Hydrolysis of OCs2-casein in solution by chymosin, plasmin, trypsin and Lactobacillus- proteinases Anne Pihlanto-Leppälä, Eero Pahkala, Minna Kamala and Veijo Antila Pihlanto-Leppälä, A., Pahkala, E., Kahala, M. & Antila,V. 1993. Hydrolysis of ots2 -casein in solution by chymosin, plasmin, trypsin and Lartoftaa7/«s-proteinases. Agric. Sei. Finl. 2: 133-139. (Agric. Res. Centre of Finland, Food Res. Inst., FIN-31600 Jokioinen, Finland.) The aim of thisstudy was toexamine the enzymic hydrolysis ofOkz-casein by isolating and identifyingthe released peptides. The enzymes applied in the study were chymosin, plasmin and trypsin as well as cell free extracts from three strains of Lactobacillus helveticus and nine strains of L. casei. The findings showed that chymosin had weak proteolytic activity on cts2-casein. Plasmin, on the other hand, released numerous peptides under the used conditions. The majority of the identified fragments were released from the C terminal end of the substrate. Plasmin hydrolysed mainly Lys-X bonds. The third enzyme, trypsin, hydro- lysed several bonds ofaS 2-casein. Peptides were released from almost all regions of the protein. Trypsin acted on the carboxyl sides of arginyl and lysyl residues. Cell free extract of lactobacilli had little activity on aS2-casein. Key words: (fe-casein, enzymatic hydrolysis, peptides Introduction aS2-Casein is the most recently discovered casein component of milk, and that is perhaps why it is also the least wellknown. It constitutes around 10% of the total casein fraction of milk (Davies and Law 1977). a S2-Casein occurs in different forms depending on the extent of phosphorylation. Its primary structure consists of 207 amino acids in a linear chain, with 10-13 phosphoseryl residues and two cysteinyl residues. aS 2-Casein contains few prolyl residues but numerous lysyl residues. The C terminal of the molecule is hydrophobic and its N terminal is hydrophilic (Brignon et al. 1977). The ots2-casein in milk is hydrolysed rapidly by plasmin and the peptides released have been identi- fied (Le Bars and Gribon 1989, Visser et al. 1989). However, the effect of other proteolytic en- zymes on the hydrolysis of as 2-casein has not been studied. The aim of this research was to examine the hydrolyses of aS2-casein by proteolytic enzymes related to cheese ripening, by isolating and identi- fying the released peptides. Material and methods Substrate and enzymes The «s2-casein was isolated using the method of Vreeman and VanRiel(l99o). The enzymes used 133 Agric. Sei. Finl. 2 (1993) https://www.c-info.fi/en/info/?token=x06402mQxygDE7ER.YwH8rdrb--hWZqDW53uX0g.x0S_Oj0u03H4TizcbXKsqVK17Us5zu5K1Ampf9lIzmxWZ9zLlO_S8xyNQ8WN8G4OBx3f7h1mt5WJkuYiSfllbtSY1RxDpjtsby96UpFxKSXcMtfbChvHwQkaCU6dvseyztTks5-HHal9GtAxROK9peb9WZRWxP76I682GpyshHYVVgWVLnG5X99MhaLd3vmvbejecd86N0OFYFc8woYb-h2zzoY2wTZpQE4f78c1xdNJ7JSmzWdnqVZWATAJrlxS2RdQdCUDQznoZvzfTTxCbkWccY11p48efObH2wxZ56_gbfO4mB6J0ZaDMjwahv3Hi9ZUVtkKkJuW18xcSVWH9yMZuw in the study were chymosin (Chr. Hansen, from calf), plasmin (Sigma, bovine plasma) and TPCK trypsin (Sigma). The selection of lactobacilli was based on their differing peptidase and caseinolytic activity (Pahkala and Antila 1987). The follow- ing lactobacilli were included: Lactobacillus helveticus LHI, LHS and LH7 Lactobacillus casei G2, S9, EB, P3, PB, Al and A5 Lactobacillus casei subsp. rhamnosus Ml and M9. The bacteria were grown, isolated and disinte- grated according to the method used by Pahkala et al. (1986). After overnight cultivation (200 ml) the cells were centrifuged and washed twice with distilled water. The cells were then suspended in distilled water (25 ml) and autolysed for 48 hours at 42°C. The autolysis suspension was cooled to -20°C, thawed and homogenized in an Ultra-Turrax for 10 min in cold water. The suspension was then centrifuged (20,000 x g, 4°C, 15 min), cell debris were washed with distilled water and finally sus- pended in 10 ml of distilled water. This suspension was used for the hydrolysis. Hydrolysis The enzymes (0.015 -3% in water) were added into 1.5% (w/v) ots2-casein solution (0.05 M phosphate buffer, pH 6.0). The ratios of enzyme to substrate were: E : Senzyme 1 : 50chymosin plasmin trypsin 1 : 200 1 : 10000 Lactobacilli cell extract (200 pi) was added to 2 ml of 1.5% protein solution. The mixtures were incubated at 40°C. After the reaction period, TFA to 1.1% was added to the mixture. The mixture was filtered (0.45 pm) and the filtrate was stored at -20°C until analysed. Separation of peptides in protein hydrolysates FPLC equipment (Pharmacia LKB, Sweden) was used in peptide analyses. The