was purified by CM-Sepharose column chromatography and approximately 104mg/L was obtained. The purity and conformational state of the recombinant des-pE1M-brazzein were confirmed using SDS–PAGE, HPLC, and circular dichroism. The identity of the recombinant protein was also confirmed by N-terminal amino acid analysis and taste testing. The purified recombinant des-pE1M-brazzein had an intrinsic sweetness in its minor form, approximately 2130 times sweeter than sucrose on a weight basis. These results demonstrate that the K. lactis expression system is useful for producing the recombinant brazzein in active form at a high yield with attributes useful in the food industry.

Expression of the sweet-tasting protein brazzein in Lactobacillus spp.

As sucrose has been implicated in healthrelated problems such as obesity and diabetes, awareness of a safe natural non-sugar sweetener has increased. Brazzein is the intensely sweet-tasting plant protein which consists of 54 amino acids. In this study, brazzein was expressed in Lactobacillus spp., probiotics, which are widely used in food industry. Three Lactobacillus spp. (Lb. paracasei KLB58, Lb. plantarum KLB213, and Lb. reuteri KCTC3594) were electrotransformed with pMSP3545::Braht which contains brazzein gene along with nisin-controlled expression (NICE) system. Brazzein expression in Lactobacillus spp. was confirmed by Western blot analysis. Overall, higher expression rate was obtained with nisin concentration of 25–40 ng/mL.

Residue mutations in the sweetness loops for the sweet-tasting protein brazzein

To identify critical residues, important for sweetness, of the sweet protein brazzein, 11 mutants of the residues in three loops of brazzein were constructed by site-directed mutagenesis. We found that mutations of Glu41 to Ala, Lys, or Arg at position 41 in loop 40–43 made the molecules significantly sweeter than brazzein, while mutations at two distant residues (changing Arg43 to Lys or Glu) decreased sweetness. A similar pattern occurred at loop 30–33, where mutation of the His31 to Arg significantly increased sweetness, while mutations at positions 30 or 33 in the immediate vicinity of this region significantly decreased sweetness. In addition, a Gln17 residue in the loop 9–19 was necessary for structural integrity. From these results, we suggest that the loops containing His31 and Glu41 are critical regions of the molecule for eliciting sweetness, and the charge and/or structure of the side chain of these residues play an important role in the multi-point interactions between brazzein and the sweet-taste receptor.

Optimization of fermentation conditions for the expression of sweet-tasting protein brazzein in Lactococcus lactis

To improve the production of sweet-tasting protein brazzein in Lactococcus lactis using controlled fermentation conditions. The nisin-controlled expression system was used for brazzein expression. The concentration of nisin for induction and the optical density (OD) at induction were therefore optimized, together with growth conditions (medium composition, pH, aerobic growth in the presence of hemin). Brazzein was assayed with ELISA on Ni-NTA plates and Western blot. Use of the M-17 medium, containing 2.5% glucose, anaerobic growth at pH 5.9 and induction with 40 ng ml(-1) nisin at OD 3.0 led to an approx. 17-fold increase in brazzein per cell production compared to non-optimized starting conditions. Aerobic growth in the presence of hemin did not increase the production. Considerable increase in brazzein per cell production was obtained at optimized fermentation conditions. Optimized growth conditions could be used in application of brazzein expression in L. lactis. The importance of pH and OD at induction contributes to the body of knowledge of optimal recombinant protein expression in L. lactis. The new assay for brazzein quantification was introduced.

Solution structure of the thermostable sweet-tasting protein brazzein.

The fruit of Pentadiplandra brazzeana Baillon contains a small, sweet-tasting protein named brazzein. The structure of brazzein in solution was determined by proton nuclear magnetic resonance spectroscopy at pH 5.2 and 22 degrees C. The brazzein fold, which contains one alpha-helix and three strands of antiparallel beta-sheet, does not resemble that of either of the other two sweet-tasting proteins with known structures, monellin and thaumatin. Instead, the structure of brazzein resembles those of plant gamma-thionins and defensins and arthropod toxins. Sequence comparisons predict that members of a newly-identified family of serine proteinase inhibitors share the brazzein fold.