Essen Rivesta Issue 26
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Functional Expression of the Sweet-Tasting Protein Brazzein In
a ISSN 0101-2061 (Print) Food Science and Technology ISSN 1678-457X (Online) DOI: https://doi.org/10.1590/fst.40521 Functional expression of the sweet-tasting protein brazzein in transgenic tobacco Hyo-Eun CHOI1, Ji-In LEE1, Seon-Yeong JO1, Yun-Cheol CHAE2, Jeong-Hwan LEE3, Hyeon-Jin SUN4, Kisung KO3, Sungguan HONG1* , Kwang-Hoon KONG1* Abstract 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. Keywords: alternative sweetener; brazzein; Agrobacterium-mediated transformation; transgenic tobacco plant; protein purification. Practical Application: Our study provides an economic mass-production system for this sweet-tasting protein, brazzein, with potential use as an alternative sweetener in the food industry. 1 Introduction The appeal sweet food is undeniable, however, excessive (Assadi-Porter et al., 2000). -
Enhancing Sweetness and Stability of the Single Chain Monellin MNEI
www.nature.com/scientificreports OPEN Sweeter and stronger: enhancing sweetness and stability of the single chain monellin MNEI through Received: 08 July 2016 Accepted: 07 September 2016 molecular design Published: 23 September 2016 Serena Leone1, Andrea Pica1, Antonello Merlino1, Filomena Sannino1, Piero Andrea Temussi1,2 & Delia Picone1 Sweet proteins are a family of proteins with no structure or sequence homology, able to elicit a sweet sensation in humans through their interaction with the dimeric T1R2-T1R3 sweet receptor. In particular, monellin and its single chain derivative (MNEI) are among the sweetest proteins known to men. Starting from a careful analysis of the surface electrostatic potentials, we have designed new mutants of MNEI with enhanced sweetness. Then, we have included in the most promising variant the stabilising mutation E23Q, obtaining a construct with enhanced performances, which combines extreme sweetness to high, pH-independent, thermal stability. The resulting mutant, with a sweetness threshold of only 0.28 mg/L (25 nM) is the strongest sweetener known to date. All the new proteins have been produced and purified and the structures of the most powerful mutants have been solved by X-ray crystallography. Docking studies have then confirmed the rationale of their interaction with the human sweet receptor, hinting at a previously unpredicted role of plasticity in said interaction. Sweet proteins are a family of structurally unrelated proteins that can elicit a sweet sensation in humans. To date, eight sweet and sweet taste-modifying proteins have been identified: monellin1, thaumatin2, brazzein3, pentadin4, mabinlin5, miraculin6, neoculin7 and lysozyme8. With the sole exception of lysozyme, all sweet proteins have been purified from plants, but, besides this common feature, they share no structure or sequence homology9. -
Sweet Sensations by Judie Bizzozero | Senior Editor
[Confections] July 2015 Sweet Sensations By Judie Bizzozero | Senior Editor By R.J. Foster, Contributing Editor For many, terms like “reduced-sugar” or “sugar-free” do not go with the word “candy.” And yet, the confectionery industry is facing growing demand for treats that offer the taste people have grown to love without the adverse health effects they’re looking to avoid. Thankfully, there is a growing palette of ingredients from which candy makers can paint a new picture of sweetness that will be appreciated by the even most discerning of confectionery critics. SUGAR ALCOHOLS Also referred to as polyols, sugar alcohols are a common ingredient in reduced-sugar and sugar-free applications, especially confections. Funny thing, they’re not sugars or alcohols. Carbohydrate chains composed of monomeric, dimeric and polymeric units, polyols resemble both sugars and alcohols, but do not contain an ethanol molecule. All but two sugar alcohols are less sweet than sugar. Being only partially digestible, though, replacing a portion of a formulation’s sugar with a sugar alcohol reduces total calories without losing bulk (which can occur when replacing sugar with high-intensity sweeteners). Unique flavoring, texturizing and moisture-controlling effects also make polyols well-suited for confectionery products. Two very common and very similar monomeric polyols are sorbitol and mannitol. Present in a variety of fruits and vegetables, both are derived from products of cornstarch hydrolysis. Sorbitol is made via hydrogenation of glucose, which is why sorbitol is sometimes referred to as glucitol. Mannitol is created when fructose hydrogenation converts fructose into mannose, for which the final product, mannitol, is named. -
