In yeast

Biochemistry resulted from the early elucidation of the pathway of enzymatic conversion of glucose to ethanol by yeasts and its relation to carbohydrate metaboHsm in animals. The word enzyme means "in yeast," and the earfler word ferment has an obvious connection. Partly because of the importance of wine and related products and partly because yeasts are relatively easily studied, yeasts and fermentation were important in early scientific development and stiU figure widely in studies of biochemical mechanisms, genetic control, cell characteristics, etc. Fermentation yeast was the first eukaryote to have its genome elucidated. 

Miscellaneous Alkaloids. Stukimic acid (57) is a precursor of anthranihc acid (28) and, in yeasts and Escherichia coli (a bacterium), anthranHic acid (o-aminobenzoic acid) is known to serve as a precursor of tryptophan (26). A similar but yet unknown path is presumed to operate in higher plants. Nonetheless, anthranHic acid itself is recognized as a precursor to a number of alkaloids. Thus damascenine [483-64-7] (134), C qH NO, from the seed coats of JSHgella damascena has been shown (95) to incorporate labeled anthranHic acid when unripe seeds of the plant are incubated with labeled precursor. 

Benzoates. The sodium and potassium salts of ben2oic acid [65-85-0], C2H 02, ate most effective against yeast and mold. They ate used in beverages, fmit products, chemically leavened baked goods, and condiments. Owing to their inhibitory effect on yeast, they cannot be used in yeast-leavened products. Potassium ben2oate was developed for use in reduced-sodium products. Ben2oates ate permitted for use in foods up to a level of 0.1% (76). 

Propionates. Propionic acid [79-09-4], C2H 02, and its calcium and sodium salts ate effective mold inhibitors. They ate particularly useful in yeast-leavened baked products because they do not affect the activity of yeast. In addition to being widely used in baked goods, they ate used as mold inhibitors in cheese foods and spreads (77). 

Nucleic acid contents of SCP products, which range up to 16% in bacteria and 6—11% in yeasts, must be reduced by processing so that intakes are less than 2 g/d to prevent kidney stone formation or gout. Adverse skin and gastrointestinal reactions have also been encountered as a result of human consumption of some SCP products (87). 

Many plant substances possess antivitamin D activity but the mode of action and in most cases the identity remain unknown. Rachitogenic factors have been observed in yeast. Because of the metaboHc interrelationships that exist between vitamin D, Ca, and P, it is sometimes difficult to differentiate between chelators of mineral elements and tme antivitamins. One reported vitamin D antagonist in oats was later identified as phytic acid (72). 

Yeast. The advantages of expression in yeast include potentially high level production of proteins, the abiUty to have expressed proteins secreted into the media for ease of purification, and relatively low cost, easy scale-up. A disadvantage is that plasmid instabiUty may be a problem which can lead to low product yield. Whereas post-translational modification occurs in yeast, proteins are quite often hyperglycosylated. This is generally a problem with expression in Saccharomyces cerevisiae but not for the more recently used yeast host Pichiapastoris (25) (see Yeasts). 

Bakery Products. Sorbates are used in and/or on yeast-raised and chemically leavened bakery products. The internal use of sorbates in yeast-raised products at one-fourth the amount of calcium—sodium propionate that is normally added provides a shelf life equal to that of propionate without adversely affecting the yeast fermentation. Sorbates added at one-tenth the propionate level reduce the mix time by 30% (126). This internal treatment combined with an external spray of potassium sorbate can provide the same or an increased shelf life of pan breads, hamburger and hot-dog buns, English muffins, brown-and-serve roUs, and tortillas. The total sorbate useful in or on these baked goods ranges from 0.03 wt % for pan breads to 0.5 wt % for tortillas 0.2—0.3 wt % sorbic acid protects chemically leavened yellow and chocolate cakes (127). Emit-pie fillings and icings can be protected with 0.03—0.1 wt % sorbates. 

Flavin mononucleotide was first isolated from the yellow en2yme in yeast by Warburg and Christian in 1932 (4). The yellow en2yme was spHt into the protein and the yellow prosthetic group (coen2yme) by dialysis under acidic conditions. Flavin mononucleotide was isolated as its crystalline calcium salt and shown to be riboflavin-5Lphosphate its stmeture was confirmed by chemical synthesis by Kuhn and Rudy (94). It is commercially available as the monosodium salt dihydrate [6184-17 /, with a water solubiUty of more than 200 times that of riboflavin. It has wide appHcation in multivitamin and B-complex solutions, where it does not require the solubili2ers needed for riboflavin. 

