[Sweet protein brazzein as a promising sweetener].

The excessive consumption of sugar-containing foods contributes to the development of a number of diseases, including obesity, diabetes mellitus, etc. As a substitute for sugar, people with diabetes mellitus and obesity most often use sweeteners. Sweet proteins, in particular brazzein, are an alternative to synthetic sweeteners that have natural origin, are broken down in the intestines along with food proteins, and do not affect blood sugar and insulin levels. The purpose of the review was to analyze the available data on the sweet protein brazzein, its physical and chemical properties, existing biotechnological methods of production, and prospects for application in the food industry in order to further develop an optimized heterologous expression system. Material and methods. Google Scholar, Scopus, Web of Science, PubMed, RSCI and eLibrary.ru databases were used for collecting and analyzing literature. Search depth - 30 years. Results. Numerous studies of the physical and chemical properties of brazzein have demonstrated its high potential for use in the food industry. In particular, a short amino acid sequence, thermal stability, the ability to maintain its structure and sweet properties in a wide pH range, hypoallergenicity, lack of genotoxicity, and an extremely high level of sweetness compared to sucrose allow us to conclude that its use is promising. Mutant variants of brazzein have been generated, the sweetest of which (with three amino acid substitutions H31R/E36D/E41A) exceeds sucrose sweetness by 22 500 times. To date, various systems for the expression of recombinant brazzein have already been developed, in which bacteria (Escherichia coli, Lactococcus lactis, Bacillus licheniformis), yeast (Komagataella phaffii, Kluyveromyces lactis, Saccharomyces cerevisiae), plants (Zea mays, Oryza sativa, Lactuca sativa, Nicotiana tabacum, Daucus carota) and animals (Mus musculus) have been used. Conclusion. Due to its high sweetness, organoleptic properties and long history of human consumption, brazzein can be considered as a promising natural sweetener. Despite the short peptide sequence, the production of the recombinant protein faced a number of problems, including low protein yield (for example, it could only be detected in mouse milk by Western blot hybridization) and loss of sweetness. Thus, further optimization of the process is necessary for widespread brazzein use in the food industry, which includes the selection of an adequate producer and the use of extracellular expression systems to reduce the final cost of the product.

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 107 mg L-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.

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.

Importance of Glu53 in the C-terminal region of brazzein, a sweet-tasting protein.

BACKGROUND: The sweetness of brazzein, one of the known sweet proteins, is dependent on charges and/or structures of its specific amino acid side chains. As the residues in the C-terminus of brazzein are known to play a critical role in sweetness, the currently unknown function of Glu53 requires further study. RESULTS: To identify important residues responsible for the sweetness of the protein brazzein, four mutants of the Glu53 residue in the C-terminal region of des-pE1M-brazzein, which lacks the N-terminal pyroglutamate, were constructed using site-directed mutagenesis. Mutations of Glu53 substitution to Ala or Asp significantly decreased the sweetness. On the other hand, a Lys mutation resulted in a molecule with sweetness similar to that of des-pE1M-brazzein. Mutation of Glu53 to Arg resulted in a molecule significantly sweeter than des-pE1M-brazzein, which agrees with previous findings showing that mutation with positively charged residues results in a sweeter protein. CONCLUSION: Our results suggest that the residue at position 53 is crucial for the sweetness of brazzein, which may be interacting with the sweet-taste receptor. © 2015 Society of Chemical Industry.

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.

The structure of brazzein, a sweet-tasting protein from the wild African plant Pentadiplandra brazzeana.

Brazzein is the smallest sweet-tasting protein and was isolated from the wild African plant Pentadiplandra brazzeana. The brazzein molecule consists of 54 amino-acid residues and four disulfide bonds. Here, the first crystal structure of brazzein is reported at 1.8 Å resolution and is compared with previously reported solution structures. Despite the overall structural similarity, there are several remarkable differences between the crystal and solution structures both in their backbone folds and side-chain conformations. Firstly, there is an additional α-helix in the crystal structure. Secondly, the atomic r.m.s.d.s between the corresponding C(α)-atom pairs are as large as 2.0-2.2 Å between the crystal and solution structures. Thirdly, the crystal structure exhibits a molecular shape that is similar but not identical to the solution structures. The crystal structure of brazzein reported here will provide additional information and further insights into the intermolecular interaction of brazzein with the sweet-taste receptor.