Essen Rivesta Issue 26
ISSUE NO 27 FEB ‘19 2 ABOUT THE EDITION, 3 SWEETNER FOR SUGAR INDUSTRY SWEET NEWS FOR FARMERS: NOW, ELECTION REPORT: ‘LOAN OF A DISEASE-RESISTANT SUGARCANE ₹12,000 CRORES’ Sujakumari M Keerthiga R R Indira Gandhi Krishi Vishwavidyalaya has The Narendra Modi government is looking at yet produced tissue culture saplings of disease-free another relief package for sugar companies, and this sugarcane plant with naturally high level of is going to be twice the size of one announced in sweetness, which will translate into good quality September 2018.This relief package facilitates the sugar in mills. This is the first time such a sapling has loan which is nearly ₹12,000 crore for which the ex- been produced. chequer will bear 5-6% interest subvention for 5 IGKV has four lakh such saplings available for sale years. The loans will be granted for enhancing at a rate of ₹8 per piece. The IGKV tissue culture lab ethanol production. The package is being finalised by developed the variety using sugarcane from the Prime Minister’s Office, Finance Ministry, Coimbatore. Lab in charge, Dr SL Verma said, Agriculture Ministry and the Food Ministry. farmers generally sow sugarcane either as a mature India is staring at a second consecutive year of step bud shoots, or by extracting buds by a chipping surplus sugar production this season. Indian Sugar machine and sowing them directly in the soil. “The Mills Association has estimated the country’s sugar practice however requires massive quantity of buds output in 2018-19 at 31.5-32 million tonnes. -
A Biobrick Compatible Strategy for Genetic Modification of Plants Boyle Et Al
A BioBrick compatible strategy for genetic modification of plants Boyle et al. Boyle et al. Journal of Biological Engineering 2012, 6:8 http://www.jbioleng.org/content/6/1/8 Boyle et al. Journal of Biological Engineering 2012, 6:8 http://www.jbioleng.org/content/6/1/8 METHODOLOGY Open Access A BioBrick compatible strategy for genetic modification of plants Patrick M Boyle1†, Devin R Burrill1†, Mara C Inniss1†, Christina M Agapakis1,7†, Aaron Deardon2, Jonathan G DeWerd2, Michael A Gedeon2, Jacqueline Y Quinn2, Morgan L Paull2, Anugraha M Raman2, Mark R Theilmann2, Lu Wang2, Julia C Winn2, Oliver Medvedik3, Kurt Schellenberg4, Karmella A Haynes1,8, Alain Viel3, Tamara J Brenner3, George M Church5,6, Jagesh V Shah1* and Pamela A Silver1,5* Abstract Background: Plant biotechnology can be leveraged to produce food, fuel, medicine, and materials. Standardized methods advocated by the synthetic biology community can accelerate the plant design cycle, ultimately making plant engineering more widely accessible to bioengineers who can contribute diverse creative input to the design process. Results: This paper presents work done largely by undergraduate students participating in the 2010 International Genetically Engineered Machines (iGEM) competition. Described here is a framework for engineering the model plant Arabidopsis thaliana with standardized, BioBrick compatible vectors and parts available through the Registry of Standard Biological Parts (www.partsregistry.org). This system was used to engineer a proof-of-concept plant that exogenously expresses the taste-inverting protein miraculin. Conclusions: Our work is intended to encourage future iGEM teams and other synthetic biologists to use plants as a genetic chassis. -
Application of Sweet and Taste Modifying Genes for Development in Plants: Current Status and Prospects