column was Pep RPC HR 5/5 (5 pm, 100 Å). The runs were conducted at room temperature at a flow rate of 1.0 ml/min. Solvents and gradient were prepared as described by Pahkala et al. (1989a). Peak detection was at 206 nm and the injection volume was 100 pi. Ac- cording to the peptide profile obtained in the first run, the fractions were collected manually from a second run. Identification of peptides in fractions Amino acid analyses using HPLC After collection, peptide fractions were evaporated on a Waters PICO TAG Work Station and hydro- lysed using 6 M HCI (1% phenol) in the gas phase for 24 h at 110°C. Amino acids were analysed as phenylthiocarbamate (PTC) derivatives. Derivati- zationand HPLC runs were performed according to instructions issued by Millipore Corporation (1987). The HPLC equipment consisted following components: Waters Model 510 pumps, Waters automatic sample feeder (Wisp Model 710), Phar- macia LKB VWM 2141 spectrophotometer, and data processing equipment Nec APV IV (program Baseline 810). The column was PICO-TAG (3.9 mm x 15 cm) and its temperature was held at 40°C (Waters Column Heater/Temperature Control Module). Sequence analysis N terminal amino acid sequence analysis from some of the peptides was performed using a manual method (Tarr 1986). Results Hydrolysis by proteolytic enzymes The peptide profiles obtained from chymosin hy- drolysates ofots2-casein are shown in Figure 1. The results show that the proteolytic activity of chy- 134 Agric. Sd. Finl. 2 (1993) mosin on cts2-casein was weak. At the enzyme/sub- strate ratio 1:50,no substantial amounts of peptides were released even after incubation for 24 hours (Figure 1). In order to study the possibility that the peptides produced might precipitate on addition of TFA, the reaction was stopped by rapidly freezing the samples. Hydrophobic peptides were observed in the chromatogram, but it was not possible to identify them by the applied methods. These pep- tides were detectableafter hydrolysis for 2 hours. The hydrolysis of otS2-casein by plasmin under the given conditions released numerous peptides (Figure 2). The majority of the peptides were re- leased from the C terminal end of the protein. Only one peptide from the N terminal end (a S 2-CN 1-24) was identified. The peptides released after 2 and 4 hour were approximately the same, and their con- centration increased with continued incubation. Figure 3 shows that several bonds in ot S2-casein were hydrolysed by trypsin. Peptides were released almost all along the length of the amino acid chain. Altogether, 17 cleaved sites were identified. The N terminal amino acid sequence was determinedfrom the fragments No 5,9, 10, 13 and 14. Hydrolysis by lactobacilli-proteases The proteolytic effect of lactobacilli, namely L. helveticus LHS, L. casei E 8 and L. casei subsp. rhamnosus Ml, on ot S2-casein is presented in Fig- ure 4. It can be seen that these lactobacilli had limited ability to hydrolyse this protein. The hydro- lysis ofots 2-casein effected by the otherLactobacil- /«.v-strains was closely similar to that shown in Figure 4. Fig. 1. Peptide profile of a24 h hydrolysate of aS2-casein by chymosin (E:S = 1:50), without TFA (A) and with TFA (B). Detection at 206 nm, injection volume of 100pi. Fig. 2. Peptide profile of Os2-casein after hydrolysis by plas- min(E:S = 1:200) for4 (A) and 24 (B) hours. Detection at 206 nm, injection volume 100 pi. Identified fragments: 1. 182- 188; 2. 167-173; 3. 166-173; 4.71-80; 5. 1-24; 6. 150-165; 7. 151-165; 8. 115-150; 9. 115149; 10. 174-181; 11. 198-207; 12. 153-170+ 182-197; 13. 153-207. 135 Agric. Sei. Fint. 2 (1993) Discussion Chymosin was found to have weak proteolytic ac- tivity on ot s2-casein. Under the conditions used and with a relatively high proportion of enzyme (E;S = 1:50), peptides were precipitated when TFA was added. It may be presumed that the peptides re- leased were long and hydrophobic. Unhydrolysed casein and high molecular weight peptides are known to precipitate at pH 4.6. Precipitation of peptides by trichloroacetic acid (TCA) is influ- enced by hydrophobicity, in particular, and also to a certain extent by length (Yvon et al. 1989). The addition ofTFA (1.1%) reduces the pH below 4.6, causing the large peptides to precipitate. The hydro- phobicity of peptides may also affect their solubil- ity on addition of TFA. In previous studies, the proteolytic activity of chymosin on 6-casein was found to be weak. In the case of 6-casein, peptides are released primarily from its hydrophobic C terminal region (VISSER and Slangen 1977, Pahkala et al. 1989b). Plasmin readily hydrolyses