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 96 h at 30°C. The expressed recombinant des-pE1M-brazzein was purified by CM-Sepharose column chromatography and approximately 104 mg/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.

Brazzein and Monellin: Chemical Analysis, Food Industry Applications, Safety and Quality Control, Nutritional Profile and Health Impacts.

Recently, customers have been keener to buy products manufactured using all-natural ingredients with positive health properties, but without losing flavor. In this regard, the objective of the current study is to review the consumption of brazzein and monellin, their nutritional profiles and health effects, and their potential applications in the food industry. This poses challenges with sustainability and important quality and safety indicators, as well as the chemical processes used to determine them. To better understand the utilization of brazzein and monellin, the chemical analysis of these two natural sweet proteins was also reviewed by placing particular emphasis on their extraction methods, purification and structural characterization. Protein engineering is considered a means to improve the thermal stability of brazzein and monellin to enhance their application in food processing, especially where high temperatures are applied. When the quality and safety of these sweet proteins are well-investigated and the approval from safety authorities is secured, the market for brazzein and monellin as food ingredient substitutes for free sugar will be guaranteed in the future. Ultimately, the review on these two natural peptide sweeteners increases the body of knowledge on alleviating problems of obesity, diabetes and other non-communicable diseases.

A sweeter future: Using protein language models for exploring sweeter brazzein homologs.

With growing concerns over the health impact of sugar, brazzein offers a viable alternative due to its sweetness, thermostability, and low risk profile. Here, we demonstrated the ability of protein language models to design new brazzein homologs with improved thermostability and potentially higher sweetness, resulting in new diverse optimized amino acid sequences that improve structural and functional features beyond what conventional methods could achieve. This innovative approach resulted in the identification of unexpected mutations, thereby generating new possibilities for protein engineering. To facilitate the characterization of the brazzein mutants, a simplified procedure was developed for expressing and analyzing related proteins. This process involved an efficient purification method using Lactococcus lactis (L. lactis), a generally recognized as safe (GRAS) bacterium, as well as taste receptor assays to evaluate sweetness. The study successfully demonstrated the potential of computational design in producing a more heat-resistant and potentially more palatable brazzein variant, V23.

Bioprospecting and biotechnological insights into sweet-tasting proteins by microbial hosts-a review.

Owing to various undesirable health effects of sugar overconsumption, joint efforts are being made by industrial sectors and regulatory authorities to reduce sugar consumption practices, worldwide. Artificial sweeteners are considered potential substitutes in several products, e.g., sugar alcohols (polyols), high-fructose corn syrup, powdered drink mixes, and other beverages. Nevertheless, their long-standing health effects continue to be debatable. Consequently, growing interest has been shifted in producing non-caloric sweetenersfrom renewable resources to meet consumers' dietary requirements. Except for the lysozyme protein, various sweet proteins including thaumatin, mabinlin, brazzein, monellin, miraculin, pentadin, and curculin have been identified in tropical plants. Given the high cost and challenging extortion of natural resources, producing these sweet proteins using engineered microbial hosts, such as Yarrowia lipolytica, Pichia pastoris, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Pichia methanolica, Saccharomyces cerevisiae, and Kluyveromyces lactis represents an appealing choice. Engineering techniques can be applied for large-scale biosynthesis of proteins, which can be used in biopharmaceutical, food, diagnostic, and medicine industries. Nevertheless, extensive work needs to be undertaken to address technical challenges in microbial production of sweet-tasting proteins in bulk. This review spotlights historical aspects, physicochemical properties (taste, safety, stability, solubility, and cost), and recombinant biosynthesis of sweet proteins. Moreover, future opportunities for process improvement based on metabolic engineering strategies are also discussed.

Expression of a triple mutational des-pGlu brazzein in transgenic mouse milk.