J Plant Biotechnol (2016) 43:397–404 ISSN 1229-2818 (Print) DOI:https://doi.org/10.5010/JPB.2016.43.4.397 ISSN 2384-1397 (Online) Review Application of sweet and taste modifying genes for development in plants: current status and prospects Shahina Akter ・ Md. Amdadul Huq ・ Yu-Jin Jung ・ Yong-Gu Cho ・ Kwon-Kyoo Kang Received: 12 December 2016 / Revised: 20 December 2016 / Accepted: 20 December 2016 ⓒ Korean Society for Plant Biotechnology Abstract Sweet and taste modifying proteins are natural diseases and to add nutritional value. Therefore, the use of alternatives to synthetic sweeteners and flavor enhancers, sweet and taste modifying genes for development of different and have been used for centuries in different countries. Use crop varieties like maize, tomato, rice, potato, wheat, barley, of these proteins is limited due to less stability and availability. or different fruits would be a good way. The necessity of healthy, However, recent advances in biotechnology have enhanced natural and low calorie sweeteners production is increasing their availability. These include production of sweet and taste (Faus 2000) because every day many of world population are modifying proteins in transgenic organisms, and protein attacked by many diseases like caries, hyperlipemia, obesity engineering to improve their stability. Their increased availability and type II diabetes as a result of consumption of high caloric in the food, beverage or medicinal industries as sweeteners food many of which are comprised of sugars as well as and flavor enhancers will reduce the dependence on artificial carbohydrates. Sometimes these substances, cause other side alternatives. Production of transgenic plants using sweet and effects such as, brain tumors, bladder cancer, heart failure, even mental disorders (Kant 2005; Sun et al. -
Large-Scale All-Electron Quantum Chemical Calculation Toward a Sweet-Tasting Protein, Brazzein, and Its Mutants
Large-Scale All-Electron Quantum Chemical Calculation Toward a Sweet-Tasting Protein, Brazzein, and Its Mutants Yoichiro Yagi 1,2 and Yoshinobu Naoshima 1,2 1 Institute of Natural Science, Okayama University of Science, Japan 2 Graduate School of Informatics, Okayama University of Science, Japan 1 Introduction It had been recognized for many years that only small method program, ProteinDF. The former mutant is sweeter than molecules were capable of causing a sweet taste. The search for the brazzein and the latter mutant has a taste like water. sweeteners, however, found out naturally occurring sweet- tasting macromolecules, namely sweet proteins, in a variety of 2 Computational Methods West African and South Asian fruits. Thaumatin was first The NMR structure of brazzein was downloaded from identified as one of the sweet proteins, and then monellin, Protein Data Bank (PDB code: 2brz) and the structure of des - mabinlin, pentadin, curculin, brazzein, and neoculin were pGlu brazzein (Fig. 1(a)) obtained by removal of N-terminal isolated sequentially. Sweet-tasting proteins are expected to be a pyro-grutamate from brazzein. Since the structures of two potential replacement for natural sugars and artificial sweeteners . different mutants Glu41Lys and Arg43Ala are not available in The human sweet taste receptor is a heterodimer of two G- the Protein Data Bank, we mutated the amino acid residues protein coupled receptor subunits, T1R2 and T1R3, and broadly Glu41 and Arg43 in des -pGlu brazzein to Lys and Ala, responsive to natural sugars, artificial sweeteners, D-amino acids, respectively, by using ProteinEditor implemented in ProteinDF and sweet-tasting proteins. -
Sweeteners and Sweet Taste Enhancers in the Food Industry Monique CARNIEL BELTRAMI1, Thiago DÖRING2, Juliano DE DEA LINDNER3*
