Artificial sweeteners Enzymes

Neotame is an artificial sweetener designed to overcome some of the problems with aspartame. The dimethylbutyl part of the molecule was added to block the action of peptidases, enzymes that break the peptide bond between the two amino acids aspartic acid and phenylalanine. This reduces the availability of phenylalanine, eliminating the need for a warning on labels directed at people who cannot properly metabolize phenylalanine. 

Nowadays a wide variety of food ingredients are already produced in an encapsulated form. These comprise artificial sweeteners (aspartame), flavouring agents such as oils or spices (with desirable flavour but possibly undesirable odour), natural colorants (e.g., p-carotene, turmeric), preservatives, acids (citric, lactic and ascorbic), bases, buffers, enzymes, lactic acid bacteria, and some antioxidants (Kirby, 1991 Gibbs et al, 1999 Chen et al, 2006b Ubbink and Kruger, 2006 Augustin and He-... 

The food industry is a fertile area for biocatalysis applications high-fructose corn syrup (HFCS) from glucose with glucose isomerase, the thermolysin-catalyzed synthesis of the artificial sweetener Aspartame , hydrolysis of lactose for lactose-intolerant consumers, and the synthesis of the nutraceutical i-camitine in a two-enzyme system from "ybutyrobetaine all serve as examples. 

In the mid to late 1980s, many research groups focused on methods and processes to prepare L-phenylalanine (Chapter 3). This was a direct result of the demand for the synthetic, artificial sweetener aspartame. One of the many routes studied was the use of phenylalanine dH (Scheme 19.4, R = C6H5CH2) with phenylpyruvate (PPA) as substrate.57-58 This enzyme from Bacillus sphaericus shows a broad substrate specificity and, thus, has been used to prepare a number of derivatives of L-phenylalanine.59 A phenylalanine dH isolated from a Rhodococcus strain M4 has been used to make L-homophenylalanine (.S )-2-amino-4-pheny I butanoic acid], a key, chiral component in many angiotensin-converting enzyme (ACE) inhibitors.40 More recently, that same phenylalanine dH has been used to synthesize a number of other unnatural amino acids (UAAs) that do not contain an aromatic sidechain.43... 

Aspartame monitoring represents a second interesting task for the ET. Immobilized a-chymotrypsin hydrolyzes the artificial sweetener under release of protons which are easily detected via a tris/HCl-buffer due to its high protonation enthalpy. The immobilized enzyme unfortunately causes an unspecific conversion. However, the procedure might be interesting for monitoring aspartame during its industrial production within the Tosoh-process. 

The enzyme has also been used in the production of several natural amino acids such as L-serine from glycine and formaldehyde and L-tryptophan from glycine, formaldehyde, and indole [77-79], In addition, SHMT has also been used for the production of a precursor, 20, to the artificial sweetener aspartame (21) through a non-phenylalanine-requiring route (Scheme 14) [80-83]. Glycine methyl ester (22) is condensed with benzaldehyde under kinetically controlled conditions to form L-enY/ ra-p-phenylserine (23). This is then coupled enzymatically using thermolysin with Z-aspartic acid (24) to form A -carbobcnzyloxy-L-a-aspartyl-L-eryt/zro-p-phenylserine (20). and affords aspartame upon catalytic hydrogenation. 

Decarboxylases of phenylalanine, tyrosine, and lysine and ammonia lyases of histidine, glutamine, and asparagine are also highly selective. Guilbault et al. (1988) described a potentiometric enzyme sensor for the determination of the artificial sweetener aspartame (L-aspartyl-L-phen-ylalanine methylester) based on L-aspartase (EC 4.3.1.1). The ammonia liberated in the enzyme reaction created a slope of 30 mV/decade for the enzyme-covered ammonia sensitive electrode. The specificity of the sensor was excellent however, the measuring time of 40 min per sample appears not to be acceptable. The measuring time has been decreased to about 20 min by coimmobilizing carboxypeptidase A with L-aspartase (Fatibello-Filho et al., 1988). 

This paper has two primary objectives. The first Is to report on the use of an enzyme-containing reversed micellar medium for organic synthesis specifically the formation of a model dlpeptlde. The feasibility of peptide synthesis In reversed micelles has been previously demonstrated ( ). Small peptides are used In a variety of applications, such as artificial sweeteners, pesticides and pharmaceuticals. Since peptide bonds between an aromatic amino acid and a hydrophilic amino acid are of considerable Interest (for Instance In peptide analgesics), the synthesis of a tyrosine-glycine dlpeptlde was chosen for the model reaction. 

Acetylcholine Active Site Allosteric Enzymes Amino Acid Antibiotics Artificial Sweeteners Base Pairing Bioluminescence Caffeine Carbohydrates Cellulose... 

Chapter 15 presents the fundamental chemical principles of some important biochemical phenomena in a straightforward manner. This edition of the text has an expanded discussion of optical activity and stereoisomers that includes more details of the thahdomide tragedy and current use of drugs. Chair conformations of simple sugars are introduced. There is a discussion of the use of Splenda as an artificial sweetener. Steroids and steroid abuse are discussed in more detail. The discussion of soaps and how they work has been improved. The discussion of proteins has been expanded to include prosthetic groups, cofactors, and coenzymes, and the discussion of sickle cell anemia has been enlarged. The treatment of enzyme action using lysozyme, as an example, has been improved. 