Brazzein has excellent potential for use as a sweetener because of its high level of sweetening potency and stability against extreme temperature and pH. It is extracted from the tropical and difficult-to-cultivate African plant Pentadiplandra brazzeana, which hampers its commercial viability. Here we report the mammary-specific expression of wildtype or triple mutational (H31R/E36D/E41A) des-pGlu brazzeins in the milk of transgenic mice. Using enzyme-linked immunoassay (ELISA), western blot, and sweetness intensity testing, we confirmed that the triple mutation made the des-pGlu brazzein molecule 10,000 times sweeter than sucrose in a weight base, even after 10 min of incubation at 100 °C; in addition, the triple mutant was also significantly sweeter than the wildtype des-pGlu brazzein. This study provides new insights for producing brazzein or brazzein-sweetened milk from animals for use in food and healthcare applications.

Comparison of Different Signal Peptides for the Efficient Secretion of the Sweet-Tasting Plant Protein Brazzein in Pichia pastoris.

Brazzein is a small sweet-tasting protein found in the red berries of a West African evergreen shrub, Pentadiplandra brazzeana Baillon. Brazzein is highly soluble and stable over a large pH range and at high temperatures, which are characteristics that suggest its use as a natural sweetener. However, Pentadiplandra brazzeana culture is difficult at a large scale, limiting the natural source of brazzein. Heterologous expression of brazzein has been established in numerous systems, including bacteria, yeast, and transgenic plants. Brazzein requires four disulfide bonds to be active in eliciting an intense sweet taste, and the yeast Pichia pastoris appears to be one of the best options for obtaining functional brazzein in high quantities. Employing yeast secretion in the culture medium allows us to obtain fully active brazzein and facilitate purification later. To increase yeast secretion, we compared seven different signal peptides to successfully achieve brazzein secretion using the yeast P. pastoris. The brazzein proteins corresponding to these signal peptides elicited activation of the sweet taste receptor functionally expressed in a cellular assay. Among these tested signal peptides, three resulted in the secretion of brazzein at high levels.

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 1.5 µg/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 â†’ Arg, 9Glu â†’ Lys and 9Glu â†’ 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.

3M-Brazzein as a Natural Sugar Substitute Attenuates Obesity, Metabolic Disorder, and Inflammation.

Obesity is a global chronic disease linked to various diseases. Increased consumption of added sugars, especially in beverages, is a key contributor to the obesity epidemic. It is essential to reduce or replace sugar intake with low-calorie sweeteners. Here, a natural sweet protein, 3M-brazzein, was investigated as a possible sugar substitute. Mice were exposed to 3M-brazzein or 10% sucrose of equivalent sweetness, in drinking water to mimic human obesity development over 15 weeks. Consumption of 3M-brazzein in liquid form did not cause adiposity hypertrophy, resulting in 33.1 ± 0.4 g body weight and 0.90 ± 0.2 mm fat accumulation, which were 35.9 ± 0.7 g (p = 0.0094) and 1.53 ± 0.067 mm (p = 0.0031), respectively, for sucrose supplement. Additionally, 3M-brazzein did not disrupt glucose homeostasis or affect insulin resistance and inflammation. Due to its naturally low-calorie content, 3M-brazzein could also be a potential sugar substitute that reduces adiposity.

Expression of Brazzein, a Small Sweet-Tasting Protein in Saccharomyces cerevisiae: An Introduction for Production of Sweet Yeasts.

BACKGROUND: The replacement of carbohydrate sweeteners with protein sweeteners from plants has attracted the interest of researchers because these proteins don't trigger the insulin response and are more nutritive for consumption in food. Brazzein (Braz) is a small and heat- stable sweet protein that has been originally derived from African plant Pentadiplandra brazzeana. In the present work the solubility, sweetness and yield of recombinant forms of Braz in two expression hosts, E. coli and S. cerevisiae were comprised. METHODS: The codon-optimized gene of Braz was cloned in expression vectors pET28a and pET41a and GPD. The resulted vectors pET28a-Braz and pEt41a-Braz were transformed into Escherichia coli strain Rosetta (DE3) and the vector GPD-Braz was transformd to S. cerevisiae. The expression of Braz in different systems was analyzed by SDS-PAGE and western blotting. RESULTS: The results verified the heterologous expression of Braz in S. cerevisiae carrying GPDBraz. Also the expression of Braz as carboxy-terminal extensions of His-tag and Glutathione-STransferase (GST) were verified in transgenic E. coli containing pET28a-Braz and pET41a-Braz, respectively. CONCLUSION: Although the yield of GST-Braz was higher than His-Braz and Braz expressed in S. cerevisiae, but the higher solubility, sweetness, safety (GRAS) are important advantages of the use of S. cerevisiae as expression host for production of Braz. Therefore the result of present work opens new insights for providing the new sweet yeasts that can be used as food additives.