a OSSN 0101-2061 (Print) Food Science and Technology OSSN 1678-457X (Dnline) DDO: https://doi.org/10.1590/fst.31117 Sweeteners and sweet taste enhancers in the food industry Monique CARNOEL BELTRAMO1, Thiago DÖRONG2, Juliano DE DEA LONDNER3* Abstract The search for new sweeteners technologies has increased substantially in the past decades as the number of diseases related to the excessive consumption of sugar became a public health concern. Low carbohydrates diets help to reduce ingested calories and to maintain a healthy weight. Most natural and synthetic high potency non-caloric sweeteners, known to date, show limitations in taste quality and are generally used in combination due to their complementary flavor characteristics and physicochemical properties in order to minimize undesirable features. The challenge of the food manufacturers is to develop low or calorie-free products without compromising the real taste of sugar expected by consumers. With the discovery of the genes coding for the sweet taste receptor in humans, entirely new flavor ingredients were identified, which are tasteless on their own, but potentially enhance the taste of sugar. These small molecules known as positive allosteric modulators (PAMs) could be more effective than other reported taste enhancers at reducing calories in consumer products. PAMs could represent a breakthrough in the field of flavor development after the increase in the knowledge of safety profile in combination with sucrose in humans. Keywords: positive allosteric modulators; sweet taste receptor; sugar; non-caloric sweeteners. Practical Application: The food industry uses more and more sweeteners to supply the demand for alternative sugar substitutes in products with no added, low or sugar free claims. -
Characterization of the Sweet Taste Receptor Tas1r2 from an Old World
RESEARCH ARTICLE Characterization of the Sweet Taste Receptor Tas1r2 from an Old World Monkey Species Rhesus Monkey and Species-Dependent Activation of the Monomeric Receptor by an Intense Sweetener Perillartine Chenggu Cai1, Hua Jiang2, Lei Li1, Tianming Liu1, Xuejie Song1,BoLiu2,3* a11111 1 Department of Bioengineering, Qilu University of Technology, Jinan, Shandong, 250353, P.R. China, 2 Department of Food Science and Engineering, Qilu University of Technology, Jinan, Shandong, 250353, P.R. China, 3 Department of Biochemistry and Molecular Biology, School of Medicine, Shandong University, Jinan, Shandong, 250012, P.R. China * [email protected] OPEN ACCESS Abstract Citation: Cai C, Jiang H, Li L, Liu T, Song X, Liu B (2016) Characterization of the Sweet Taste Receptor Sweet state is a basic physiological sensation of humans and other mammals which is Tas1r2 from an Old World Monkey Species Rhesus mediated by the broadly acting sweet taste receptor-the heterodimer of Tas1r2 (taste recep- Monkey and Species-Dependent Activation of the tor type 1 member 2) and Tas1r3 (taste receptor type 1 member 3). Various sweeteners Monomeric Receptor by an Intense Sweetener interact with either Tas1r2 or Tas1r3 and then activate the receptor. In this study, we cloned, Perillartine. PLoS ONE 11(8): e0160079. doi:10.1371/ journal.pone.0160079 expressed and functionally characterized the taste receptor Tas1r2 from a species of Old World monkeys, the rhesus monkey. Paired with the human TAS1R3, it was shown that the Editor: Maik Behrens, Universitat Potsdam, GERMANY rhesus monkey Tas1r2 could respond to natural sugars, amino acids and their derivates. Furthermore, similar to human TAS1R2, rhesus monkey Tas1r2 could respond to artificial Received: April 9, 2016 sweeteners and sweet-tasting proteins. -
Insights Into Carbon Content in Examining the Effect of Mutation in Brazzein, a Sweet Tasting Protein
Advances in Biological Research 9 (5): 355-362, 2015 ISSN 1992-0067 © IDOSI Publications, 2015 DOI: 10.5829/idosi.abr.2015.9.5.95243 Insights into Carbon Content in Examining the Effect of Mutation in Brazzein, a Sweet Tasting Protein Ezekiel Amri Department of Science and Laboratory Technology, Dar es Salaam Institute of Technology, P. O. Box 2958, Dares Salaam, Tanzania Abstract: Brazzein is a small sweet testing protein isolated from the fruit of the African plant, Pentadiplandra brazzeana Baillon with potential of replacement of carbohydrate sweeteners. Carbon content analysis was used to examine the effect of mutation brazzein’s two regions at residues 29–33 and 39–43 with residue 36 reported to be important in sweet tasting of the protein. Analysis for local carbon density at the mutational