Total Enzymatic Hydrolysis

Several proteolytic enzymes have a broad substrate specificity, but none are known which will hydrolyze all of the types of peptide bonds found in proteins. The S. griseus proteinase, papain, and the subtilisins extensively hydrolyze most proteins with liberation of free amino acids, but each enzyme also leaves many peptide bonds intact. For total enzymatic 

One of the earliest suggestions that total enzymatic hydrolysis was possible came from the studies of Frankel (1916), who showed that over 90 % of the bonds in several proteins could be broken when proteolysis with pepsin, trypsin, and chymotrypsin was followed by prolonged hydrolysis with the erepsin preparation of Cohnheim (1901). The recognition in later years of several peptidases in intestinal exti acts which will specifically act upon bonds that are not susceptible to the endopoptidases (Bcrg-mann, 1942) probably accounts for these obseiwations. The specific peptidases such as prolidase, iminodipeptidase (prolinase), glycylglycine dipeptidase, tripeptidase, and leucine aminopeptidase, whi( h are present in mucosa, attack many of the bonds that resist the action of endopoptidases. 

In a recent study on complete enzymatic hydrolysis (Hill and S( hmidt, 1962), methods similar to those of Frankel were employed, but other endopoptidases as well as highly purified exopeptidases were used instead 

Tower et al. (1962) have employed enzymatic hydrolysis with pancreatin preparations for liberation of glutamine and asparagine from proteins. Under the conditions employed, proteins were not hydrolyzed to an extent of more than 50-80 %, but after correcting for incomplete hydrolysis, yields of the two amides were in excellent agreement with theoretical values. 

In all probability refinement of methods such as those of Hill and 

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The hydrolysis of proteins by chemical and enzymatic methods is discussed by R. L. Hill in the second chapter. The chapter should serve as a standard source for information on the many operational techniques with which only the experienced specialist is likely to be familiar. Dr. Hill covers not only the partial hydrolysis of proteins by general and specific procedures but also tbe total enzymatic hydrolysis of proteins for the purpose of amino acid analysis. 

However, when each diastereomerically pure l-acetoxy-//-phosphinate 80 was subjected to enzymatic hydrolysis, only one of them, namely (R, Sp)-80, underwent the desired reaction, the other one, (R, Rp)-80, being totally unreactive (Scheme 7). ... 

Assay of Enzymatic Hydrolysis of Synthetic Solid Polymers. Hydrolysis of solid polymers was measured by the rate of their solubilization, and the measurement process does not necessarily involve complete hydrolysis into the constituent parts. The rate was determined by measuring the water-soluble total organic carbon (TOC) concentration at 30 °C in the reaction mixture using a Beckman TOC analyzer (Model 915-B). In the substrate and enzyme controls, enzyme or substrate was omitted from the reaction mixture. 

By comparing the chromatogram from enzymatic hydrolysis with that in the absence of enzymatic hydrolysis, the completeness of hydrolysis by enzymes and the total isoflavones in soy food extract can be determined (see Basic Protocol 3). 

Methods of extracting the fat-soluble vitamin from food matrices include alkaline hydrolysis, enzymatic hydrolysis, alcoholysis, direct solvent extraction, and supercritical fluid extraction of the total lipid component. 

Total thiamine Milk Enzymatic hydrolysis of protein with trypsin and thiamine phosphates to thiamine with claradiastase oxidation of thiamine to thiochrome using ferricyanide (derivatization stopped with sodium sulphite) thiochrome extracted with 1-butanol Analytical Nucleosil Phenyl (150 mm, 5 fi Macherey-Nagel). Isocratic methanol + acetonitrile + isobutanol + water (80 +10+10+5 v/v/v/v). 0.7 ml/min. Fluorescence 375/430 nm (ex/em). External standardization. 76 Recoveries 95% thiamine as thiochrome from milk. 

Total thiamine Baby food cereals cookies Acid hydrolysis with 0.1 M hydrochloric acid at 100°C for 30 min enzymatic hydrolysis of thiamine phosphates to thiamine with takadiastase at 47°C for 3 h weak ion-exchange (methyl-carboxylatein, add form) solid-phase extraction/ cleanup Analytical Lichrospher 100RP-18 (125 X 4 mm, 5 /u.m Merck ). Isocratic methanol + 10 mM phosphate buffer, pH 2.8 containing 5 mM sodium hexane-sulphonate + tri-ethylamine (15 + 84.9 + 0.1, v/v/v). 1.0 ml/min. UV absorbance 254 nm. External standardization. 79 Linear range = LoD to 7 /zg/ml thiamine, r = 0.9995. Reproducibility CV < 3% using food samples (n = 10). Recoveries 92.1-96.0% thiamine using baby food (n = 3). Samples spiked before hydrolysis. 

Total riboflavin Casein Enzymatic hydrolysis with Precolumn ODS... 

Due to the relative stability of the niacin vitamers, either acid or alkaline hydrolysis can be used to convert nicotinamide to nicotinic acid for quantitation of both vitamers as nicotinic acid (9,44). Acid hydrolysis is used to quantitate biologically available niacin. Alkaline hydrolysis releases both the biologically available and the unavailable vitamers and provides an estimate of the total niacin content. Because alkaline hydrolysis is much faster than acid hydrolysis, the latter is usually supplemented with enzymatic hydrolysis. The most common enzymes are takadiastase, papain, and clarase. On occasion, organic solvents such as methanol have been used to extract free nicotinic acid. 

The total reducing sugars produced from the enzymatic hydrolysis ofcellulose can be measured by the dimtrosalicylic acid (DNS) method (Miller, 1959 Andreotti, 1980), as follows ... 

Grandjean D, Pale P et al (1991) Enzymatic hydrolysis of cyclopropanes. Total synthesis of optically pure dictyopterenes A and C. Tetrahedron 47 1215-1230... 

Fig. 14 Enzymatic hydrolysis with chitosanase of (a) the layer-by-layer (LbL) assembly of dextran sulfate/chitosan (five steps, total thickness 22 nm) with a chitosan surface and (b) a comparable LbL assembly (dextran sulfate/chitosan, six steps, total thickness 55 nm) with a dextran sulfate surface...

To determine the time required for total hydrolysis, the samples were hydrolyzed for 72 h. Samples were taken after 0,1,2,3,4,5, 6, 7, 8, 12,24,48, and 72 h. The samples were centrifuged at 1400 for 10 min, and the composition of monosaccharides was analyzed by HPLC. The percentage of cellulose enzymatically converted to glucose (ECC) was calculated as a quotient of liberated glucose (g) during the hydrolysis and weight of cellulose (g) before enzymatic hydrolysis. The ECC value based on the glucose concentration measured by HPLC was calculated as follows ... 

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