Sweet-tasting Protein Research Articles

Gastric digestion of the sweet-tasting plant protein thaumatin releases bitter peptides that reduce H. pylori induced pro-inflammatory IL-17A release via the TAS2R16 bitter taste receptor

About half of the world's population is infected with the bacterium Helicobacter pylori. For colonization, the bacterium neutralizes the low gastric pH and recruits immune cells to the stomach. The immune cells secrete cytokines, i.e., the pro-inflammatory IL-17A, which directly or indirectly damage surface epithelial cells. Since (I) dietary proteins are known to be digested into bitter tasting peptides in the gastric lumen, and (II) bitter tasting compounds have been demonstrated to reduce the release of pro-inflammatory cytokines through functional involvement of bitter taste receptors (TAS2Rs), we hypothesized that the sweet-tasting plant protein thaumatin would be cleaved into anti-inflammatory bitter peptides during gastric digestion. Using immortalized human parietal cells (HGT-1 cells), we demonstrated a bitter taste receptor TAS2R16-dependent reduction of a H. pylori-evoked IL-17A release by up to 89.7 ± 21.9% (p ≤ 0.01). Functional involvement of TAS2R16 was demonstrated by the study of specific antagonists and siRNA knock-down experiments.

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.

Effect of the consumption of brazzein and monellin, two recombinant sweet-tasting proteins, on rat gut microbiota.

Sweet-tasting proteins (SPs) are proteins of plant origin initially isolated from tropical fruits. They are thousands of times sweeter than sucrose and most artificial sweeteners. SPs are a class of proteins capable of causing a sweet taste sensation in humans when interacting with the T1R2/T1R3 receptor. SP thaumatin has already been introduced in the food industry in some countries. Other SPs, such as monellin and brazzein, are promising products. An important stage in researching SPs, in addition to confirming the absence of toxicity, mutagenicity, oncogenicity, and allergenic effects, is studying their influence on gut microbiota. In this paper we describe changes in the composition of rat gut microbiota after six months of consuming one of two recombinant SPs-brazzein or monellin. A full length 16S gene sequencing method was used for DNA library barcoding. The MaAsLin2 analysis results showed noticeable fluctuations in the relative abundances of Anaerocella delicata in brazzein-fed rat microbiota, and of Anaerutruncus rubiinfantis in monellin-fed rat microbiota, which, however, did not exceed the standard deviation. The sucrose-fed group was associated with an increase in the relative abundance of Faecalibaculum rodentium, which may contribute to obesity. Overall, prolonged consumption of the sweet proteins brazzein and monellin did not significantly change rat microbiota and did not result in the appearance of opportunistic microbiota. This provides additional evidence for the safety of these potential sweeteners.

Sweet-Tasting Natural Proteins Brazzein and Monellin: Safe Sugar Substitutes for the Food Industry.

This article presents the results of a comprehensive toxicity assessment of brazzein and monellin, yeast-produced recombinant sweet-tasting proteins. Excessive sugar consumption is one of the leading dietary and nutritional problems in the world, resulting in health complications such as obesity, high blood pressure, and cardiovascular disease. Although artificial small-molecule sweeteners widely replace sugar in food, their safety and long-term health effects remain debatable. Many sweet-tasting proteins, including thaumatin, miraculin, pentadin, curculin, mabinlin, brazzein, and monellin have been found in tropical plants. These proteins, such as brazzein and monellin, are thousands-fold sweeter than sucrose. Multiple reports have presented preparations of recombinant sweet-tasting proteins. A thorough and comprehensive assessment of their toxicity and safety is necessary to introduce and apply sweet-tasting proteins in the food industry. We experimentally assessed acute, subchronic, and chronic toxicity effects, as well as allergenic and mutagenic properties of recombinant brazzein and monellin. Our study was performed on three mammalian species (mice, rats, and guinea pigs). Assessment of animals' physiological, biochemical, hematological, morphological, and behavioral indices allows us to assert that monellin and brazzein are safe and nontoxic for the mammalian organism, which opens vast opportunities for their application in the food industry as sugar alternatives.