The plastid genome of Pentadiplandra brazzeana Baillon (Pentadiplandraceae).

Pentadiplandra brazzeana is the only species of Pentadiplandraceae. This species is shrubs or tuberous liana with a natural distribution in tropical West Africa. This study firstly determined its complete plastome. The plastome is totally 156,625 bp in length, which contains a pair of 26,751-bp-long inverted repeat regions (IRs), a large single copy region of 85,318 bp, and a small single copy region of 17,805 bp. A total of 112 unique genes were identified in this plastome, of which 78 are protein-coding genes, 30 are tRNA genes, and 4 are rRNA genes. Phylogenetic analysis based on 82 genes fully resolved relationships among sampled families of Brassicales, which are consistent with previous studies.

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 ß-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.

Structural features and activity of Brazzein and its mutants upon substitution of a surfaced exposed alanine.

Brazzein (Brz) is a member of sweet-tasting protein containing four disulfide bonds. It was reported as a compact and heat-resistant protein. Here, we have used site-directed mutagenesis and replaced a surface-exposed alanine with aspartic acid (A19D mutant), lysine (A19K mutant) and glycine (A19G mutant). Activity comparisons of wild-type (WT) and mutants using taste panel test procedure showed that A19G variant has the same activity as WT protein. However, introduction of a positive charge in A19K mutant led to significant increase in Brz's sweetness, while A19D has reduced sweetness compared to WT protein. Docking studies showed that mutation at position 19 results in slight chain mobility of protein at the binding surface and changing the patterns of interactions toward more effective binding of E9K variant in the concave surface of sweet taste receptor. Far-UV CD data spectra have a characteristic shape of beta structure for all variants, however different magnitudes of spectra suggest that beta-sheet structure in WT and A19G is more stable than that of A19D and A19K. Equilibrium unfolding studies with fluorescence spectroscopy and using urea and dithiothritol (DTT) as chemical denaturants indicates that A19G mutant gains more stability against urea denaturation; while conformational stability of A19D and A19K decreases when compared with WT and A19G variants. We concluded that the positive charge at the surface of protein is important factor responsible for the interaction of protein with the human sweet receptor and Ala19 can be considered as a key region for investigating the mechanism of the interaction of Brz with corresponding receptor.

Improved Secretory Production of the Sweet-Tasting Protein, Brazzein, in Kluyveromyces lactis.

Brazzein is an intensely sweet protein with high stability over a wide range of pH values and temperatures, due to its four disulfide bridges. Recombinant brazzein production through secretory expression in Kluyveromyces lactis is reported, but is inefficient due to incorrect disulfide formation, which is crucial for achieving the final protein structure and stability. Protein disulfide bond formation requires protein disulfide isomerase (PDI) and Ero1p. Here, we overexpressed KlPDI in K. lactis or treated the cells with dithiothreitol to overexpress KlERO1 and improve brazzein secretion. KlPDI and KlERO1 overexpression independently increased brazzein secretion in K. lactis by 1.7-2.2- and 1.3-1.6-fold, respectively. Simultaneous overexpression of KlPDI and KlERO1 accelerated des-pE1M-brazzein secretion by approximately 2.6-fold compared to the previous system. Moreover, intracellular misfolded/unfolded recombinant des-pE1M-brazzein was significantly decreased. In conclusion, increased KlPDI and KlERO1 expression favors brazzein secretion, suggesting that correct protein folding may be crucial to brazzein secretion in K. lactis.