The yellow form (11) on acidification is converted to the more stable thiol form (12). On oxidation, typically with alkaline ferhcyanide, yellow form (11) is irreversibly converted to thiochrome [299-35-4] (14), a yellow crystalline compound found naturally in yeast but with no thiamine activity. In solution, thiochrome exhibits an intense blue fluorescence, a property used for the quantitative determination of thiamine. 

The pathways for thiamine biosynthesis have been elucidated only partiy. Thiamine pyrophosphate is made universally from the precursors 4-amino-5-hydroxymethyl-2-methylpytimidinepyrophosphate [841-01-0] (47) and 4-methyl-5-(2-hydroxyethyl)thiazolephosphate [3269-79-2] (48), but there appear to be different pathways ia the eadier steps. In bacteria, the early steps of the pyrimidine biosynthesis are same as those of purine nucleotide biosynthesis, 5-Aminoimidazole ribotide [41535-66-4] (AIR) (49) appears to be the sole and last common iatermediate ultimately the elements are suppHed by glycine, formate, and ribose. AIR is rearranged in a complex manner to the pyrimidine by an as-yet undetermined mechanism. In yeasts, the pathway to the pyrimidine is less well understood and maybe different (74—83) (Fig. 9). 

In the biosynthesis of the thia2ole, cysteine is the common sulfur donor. In yeasts, the C-2 and N may be suppHed by glycine, and the remaining carbons byD-ribulose-5-phosphate [108321-99-9] (50). In anaerobic bacteria, the C-2 andN maybe recmited from tyrosine and the carbons from D-l-deoxyxylulose [16709-34-5] (51), whereas in aerobic bacteria the C-2 and N maybe derived from glycine, as in yeasts 7 (74—76,83—86) (see Fig. 9). 

Biosynthesis of pyrophosphate (5) from pyrimidine phosphate (47) and thia2ole phosphate (48) depends on the activity of five en2ymes, four of them kinases (87). In yeasts and many other organisms, including humans, pyrophosphate (5) can be obtained from exogenous thiamine in a single step cataly2ed by thiamine pyrophosphokinase (88). 

Miconazole. Miconazole nitrate [22832-87-7] (Fig. 2), the 1-phenethyl-imidazole derivative first described in 1969, interferes at low doses with the cytochrome P-450 dependent ergosterol biosynthesis in yeasts and fungi. The result is accumulation of C-14 methylated sterols on the one hand and reduction of the ergosterol levels in the membranes on the other hand (12). Analogous to clotrimazole, this leads to a disturbance in the membranes it results in inhibition of ceU repHcation, mycelium development (in C. albicans) and finally, ceU death. High concentrations of miconazole, which may be achieved with topical use, disturb the orientation of phosphoHpids in the membranes, which produces leaks (13). 

Like the a2ole derivatives, it inhibits the biosynthesis of ergosterol. However, naftifine [65472-88-0] does not inhibit the cytochrome P-450 dependent C-14-demethylase, but the epoxidation of squalene. Squalene epoxidase cataly2es the first step in the conversion of squalene via lanosterol to ergosterol in yeasts and fungi or to cholesterol in mammalian cells. The squalene epoxidase in C. albicans is 150 times more sensitive to naftifine, C2 H2 N, than the en2yme in rat fiver (15). Naftifine is available as a 1% cream. 

A typical bourbon fermentation continues for 72 hours at a fermentation temperature within the 31—35°C range. Many fermentation vessels are equipped with agitation and/or cooling coils that facHitate temperature control. Significant increases in yeast numbers occur during the first 30 hours of fermentation. Over 75% of the carbohydrate is consumed and converted to ethanol. Within 48 hours, 95% or more of the ethanol production is complete. 

In 1878 the term enzyme, Greek for "in yeast," was proposed (8). It was reasoned that chemical compounds capable of catalysis, ie, ptyalin (amylase from sahva), pepsin, and others, should not be called ferments, as this term was already in use for yeast cells and other organisms. However, proof was not given for the actual existence of enzymes. EinaHy, in 1897, it was demonstrated that ceU-free yeast extract ("zymase") could convert glucose into ethanol and carbon dioxide in exactiy the same way as viable yeast cells. It took some time before these experiments and deductions were completely understood and accepted by the scientific community. 

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