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Patent Issued for Enhanced Production and Purification of a Natural High Intensity Sweetener

June 17, 2014



By a News Reporter-Staff News Editor at Life Science Weekly -- A patent by the inventors Carlson, Alfred (Terre Haute, IN); Armentrout, Richard W. (Decatur, IL); Ellis, Timothy Peter (Boxborough, MA), filed on October 1, 2009, was published online on June 3, 2014, according to news reporting originating from Alexandria, Virginia, by NewsRx correspondents (see also Tate & Lyle Technology Limited).

Patent number 8741621 is assigned to Tate & Lyle Technology Limited (London, GB).

The following quote was obtained by the news editors from the background information supplied by the inventors: "Brazzein encompasses a group of related sweet proteins extracted and purified from the fruit of the African plant, Pentadiplandra brazzeana. (Ming and Hellekant, FEBS Lett. 355: 106-108, 1994). Brazzein occurs in three naturally occurring forms, the amino acid sequences of which are known: Type I is 54 amino acids in length and has an N-terminal pyrrolidone carboxylic acid; Type II is 54 amino acids in length and has an N-terminal glutamine, and Type III is 53 amino acids in length and has an N-terminal aspartic acid (Assadi-Porter, et al., Arch. Biochem. Biophys. 376: 252-258, 2000). In addition to the natural forms of brazzein, variants of brazzein, i.e., mutated and recombinant proteins with varied sequences and properties, have been created by genetic engineering, such as those described in U.S. Pat. Nos. 5,326,580; 5,346,998; 5,527,555; 5,741,537; 7,153,535; 6,274,707 and in Jin, et al., Chemical Senses 28: 491-498; Assadi-Porter, et al., Arch. Biochem. Biophys. 376: 259-265, 2000; Assadi-Porter, et al., Chem. Senses 30 (Suppl. 1): i90-i91, 2005. Brazzein is a high-intensity natural sweetener, having an excellent taste profile, 400-500 times the sweetness of sucrose, and excellent stability at low pH and high temperatures.

"Described below are methods, using food-grade methodology and yeast fermentation, for enhanced production and improved purification of a natural, high-quality brazzein protein that enable the commercial production of brazzein. This unique protein has applications as a sweetener in the baking, beverage, and table-top product industries."

In addition to the background information obtained for this patent, NewsRx journalists also obtained the inventors' summary information for this patent: "Disclosed is an expression vector for the production of a brazzein protein by a yeast cell comprising at least two cassettes, each cassette comprising a promoter sequence, a secretion signal sequence, a brazzein protein reading frame sequence, and a termination sequence, wherein the at least two cassettes are present in an expression vector comprising an integration sequence for integration of the cassettes into the yeast genome at a specific site.

"Also disclosed is a recombinant P. pastoris cell comprising at least two copies of a gene construct comprising a pGAP promoter sequence, a secretion signal sequence, a brazzein Type II or Type III protein reading frame sequence, and a termination sequence, wherein the copies of the gene construct are present at a preselected integration site within the P. pastoris genome.

"A method for producing enhanced levels of a brazzein protein is presented, comprising the steps of a) transforming one or more cells of P. pastoris with the expression vector of claim 1; b) growing the transformed cell or cells in a fermentation medium under aerobic conditions, wherein the transformed cells produce enhanced levels of a brazzein protein compared with cells containing only one copy of the brazzein protein gene.

"A method for enhancing production of a foreign protein in recombinant Pichia is presented, comprising the steps of c) initiating a culture of the recombinant Pichia, under aerobic conditions, d) reducing oxygen levels in the culture to less than 5%, and e) maintaining oxygen levels in the culture at less than 5%.

"A method for purifying a protein from a yeast fermentation culture is also presented, comprising the ordered steps of a) adjusting the pH of the fermentation medium above or below the isoelectric point of the protein to be purified; b) removing the cells from the pH adjusted fermentation medium; c) subjecting the cell-free fermentation medium to ion-exchange chromatography and eluting the protein as a solution; d) passing the eluted protein solution through a first membrane to remove unwanted high molecular weight solutes; and e) concentrating and diafiltering the protein permeate on a second membrane to remove low molecular weight solutes."

URL and more information on this patent, see: Carlson, Alfred; Armentrout, Richard W.; Ellis, Timothy Peter. Enhanced Production and Purification of a Natural High Intensity Sweetener. U.S. Patent Number 8741621, filed October 1, 2009, and published online on June 3, 2014. Patent URL: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=8741621.PN.&OS=PN/8741621RS=PN/8741621

Keywords for this news article include: Peptides, Proteins, Amino Acids, Tate & Lyle Technology Limited.

Our reports deliver fact-based news of research and discoveries from around the world. Copyright 2014, NewsRx LLC


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Source: Life Science Weekly


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