Investigating the Effect of Substituting a Single Cysteine Residue on the Thermal Stability of an Engineered Sweet Protein, Single-Chain Monellin.

Single-chain monellin (SCM) is an engineered protein that links the two chains of monellin, a naturally sweet-tasting protein. This protein is an attractive candidate for use as a sugar replacement in food and beverages and has numerous other applications. Therefore, generating SCM mutants with improved stability is an active area of research to broaden the range of its potential applications. In this study, we focused on the Cys41 residue of SCM, which is a single cysteine residue present at a structurally important position. This residue is often substituted with Ser. However, this substitution may destabilize SCM because Cys41 is buried in the hydrophobic core of the protein. Therefore, we designed mutants that substituted Ala, Val, and Leu for this residue, namely C41A, C41V, and C41L. We characterized these three mutants, SCM C41S, and wild type (WT). Differential scanning fluorimetric analysis revealed that substituting Cys41 with Ala or Val increased the thermal stability of SCM, while substitution with Ser or Leu decreased its stability. Determination of the crystal structures of SCM C41A and C41V mutants revealed that the overall structures and main chain structures around the 41st residue of both mutants were almost identical to the WT. On the other hand, the orientations of the amino acid side chains near the 41st residue differed among the SCM variants. Taken together, our results indicate that substituting Cys41 with Ala or Val increases the stability of SCM and provide insight into the structural basis of this improvement.

The sweet protein brazzein as a promising natural sweetener

In the modern world, due to the overconsumption of sugar-containing products, the problem of obesity is relevant. Among the many sweeteners that minimize sugar intake, a group of sweet-tasting proteins is up-and-coming. Brazzein is the smallest of the sweet proteins (54 aa, 6473 Da), and it is also safe for obese and diabetic people since it does not affect blood sugar and insulin levels. Brazzein has high thermal stability over a wide pH range: from 2 to 8 [1]. To increase the level of sweetness of brazzein, mutant variants of this protein were created through site-directed mutagenesis, the sweetest of which is triple mutant H31R/E36D/E41A, which is 22,500 times sweeter than sucrose [2]. Since the content of brazzein in the fruits of the natural source (Pentadiplandra brazzeana) is extremely low (0.2%), various methods have been developed to obtain brazzein using heterologous expression systems, which used as producers: bacteria (Escherichia coli, Lactococcus lactis), yeast (Pichia pastoris, Kluyveromyces lactis, Saccharomyces cerevisiae), plants (Zea mays, Oryza sativa, Lactuca sativa, Nicotiana tabacum) and animals (Mus musculus) [35]. Despite the short peptide sequence, the industrial production of recombinant protein faced several problems, including low protein yield (e.g in mouse milk it was detectable on western blot analysis only) and loss of sweetness. Аn extremely relevant and promising way to obtain recombinant brazzein is the optimization of extracellular expression in bakers yeasts with the GRAS (Generally recognized as safe) status, since the safety of these microorganisms for human health can potentially significantly reduce the number of brazzein purification steps and thereby reduce its cost to consumers.

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.

Structure of thaumatin under acidic conditions: Structural insight into the conformations in lysine residues responsible for maintaining the sweetness after heat-treatment

Thaumatin is an intensely sweet-tasting protein. Its sweetness persists when heated under acidic conditions, but disappears when heated at a pH above 7.0. To clarify how the structural features of thaumatin resist insoluble aggregation during heating under acidic conditions, we analysed its crystal structure obtained at pH 4.0, 6.0, and 8.0. Simultaneously, the melting temperature (Tm) at these pH levels was determined using differential scanning fluorimetry. At pH 4.0, the Tm of thaumatin was substantially lower and the overall B-factor value of its structure was higher than those at pH 6.0. Interestingly, the relative B-factor values for most lysine residues decreased as the pH reduced. These results suggest that the overall structure at pH 4.0 becomes flexible but the relative flexibility of some regions is lower than that at pH 6.0. Thus, the reduction in relative flexibility might play an important role in preventing thermal aggregation, thereby maintaining the sweetness.

