Search

Sweet protein thaumatin, a promising alternative to table sugar, can overcome several drawbacks imposed by other sugar alternatives in the market today. Recombinant technology involving Komagataella pha#ii (previously known as Pichia pastoris) has the potential to facilitate a uniform and large-scale production of this protein. To ensure maximal yield from the bioprocess, this study performed a screening to identify the media composition that exhibits the highest secretion yield of recombinant thaumatin n and favourable growth of the host organism. To achieve this, the most recommended media formulations for K. pham namely, BSM, FM22, MGY, BMGY, MBSM and d'Anjou media were investigated. It was identified from this study that the highest yields of recombinant thaumatin II were achieved from BMGY, FM22 and BSM media, irrespective of not exhibiting the highest cell growth. Despite exhibiting a lower cell density than FM22, the BMGY medium resulted in 62.79 mg/L of recombinant thaumatin II, which is 1-2 times higher than the former. This observation facilitated an understanding that the optimal medium for cell growth is not necessarily optimal for recombinant protein production. Moreover, a comparison between baffled and non-baffled flasks demonstrated that the former leads to a higher secretion yield, thereby indicating the importance of sufficient oxygen transfer into the medium. Furthermore, casamino acids and vitamins were supplemented to the media, where the latter contributed to an increased yield of thaumatin. As such, the highest protein yield of 68.60 mg/L was achieved for BMGY supplemented with vitamins. [ABSTRACT FROM AUTHOR]

The manufacturing of recombinant proteins is increasing phenomenally, and yeasts serve as one of the most popular platforms for the large-scale production of these industrially relevant products. Protein analysis is a traditional step for the control and optimisation of such bioprocesses and often necessitates the combination of laborious sample treatments and expensive instruments. A hassle-free and cost-effective approach is always preferred for routine analyses and instigates a need for replacing these laborious tasks. In this study, a fast, simple, and reliable RP-HPLC method for the determination of the upcoming sweetener in the market, the sweet protein thaumatin was established. There is currently no reference method available for the quantification of this protein in fermentation samples. Due to an increasing industrial relevance foreseen for this sweetener, Pichia pastoris was used as a host for its bioproduction. The developed analytical method does not require a pre-treatment step of the biological sample and enables an easy quantification using a conventionally available HPLC system. The method facilitates the identfication and quantification of recombinant thaumatin n and was validated according to the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines(ICH, 2021). The developed technique enables the monitoring of thaumatin levels in fermentations, thereby facilitating bioprocess control and optimisation. [ABSTRACT FROM AUTHOR]

• Enhancing the thermal stability of single-chain monellin (MNEI) via PyRosetta. • Four variants with T m > 96 °C while maintaining sweetness. • Variants remained soluble after boiling for one hour. • Variants displayed stability across alkaline, acidic, and neutral environments. • Potential food industry applications and valuable protein engineering strategies. Sweet proteins offer a promising solution as sugar substitutes by providing a sugar-like sweetness without the negative health impacts linked to sugar or artificial sweeteners. However, the low thermal stability of sweet proteins has hindered their applications. In this study, we took a computational approach utilizing ΔΔG calculations in PyRosetta to enhance the thermostability of single-chain monellin (MNEI). By generating and characterizing 21 variants with single mutation, we identified 11 variants with higher melting temperature (T m) than that of MNEI. To further enhance the thermal stability, we conducted structural analysis and designed an additional set of 14 variants with multiple mutations. Among these variants, four exhibited a significant improvement in thermal stability, with an increase of at least 20 °C (T m > 96 °C) compared to MNEI, while maintaining their sweetness. Remarkably, these variants remained soluble even after being heated in boiling water for one hour. Moreover, they displayed exceptional stability across alkaline, acidic and neutral environments. These findings highlight the tremendous potential of these variants for applications in the food and beverage industry. Additionally, this study provides valuable strategies for protein engineering to enhance the thermal stability of sweet proteins. [ABSTRACT FROM AUTHOR]

