No abstract

Trypsin from the pyloric caeca of Pacific cod (Gadus macrocephalus) was easily prepared by affinity chromatography on Benzamidine Sepharose 6B and gel filtration on Superdex 75. Pacific cod trypsin was composed of three isozymes, and their molecular masses were estimated 23,756.34 Da, 23,939.62 Da, and 24,114.81 Da by desorption/ionization time-of-flight mass spectroscopy (MALDI/TOF-MS) and their isoelectric points (pIs) were approximately 5.1, 6.0, and 6.2, respectively. The isolated Pacific cod trypsin showed high similarity to other frigid-zone fish trypsins. The kinetic behavior of tryptic hydrolysis toward N-p-tosyl-L-arginine methyl ester hydrochloride (TAME), N-benzoyl-L-arginine p-nitroanilide hydrochloride (BAPA), and p-amidinophenyl ester were also analyzed. In addition, the cod trypsin-catalyzed dipeptide synthesis was investigated using twelve series of “inverse subdtrates” that is p- and m-isomer of amidinophenyl, guanidinophenyl, (amidinomethyl)phenyl, (guanidinomethyl)phenyl, and four position isomers of guanidinonaphtyl esters derived from N-(tert-butoxycarbonyl)amino acid as acyl donor components. They were found to couple with an acyl acceptor such as L-alanine p-nitroanilide to produce dipeptide in the presence of the trypsin. All inverse substrates tested in this study undergo less enantioselective coupling reaction. The p-guanidinophenyl ester was most practical substrate in twelve series tested. The enzymatic hydrolysis of the resulting products was negligible.

The capability of ficin, a cystine protease, to form peptide bonds was investigated using several types of N-Boc-amino acid phenyl and naphthyl esters as acyl donor components. Enzyme-catalyzed peptide synthesis was carried out under optimized reaction conditions of pH, acyl acceptor concentration and selection of the best yield organic solvent. It used a condensation of N-Boc-Ala-OpGu and Ala-pNA as a model reaction. The products were obtained in 72-96% yield using 10 different substrates, within a few minutes of reaction time.

A large number of biologically active peptides have been isolated recently from bacterial, fungal, plant and animal sources and characterized in some detail. In particular, shortsequence peptides play important roles in the sensory appreciation of food toward four basic taste sensations (sweet, bitter, sour and salty). 1) Such peptides sometimes contain Damino acid and other unusual amino acids. Synthetic chemistry has witnessed remarkable progress with the development of novel biologically active peptides. Enzymatic peptide synthesis has emerged as a powerful approach to the preparation of short sequences. Especially, peptide synthesis using protease-catalyzed reverse reaction has been extensively studied with a variety of amino acids and peptide derivatives as coupling components. [2][3][4][5][6][7][8] It has been reported that the protease-catalyzed peptide synthesis is superior to the chemical coupling method because it is highly stereoselective, racemization-free, and requires less side-chain protection. The major drawback of the enzymatic method, however, is the respective substrate specificity. Thus, the application of proteases for peptide synthesis has not been fully investigated synthetic possiblity of a number of biologically significant peptides containing D-amino acid or other unusual amino acids. A few previous reports have shown the enzymatic condensation of noncorded amino acids, 9,10) peptide mimetics, 11) peptide conjugates, 12) and D-amino acid. 13) Previously, we reported that the inverse substrates such as p-amidino-14) and p-guanidinophenyl esters [15][16][17] behave as specific substrates for trypsin and trypsin-like enzymes and allow the specific introduction of an acyl group carrying a non-specific residue into the enzymatic active site. The characteristics of inverse substrates suggested that they are useful for enzymatic peptide synthesis. [18][19][20][21][22][23][24] Many studies on the characterization of trypsins from cold-adapted species have been reported. 25) These trypsins display substantially higher catalytic efficiency than their mammalian counterparts. [25][26][27][28][29][30][31][32][33] We previously reported the chum salmon trypsin-catalyzed synthesis of peptides using inverse substrates. 23,24) We had obtained commercially available Atlantic cod trypsin at almost the same time. We are interested in comparing of the catalytic efficiency of Atlantic cod trypsin with chum salmon trypsin. In the present work, we investigated Atlantic cod trypsin-catalyzed peptide synthesis using p-amidino-and p-guanidinophenyl esters of N atert-butyloxycarbonyl (Boc)-amino acid as acyl donor components with two types of acyl acceptors. We have found that Atlantic cod trypsin-catalyzed peptide synthesis of the Damino acid and b-amino acid-containing products is more efficient than chum salmon trypsin-catalyzed synthesis. Results and DiscussionThe kinetic constants for the trypsin-catalyzed hydrolysis of synthetic inverse substrates were analyzed on the basis of the following scheme.Where: Eϭe...