sites for brazzein mutants with increased sweetness taste at residues 29and 41 revealed normal carbon distribution curves with increased carbon frequency peak compared to the wild-type, consequently stabilized the local structure. Brazzein mutants with reduced sweetness taste at residue position 30 33, 36 and 43 were mostly characterized by abnormal broadened distribution curve for carbon content with decreased frequency peak which destabilized the local structure and possibly leading to loss of protein functionality. Further analysis of carbon distribution profile along protein sequences of brazzein revealed a variation in carbon distribution between mutants with increased sweetness taste and those with decreased sweetness taste. Mutants with increased sweetness taste had carbon distribution profile balancing well conforming to the globular proteins which prefers to have 31.45% of carbon all along the sequence for stability. -
Mutagenesis of Critical Amino Acid Residues in Α-Helix and Β-Sheet Structures of Brazzein
4106 Bull. Korean Chem. Soc. 2011, Vol. 32, No. 11 Notes http://dx.doi.org/10.5012/bkcs.2011.32.11.4106 Mutagenesis of Critical Amino Acid Residues in α-Helix and β-Sheet Structures of Brazzein Hyun-Dong Do, Hyun-Joo Jo, Dong-Hyeon Jo, and Kwang-Hoon Kong* Biomolecular Chemistry Laboratory, Department of Chemistry, College of Natural Sciences, Chung-Ang University, Seoul 156-756, Korea. *E-mail: [email protected] Received September 17, 2011, Accepted September 26, 2011 Key Words : Brazzein, Sweet protein, Site-directed mutagenesis, Sweetness determinant, Sweetness evalua- tion There is a high demand for non-calorigenic, protein-based Table 1. Residues selected for site-directed mutagenesis and sweeteners with favorable taste properties. The optimal substituted residues design of such sweeteners requires knowledge of structure- Residue Residue Character function relationships and identification of chemical entities Position Substitution Primer that trigger the sweetness response. Brazzein is an intensely Before After Before After sweet-tasting plant protein with good stability at high 6 Lys Arg tgc aaa cgt gtt tac positive positive temperatures and over a wide pH range. Brazzein, with a 29 Asp Lys aag ctt aaa aag cat negative positive molecular mass of 6.5 kDa, is the smallest naturally occurr- Arg aag ctt aga aag cat positive ing, sweet-tasting protein described to date.1,2 36 Glu Asp tct gga gat tgc ttt negative negative The three-dimensional structures of brazzein have been determined using nuclear magnetic resonance (NMR) spectro- scopy, -
(51) International Patent Classification: (21) International Application
( 1 (51) International Patent Classification: C07K 14/43 (2006.01) (21) International Application Number: PCT/IL20 19/0505 10 (22) International Filing Date: 06 May 2019 (06.05.2019) (25) Filing Language: English (26) Publication Language: English (30) Priority Data: 62/667,532 06 May 2018 (06.05.2018) US (71) Applicant: AMAI PROTEINS LTD. [EVIL]; 2b Prof. A.D. Bergman Street, 7670504 Rehovot (IL). (72) Inventors: SAMISH, Ilan; 7 Hapartizanim St., Ness-Ziona 7403743 (IL). KASS, Itamar; 4 Topaz Street, 4035304 Kfar-Yona (IL). HECHT, Dalit; 19 Haoren Street, 76575 18 Rehovot (IL). (74) Agent: SEGAL, Dalia; Reinhold Cohn and Partners, P.O.B. 13239, 6 113 1 Tel-Aviv (IL). (81) Designated States (unless otherwise indicated, for every kind of national protection available) : AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY, BZ, CA, CH, CL, CN, CO, CR, CU, CZ, DE, DJ, DK, DM, DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, HN, HR, HU, ID, IL, IN, IR, IS, JO, JP, KE, KG, KH, KN, KP, KR, KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, NO, NZ, OM, PA, PE, PG, PH, PL, PT, QA, RO, RS, RU, RW, SA, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW. (84) Designated States (unless otherwise indicated, for every kind of regional protection available) : ARIPO (BW, GH, GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ, TZ, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, Cl, CM, GA, GN, GQ, GW, KM, ML, MR, NE, SN, TD, TG).