Enzymatic Protein Processing

Besides the proteases, which have been investigated extensively and are the only modifying enzymes currently in use commercially, there are transglutaminase, protein kinase, and peptidoglutaminase. These enzymes have only been reported for use in food protein modification on a laboratory scale. Feeney and Whitaker (1977, 1982, 1986) addressed possible 

Hydrolysis of peptide bonds causes several changes in proteins (1) the NHj and COO content of the protein increases, increasing its solubility (2) the molecular mass of the protein decreases and (3) the globular structure of the protein is altered, exposing the previously hidden hydrophobic groups. 

Proteolytic modification has special importance for the improvement of solubility of proteins. This effect becomes significant even after very limited proteolysis. Hydrolysis of casein to DH of 2 and 6.7% with Staphylococcus aureus V8 protease increased the isoelectric solubility to 25 and 50%, respectively (Chobert et al., 1988a). However, it should be noted that the solubility profiles were not identical, due to a shift of the isoelectric point of the modified proteins. Solubility of a protein hydrolysate depends on the enzyme used (Adler-Nissen, 1986a). Protamex (a Bacillus proteinase complex) hydrolysates of sodium caseinate (DH 9 and 15%) displayed 85-90% solubility between pH 4 and 5 (Slattery and FitzGerald, 1998). 

Enzymatic hydrolysis modifies the foaming properties of casein. Protamex hydrolysates of sodium caseinate (DH 0.5 and 1.0%) displayed increased foam expansion at pH 2, 8 and 10 as compared with unhydrolyzed caseinate (Slattery and FitzGerald, 1998). Hydrophobic peptides resulting from 

One of the applications of enzymes in the preparation of food gels is the production of cheese. During this process, chymosin hydrolyzes a specific bond of K-casein, resulting in the destabilization of the micelle structure followed by aggregation and formation of an insoluble coagulum. 

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Lorenzen, P.C., Mautner, A., and Schlimme, E. 2000. Enzymatic crosslinking of proteins in the production of milk products In Proceedings of the 1st International Symposium on Enzymatic Protein Processing (H. Gruppen and W. van Hartingsveldt eds), pp. 163—169. TNO Nutrition and Food Research Institute, Zeist, The Netherlands. 

The cAMP molecule serves as the second messenger, which carries out the effects of the hormone inside the cell. The primary function of cAMP is to activate protein kinase A. This kinase then attaches phosphate groups to specific enzymatic proteins in the cytoplasm. The phosphorylation of these enzymes enhances or inhibits their activity, resulting in the enhancement or inhibition of specific cellular reactions and processes. Either way, cellular... 

A major advantage of enzymes as catalysts is that they are capable of inducing very high degrees of enantioselectivity and, consequently, they are particularly useful in the synthesis of enanhomerically pure compounds. In cases where the enanh-oselectivity is less than optimum it can generally be improved using protein engineering techniques such as in vitro evolution [10]. Hence, in this chapter we shall be mainly concerned with the application of enzymatic cascade processes to the... 

Methionine can be obtained from enzymatic protein hydrolysates or from petrochemical sources. To a lesser extent than cysteine, it is a raw material in Maillard reactions for the preparation of process flavours and it can also be utilised as a precursor for the chemical preparation of the sulfide methional, which is an important flavour constituent for potato, malt, seafood and many other flavours. Methional can be reduced to methionol, which can be esterified with organic acids to, for instance, methionyl acetate and methionyl butyrate, which are useful compounds for pineapple and other fruit flavours (Scheme 13.16). 

Je, J. Y., Qian, Z. J., Byun, H. G., and Kim, S. K. (2007). Purification and characterization of an antioxidant peptide obtained from tuna backbone protein by enzymatic hydrolysis. Process Biochem. 42, 840-846. 

Enzymatic hydrolysates of various proteins have a bitter taste, which may be one of the main drawbacks to their use in food. Arai el al. [90] showed that the bitterness of peptides from soybean protein hydrolysates was reduced by treatment of Aspergillus acid carboxypeptidase from A. saitoi. Significant amounts of free leucine and phenylalanine were liberated by Aspergillus carboxypeptidase from the tetracosapeptide of the peptic hydrolysate of soybean as a compound having a bitter taste. Furthermore, the bitter peptide fractions obtained from peptic hydrolysates of casein, fish protein, and soybean protein were treated with wheat carboxypeptidase W [91], The bitterness of the peptides lessened with an increase in free amino acids. Carboxypeptidase W can eliminate bitter tastes in enzymatic proteins and is commercially available for food processing. 