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.

New Insight Into the Structure-Activity Relationship of Sweet-Tasting Proteins: Protein Sector and Its Role for Sweet Properties.

Sweet-tasting protein is a kind of biomacromolecule that has remarkable sweetening power and is regarded as the promising sugar replacer in the future. Some sweet-tasting proteins has been used in foods and beverages. However, the structure and function relationship of these proteins is still elusive, and guidelines for their protein engineering is limited. It is well-known that the sweet-tasting proteins bind to and activate the sweet taste receptor T1R2/T1R3, thus eliciting their sweetness. The “wedge-model” for describing the interaction between sweet-tasting proteins and sweet taste receptor to elucidate their sweetness has been reported. In this perspective article, we revealed that the intramolecular interaction forces in sweet-tasting proteins is directly correlated to their properties (sweetness and stability). This intramolecular interaction pattern, named as “protein sector,” refers to a small subset of residues forming physically connections, which cooperatively affect the function of the proteins. Based on the analysis of previous experimental data, we suggest that “protein sector” of sweet-tasting proteins is pivotal for their sweet properties, which are meaningful guidelines for the future protein engineering.

Thaumatin-like genes function in the control of both biotic stress signaling and ABA signaling pathways

Thaumatin was isolated as a sweet-tasting protein. Arabidopsis has over 20 Thaumatin-Like Protein (TLP)/Osmoti-Like Protein (OLP) genes that belong to the PR5 family. Although biotic stress-related functions of TLPs have been reported from transgenic lines expressing TLPs, it is nonetheless necessary to investigate genetic phenotypes produced by defects in the TLP genes. In this report, four TLP genes were selected based on sequence similarities (Thau1/2/3/4), and the corresponding mutant thau1/2/3/4 was examined for biotic and abiotic stress responses. The thau1/2/3/4 mutant showed increased susceptibility to the Pseudomonas syringae pv. tomato DC3000 infection, and reduced sensitivity to the ABA and drought stress treatments. Each of the four thaumatin genes showed different gene expression patterns for ABA treatment. Moreover, ABA-inductions of Thau1/2/3/4 were largely dependent on the intact ABA signaling pathway mediated by PYR/PYL receptors. Among the many ABA-responsive genes affected by the defects of Thau1/2/3/4, reduced expression of P5CS1 with decreased accumulation phenotype of prolines indicates that compromised proline synthesis may be associated with the stress phenotypes of thau1/2/3/4. Our data suggest that Thau1/2/3/4 have a function in both biotic stress and abiotic stress signal transduction through the regulation of proline synthesis.

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.

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.

Potential improvement of the thermal stability of sweet-tasting proteins by structural calculations

The low thermal stability of the sweet-tasting proteins limited their applications in food industry. Improve their thermal stability is the key to developing their applications in food processing. In the present study, saturation mutagenesis was performed on 4 sweet-tasting proteins, brazzein (988 mutations), curculin (2109 mutations), monellin (1824 mutations) and thaumatin (3933 mutations), using structural calculations in order to find more thermal stable mutations. The obtained results indicated that our calculated ΔΔG value (ΔΔG < 0 stabilizing, ΔΔG > 0 destabilizing) was a good predictor for predicting changes in thermal stability caused by mutations. Moreover, mutating the negatively charged residues to the other non-negatively charged amino acids was an efficient way to improve the thermal stability of the investigated sweet-tasting proteins. In addition, some promising mutations sites were identified for improving thermal stability using mutagenesis. This study provides useful information for future protein engineering to improve the thermal stability of the sweet-tasting proteins.

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.

Effect of Transgenesis on mRNA and miRNA Profiles in Cucumber Fruits Expressing Thaumatin II.

