Formal Report on Enzymatic Hydrolysis-1

Enzymatic Hydrolysis of Intact Casein and Albumin from Skimmed Milk Roxan Angelie P. Cruz, Ahmad M. Daji, Ysabel Ann C. de Ocampo, Diane Lorrien E. de Pablo, Celine Aliana S. Dungca, Romely H. Escobido Abstract Enzymatic hydrolysis is defined as the incomplete and selective hydrolysis of peptide bonds by enzymes. Isolated casein and albumin (intact proteins) from skimmed milk was used in the experiment. It was employed the addition of 10 mL of hydrolases (chymotrypsin and trypsin) present in 2% pancreatin (aq.) to 10 mL of 1 g/100 mL distilled water and protein mixture. Na 2CO3 was also added as buffer to maintain the mixture at optimum pH and 10% thymol in ethanol as preservative and disinfectant. The reaction mixture was then incubated in a water bath at 37oC to carry out the breakdown of proteins into amino acids. Dark brown-colored solutions (hydrolysates) were obtained and cooled before used for qualitative color reactions.

Introduction The objectives of the enzymatic hydrolysis of intact casein and albumin (isolated from skimmed milk) is to be able to analyze the products to obtain information about their compositions and to enumerate the advantages and disadvantages of the said type of hydrolysis over the other types (acidic and alkaline). Enzymatic hydrolysis is the catalytic decomposition of a chemical compound by reaction with water by the addition of specific enzymes [1]. Enzymes employed in the experiment are proteolytic enzymes (proteases or peptidases) meaning they cut peptide bonds of proteins at specific amino acid residues producing peptide fragments and some free amino acids [2]. Specifically, pancreatin was the enzyme used in the experiment. Present in the pancreatin are chymotrypsin and trypsin which are both endopeptidases. Chymotrypsin, is an enzyme that cleaves peptide bonds preferentially at the aromatic amino acids: tyrosine, tryptophan, and phenylalanine. The aromatic amino acid ends up at the Cterminal ends of the peptides produced by the reaction [5]. Trypsin, on the other hand, cleaves bonds preferentially at amino acids that have positively charged R groups, such as lysine and arginine. The cleavage takes place in such a way that the amino acid with the charged side chain nds up at the C-terminal end of one of the peptidases produced by the reaction.

The proteins used in the experiment were intact casein and albumin both obtained from the isolation of skimmed milk. Casein is a phosphoprotein, which has phosphate groups are attached to some of the amino acid sidechains. It is exists in milk as the calcium salt, calcium caseinate which has a complex structure [4]. Albumins, on the other hand, are globular proteins that are soluble in water and in dilute salt solutions. They are, however, denatured and coagulated by heat. These are the second most abundant protein types in milk. Once caseins have been removed, and the solution has been made acidic, these can be isolated by heating the mixture to precipitate them [4]. Experimental A. Compounds tested (or Samples used)

The compounds tested in the experiment were intact casein and albumin both isolated from skimmed milk. B. Procedure The experiment was started when a 1 g/100 mL distlled water and protein mixture was prepared. Ten milliliters of the mixture was mixed with ten milliliters of 2% pancreatin (aq.). Sodium carbonate (Na2CO3) was added to buffer the mixture at optimum pH which was pH 7.5. Ten percent thymol in ethanol was also added and used as preservative and disinfectant. The reaction mixture was then incubated in a water bath at 37oC (optimum temperature). After the breakdown of the proteins to amino acids has been carried out, casein hydrolysate and albumin hydrolysate were produced and cooled, ready for use for qualitative color reactions and separation and identification of amino acids by chromatography. Results and Discussion After 10 mL of the 1 g/100 mL distilled water and protein mixture was mixed with 2% pancreatin (aq.) of the same amount, Na2CO3 and 10% thymol in ethanol was added, and incubated at 37oC, the enzymatic hydrolysis of intact casein and albumin (isolated from skimmed milk) yielded a dark brown solution for both proteins which will be used for further experimentations like the qualitative color reactions and chromatography. Enzymatic hydrolysate contains a mixture of L-amino acids and smaller peptide compounds. All the amino acids are recovered quantitatively. In general, enzymatic hydrolysis is not complete and not all peptide bonds are broken [3]. For enzymatic hydrolysis, all amino acids are recovered quantitatively but it is not complete. On the other hand; acidic and basic hydrolysis may be complete, but not all amino acids are recovered quantitatively. Results obtained from qualitative analysis of the hydrolysates are correlated in determining the amino acid composition of the protein sample [3].