Figure 2. A process of enzymatic protein degradation and resynthesis for producing a plastein with improved acceptability and an improved amino acid composition (11)...

Compound 25 (Fig. 18.9), a prodrug of 9-P-D-arabinofuranosyl guanine (26), was developed for the potential treatment of leukemia. Compound 24 is poorly soluble in water and its synthesis by conventional techniques is difficult. An enzymatic demethoxylation process was developed using adenosine deaminase (Mahmoudian et al., 1999, 2001). Compound 25 was enzymatically prepared from 6-methoxyguanine (27) and ara-uracil (28) using uridine phosphorylase and purine nucleotide phosphorylase. Each protein was cloned and overexpressed in independent Escherichia coli strains. Fermentation conditions were optimized for production of both enzymes and a co-immobilized enzyme preparation was used in the biotransformation process at 200 g/L substrate input. Enzyme was recovered at the end of the reaction by filtration and reused in several cycles. A more water soluble 5 -acetate ester of compound 26 was subsequently prepared by an enzymatic acylation process using immobilized Candida antarctica lipase in 1,4-dioxane (100 g/L substrate) with vinyl acetate as the acyl donor (Krenitsky et al., 1992). 

Cho, Y.H., Shim, H.K., and Park, J. (2003). Encapsulation of fish oil by an enzymatic gelation process using transglutaminase cross-linked proteins. J. FoodSci. 68, TlYl-212 i. 

Solutions containing active enzymatic proteins (protease, lipase, trypsin, pepsin, prophase, or cellulase) or their mixtures, adjusted to the nature of the matrix of the solid material of biological origin [79, 83, 84]. The aim of the procedure is to break up proteins, polysaccharides, or fat chains and release the constituent amino acids, sugars, or short aliphatic chains. Enzymatic decomposition of the matrix can be considerably enhanced by application of ultrasound the process can, for example, increase the efficiency of disintegration of cell walls in yeast and thus improve the recovery of selenium by as much as 20 % [85]. 

H-T, it was observed that the reaction barriers for the ratedetermining steps were well above the general kinetic requirements of an enzymatic catalytic process (15-20 kcal/mol). Then, the three water molecules (W362, W615, W213) in the catalysis area were also taken into account by adding them to the contents of Model B so that the Ca first-shell coordination sphere is complete and tested upon methanol oxidation in protein environment to provide extended analysis on the oxidation mechanisms. 

In gas-phase for Models A and B, the free energy barriers for this step is 29 and 33 kcal/mol respectively (Table 2) which is well above the general kinetic requirements of an enzymatic catalytic process (15-20 kcaFmol). But in the presence of water and protein environment effects (Model B), the free energy barrier for this step is 19 kcal/mol (Table 2), which is kinetically plausible (Fig. 4). As discussed earlier, the Ca coordinating with only three atoms of PQQ as opposed to seven-coordination from the x-ray... 

Many different measurements of enzymes are undertaken to acquire different types of information. For example, the presence of genes for an enzyme has been used to infer whether organisms are capable of performing particular functions, the expression of those genes or the appearance of the enzyme protein is used to indicate if and under what conditions the gene is functioning, while assay of the activity of the enzyme has been used to infer rates of particular processes (see Section 2). In fact, virtually all measurements classified as molecular and most rate measurements of uptake are, in fact, measurements of enzymes. Because strictly molecular methods (i.e., the capacity to perform a reaction see Chapter 30 by Zehr and Jenkins, this volume) and N uptake (i.e., the net result of enzymatically mediated processes see Chapter 6 by Mulholland and Lomas, this volume) are discussed elsewhere in this book in this chapter, we will focus primarily on measurements of enzyme activities. 

Recently, MALDI-TOF (matrix-assisted laser desorption/ionisation-reflection time-of-flight) mass spectrometry was introduced as a new approach for the investigation of pellicle composition [39], Using mass spectrometry for compositional analysis, it was found that more intact salivary protein species were present in an in vitro-formed pellicle compared to an in vivo-formed pellicle [39], This finding suggests that the in vivo pellicle is an entity formed with components undergoing more extensive enzymatic (proteolytic) processing than in the in vitro pellicle. Therefore, in vitro-formed pellicle layers cannot completely mirror what occurs within the oral cavity [39], This difference may be due to differences in the proteolytic capacity of the saliva supernatant used for in vitro pellicle formation and that of the oral environment. In addition, a particular saliva sample used for in vitro pellicle formation is a closed system, whereas the oral environment is an open system with continuous influx and clearance of oral fluids [39]. 

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