Transgenic plants are commonly used in breeding programs because of the various features that can be introduced. However, unintended effects caused by genetic transformation are still a topic of concern. This makes research on the nutritional safety of transgenic crop plants extremely interesting. Cucumber (Cucumis sativus L.) is a crop that is grown worldwide. The aim of this study was to identify and characterize differentially expressed genes and regulatory miRNAs in transgenic cucumber fruits that contain the thaumatin II gene, which encodes the sweet-tasting protein thaumatin II, by NGS sequencing. We compared the fruit transcriptomes and miRNomes of three transgenic cucumber lines with wild-type cucumber. In total, we found 47 differentially expressed genes between control and all three transgenic lines. We performed the bioinformatic functional analysis and gene ontology classification. We also identified 12 differentially regulated miRNAs, from which three can influence the two targets (assigned as DEGs) in one of the studied transgenic lines (line 224). We found that the transformation of cucumber with thaumatin II and expression of the transgene had minimal impact on gene expression and epigenetic regulation by miRNA, in the cucumber fruits.

The Flexible Loop is a New Sweetness Determinant Site of the Sweet-Tasting Protein: Characterization of Novel Sweeter Mutants of the Single-Chain Monellin (MNEI).

The single-chain monellin (MNEI) displays same sweet potency as the natural monellin protein. To identify critical residues determining its sweetness, residues located at the loops region were selected for mutagenesis analysis. Mutations of positive-charge residues R31, R53, and R82 consistently led to obvious decrease of sweetness, whereas mutations of negative-charge residues resulted in variable sweet potency. Of note, the E50N mutant in the loop region linking the 2 natural chains showed significantly increased sweetness. Mutations of this residue to M or K led to similar effects, in accordance with the so-called wedge model for explanation of the sweet protein-receptor interaction. Homology modeling was carried out with the firstly reported crystal structure of sweet taste receptor (from medaka fish) as the template, and molecular docking and dynamics simulations suggested that flexible conformations of specific residues located in the loops region play essential roles for the interaction with the receptor and the sweetness of the protein. Moreover, obvious additive effects were found for the sweetness as 2 double-site mutants (E50N/Y65R and E2N/E50N) displayed increased sweetness than their single-site mutants. Our results revealed the flexible loop L23 linking the 2 natural chains as a novel sweetness determinant site of the sweet protein monellin and raised a series of new sweeter mutants, which could provide helpful guidance for molecular designing the sweet-tasting proteins.

Atomistic mechanisms underlying the activation of the G protein-coupled sweet receptor heterodimer by sugar alcohol recognition

The human T1R2-T1R3 sweet taste receptor (STR) plays an important role in recognizing various low-molecular-weight sweet-tasting sugars and proteins, resulting in the release of intracellular heterotrimeric G protein that in turn leads to the sweet taste perception. Xylitol and sorbitol, which are naturally occurring sugar alcohols (polyols) found in many fruits and vegetables, exhibit the potential caries-reducing effect and are widely used for diabetic patients as low-calorie sweeteners. In the present study, computational tools were applied to investigate the structural details of binary complexes formed between these two polyols and the T1R2-T1R3 heterodimeric STR. Principal component analysis revealed that the Venus flytrap domain (VFD) of T1R2 monomer was adapted by the induced-fit mechanism to accommodate the focused polyols, in which residues 233–268 moved significantly closer to stabilize ligands. This finding likely suggested that these structural transformations might be the important mechanisms underlying polyols-STR recognitions. The calculated free energies also supported the VFD of T1R2 monomer as the preferential binding site for such polyols, rather than T1R3 region, in accord with the lower number of accessible water molecules in the T1R2 pocket. The E302 amino acid residue in T1R2 was found to be the important recognition residue for polyols binding through a strongly formed hydrogen bond. Additionally, the binding affinity of xylitol toward the T1R2 monomer was significantly higher than that of sorbitol, making it a sweeter tasting molecule.

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.