Background MNEI and its variant Y65R-MNEI are sweet proteins with potential applications as sweeteners in food industry. Also, they are often used as model systems for folding and aggregation studies. Methods X-ray crystallography was used to structurally characterize Y65R-MNEI at five different pHs, while circular dichroism and fluorescence spectroscopy were used to study their thermal and chemical stability. ThT assay and AFM were used for studying the kinetics of aggregation and morphology of the aggregates. Results Crystal structures of Y65R-MNEI revealed the existence of a dimer in the asymmetric unit, which, depending on the pH, assumes either an open or a closed conformation. The pH dramatically affects kinetics of formation and morphology of the aggregates: both MNEI and Y65R-MNEI form fibrils at acidic pH while amorphous aggregates are observed at neutral pH. Conclusions The mutation Y65R induces structural modifications at the C-terminal region of the protein, which account for the decreased stability of the mutant when compared to MNEI. Furthermore, the pH-dependent conformation of the Y65R-MNEI dimer may explain the different type of aggregates formed as a function of pH. General significance The investigation of the structural bases of aggregation gets us closer to the possibility of controlling such process, either by tuning the physicochemical environmental parameters or by site directed mutagenesis. This knowledge is helpful to expand the range of stability of proteins with potential industrial applications, such as MNEI and its mutant Y65R-MNEI, which should ideally preserve their structure and soluble state through a wide array of conditions. [ABSTRACT FROM AUTHOR]

[Display omitted] • Protein sweeteners are healthier than synthetic ones. • Synthetic dipeptide sweeteners alter gut micro biota. • Sweeteners prevent dental caries. • Sweeteners decrease calories intake. • Aspartame metabolites lead to several drawbacks. Artificial sweeteners have become increasingly popular worldwide owing to their lower calorie content in addition to the claims of health benefits such as weight control, blood glucose level regulation in diabetics, and protection against dental caries. Nevertheless, there is still controversy regarding their safety, especially when administered over the long term, taking into account that most of the safety studies are based on animal models and only a few human studies. This review focuses on low-calorie protein/peptide sweeteners. These include artificial sweeteners, i.e. aspartame, advantame, neotame, and alitame which are synthetic, versus those of natural origin such as thaumatin, monellin, brazzein, pentadin, mabinlin, curculin, and egg white lysozyme. We conducted a systematic literature survey to ensure the accuracy of the data regarding the chemical properties, synthesis, and industrial applications. The health benefits and safety of these sweeteners in humans are presented for the first time in context to their metabolic profiles and gut interaction. [ABSTRACT FROM AUTHOR]

Sweet proteins represent a class of natural molecules, which are extremely interesting regarding their potential use as safe low-calories sweeteners for individuals who need to control sugar intake, such as obese or diabetic subjects. Punctual mutations of amino acid residues of MNEI, a single chain derivative of the natural sweet protein monellin, allow the modulation of its taste. In this study we present a structural and functional comparison between MNEI and a sweeter mutant Y65R, containing an extra positive charge on the protein surface, in conditions mimicking those of typical beverages. Y65R exhibits superior sweetness in all the experimental conditions tested, has a better solubility at mild acidic pH and preserves a significant thermal stability in a wide range of pH conditions, although slightly lower than MNEI. Our findings confirm the advantages of structure-guided protein engineering to design improved low-calorie sweeteners and excipients for food and pharmaceutical preparations. [ABSTRACT FROM AUTHOR]

Abstract: The quality of fruits and vegetables is mainly dependant on the sweetness determined by the level of soluble sugars such as glucose, fructose and sucrose. Other fruit quality parameters include Brix content, acidity, aroma, color, size and shape. Total sugar content in fruits and vegetables is a function of genetic, nutritional, environmental and developmental factors. Understanding the factors controlling sweetness is important to design strategies for enhancing quality of fruits and vegetables. Modifying the activity of enzymes in carbohydrate metabolism such as sucrose synthase (SuSy), acid invertase, ADP-glucose pyrophosphorylase (AGPase), sucrose phosphate synthase (SPS) and sucrose transporters were found to influence carbohydrate partitioning and sucrose accumulation in sink tissues of several food crops. Plant based taste-modifying sweet proteins such as brazzein, cucurmin, mabinlin, monellin, miraculin, neoculin and thaumatin have potential application for developing transgenic plants to improve the sweetness and quality of fruits and vegetables. The present review envisages various cultural, breeding and molecular approaches used for enhancing sugar content and sweetness in fruits and vegetables. [Copyright &y& Elsevier]