In recent years numerous attempts were made to apply enzymes for the systematic building of peptide chains and some of these syntheses were fairly successful . For instance the artificial sweetener, aspartame could be prepared without blocking the jS-carboxyl group of aspartic acid. In the thermolysine catalyzed reaction between benzyloxycarbonyl-L-aspartic acid and L-phenyl-alanine methyl ester, used in excess, at pH 7 the equilibrium is shifted to the right... 

Many other compounds have potential as artificial sweeteners. For example, l sugars are also sweet, and they presumably would provide either zero or very few calories because our enzymes have evolved to selectively metabolize their enantiomers instead, the d sugars. Although sources of L sugars are rare in nature, all eight L-hexoses have been synthesized by S. Masamune and K. B. Sharpless using the Sharpless asymmetric epoxidation (Sections 11.13 and 22.11) and other enantioselective synthetic methods. 

Thermolysin, which is another protease, will also catalyse peptide synthesis, and a new plant will shortly use this enzyme for the manufacture of the artificial sweetener Aspartame, at a scale of2,000 tonnes per year. In this reaction the L-enantiomer of racemic phenylalanine methyl ester reacts specifically with the a-carboxyl group of JV-protected L-aspartate (Scheme 6.26). Thus both the separation of the enantiomers of the phenylalanine and the protection of the y-carboxyl group of the L-aspartate are unnecessary, which simplifies the synthesis. Although the equilibrium favours hydrolysis rather than synthesis, the peptide product, which is the JV-protected precursor ester of Aspartame, forms an insoluble salt with the... 

However, China lacks the capabihty to produce certain fine chemicals that are required only in small amounts but are nonetheless vital to the national economy. Examples are methionine, lysine, pantothenic acid, calcium, vitamins E, A and D, L-lactic acid, behenic acid, nucleic acid, artificial sweeteners, new types of enzyme, biodegradable polymers, long-chain fatty acids and new biotech-based pesticides. Most of China s fine chemicals are currently produced in small qnantities, and in relative technical and geographical isolation. This sector can only be developed if China s scientific and technological base is upgraded, especially in chemical engineering. 

Another artificial sweetener, Neotame, is a modification of the aspartame structure. The addition of a large alkyl group to the amine group prevents enzymes from breaking the amide bond between aspartic acid and phenylalanine. Thus, phenylalanine is not produced when Neotame is used as a sweetener. Very small amounts of Neotame are needed because it is about 10 000 times sweeter than sucrose. 

In order to develop an enzymatic method to S5mthesize D-alanine N-alkyl amide, which is found in the structure of an artificial sweetener, alitame (L-a-aspartyl-N-(2,2,4,4-tetramethyl-3-thietanyl)-D-alanine amide), we screened microorganisms for an enzyme that catalyzes o-stereospecific amino acid amide hydrolysis [1]. 

There are thousands of breweries worldwide. However, the number of companies using fermentation to produce therapeutic substances and/or fine chemicals number well over 150, and those that grow microorganisms for food and feed number nearly 100. Lists of representative fermentation products produced commercially and the corresponding companies are available (1). Numerous other companies practice fermentation in some small capacity because it is often the only route to synthesize biochemical intermediates, enzymes, and many fine chemicals used in minor quantities. The large volume of L-phenylalanine is mainly used in the manufacture of the artificial dipeptide sweetener known as aspartame [22389-47-0]. Prior to the early 1980s there was httle demand for L-phenyl alanine, most of which was obtained by extraction from human hair and other nonmicrobiological sources. 

MoneUin is a sweet protein which could be used as artificial noncarbohydrate sweetener. But it denatures at high temperature and becomes unsuitable for food processing. It is a smaU protein of 11 kDa containing a single tryptophan residue, a 17 residue a-helix, and an antiparallel (3 sheet formed by five (3 strands (Fig. 10.4a). Thus, it is interesting both from application and structural point of view to study the thermal stability of moneUin in ILs since ILs are known to have unconventional properties toward preservation of enzyme structure as observed above for lipases. 

Production of the artificial low-calorie sweetener aspartame from Z-L-aspartate and D/L-phenylalanine methylester by peptide bond formation with immobilized thermolysin from Bacillus thermoproteolyticus (Tosoh Corp., Ajinomoto, Toyo-Soda, DSM, annual world production approx. 10000 tons). Aspartame is about 200 times as sweet as sucrose, and is used in drinks such as Coca Cola and Pepsi Cola Light. In contrast to the older chemical process, the enzymatic process can - due to the L-selectivity of the enzyme - use the cheaper D/L-phenylalanine methylester instead of the pure L-form. The enzymatic process (Fig. 15) yields a-aspartame exclusively, whereas the chemical route yields a mixture of a-aspartame and bitter-tasting (5-aspartame, thus requiring an additional separation step. 

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