Hydrolysis of OCs 2-casein in solution by chymosin , plasmin , trypsin and Lactobacillus-proteinases

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, hydrolysed 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.

Introduction aS2-Casein is the most recently discovered casein component of milk, and that is perhaps why it is also the least well known.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.a S 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 andGribon 1989, Visser et al. 1989).However, the effect of other proteolytic en- zymes on the hydrolysis of a s 2-casein has not been studied.
The aim of this research was to examine the hydrolyses of a S 2-casein by proteolytic enzymes related to cheese ripening, by isolating and identi- fying the released peptides.
The bacteria were grown, isolated and disintegrated 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: 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 hydrolysed 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- zation and 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).

Hydrolysis by proteolytic enzymes
The peptide profiles obtained from chymosin hydrolysates of ots2-casein are shown in Figure 1.The results show that the proteolytic activity of chy-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 peptides were detectableafter hydrolysis for 2 hours.
The hydrolysis of ot S2-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.

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 hydrophobicity of peptides may also affect their solubil- ity on addition of TFA.
rhamnosus Ml (C) strains.Detection at 206 nm, injection volume 100 u.l.hydrolysed, 12 of which were Lys-X bonds and 2 were Arg-X.This result supports previous data con- cerning the specificity of plasmin, since plasmin has been found to favor Lys-X but it is also capable of hydrolysing Arg-X bonds slowly (WEINSTEIN  and Doolittle 1972).The cleaved sites identified in this study are consistent with the findings of previous studies (Le Bars and Gripon 1989, Vis-  ser et al. 1989).
Only one peptide (aS2-CN 1-24) from the N ter- minal was identified in the plasmin hydrolysates of ots2-casein.This peptide was contaminated with peptide which could not be identified by the methods used.Previous studies (Le Bars and Gri- pon 1989, Visser et al. 1989, Pahkala et ai. 1989 a) showed that fragments 1-24 and 1-21 occur one after another and thus it is possible that the unidentified fragment was 1-21.
VISSER et al. ( 1989) noted the reduction in the concentration offragments 182-207, 189-207, 115-207/150-207 and 151-207 during incubation for 1 to 4 hours.These peptides were not observed after hydrolysis periods of 4 and 24 hours, and it is likely that they had been hydrolysed to shorter fragments.New sites sensitive to plasmin were found within the sequence 150-207 (Lys 153-Leu 154, Lys 155-Lys 156, Lys 156-Ile 157, Arg 170-Tyr 171, Lys 173-Phe 174).Previous research failed to show the formation of peptides from the segment 25-114, and it was presumed that these peptides were to- gether with some intact a S 2-casein (Visser et al.   1989).In the present study, however, fragment 71-80, was found (Figure 2, peak No. 4).The fragment was clearly detectable only after hydrolysis for 24 hours which indicates that the release of peptides from the central part of the protein requires a relat- ively long hydrolysis time.
Trypsin hydrolysed mainly bonds on the car- boxyl side of arginine and lysine residues.This is consistent with the previous findings concerning the specificity oftrypsin (Adler-Nissen 1986).On the hasis of the 16 hydrolysis products that were characterized from the trypsin hydrolysates, it was established that 19 of the 30 trypsin-sensitive bonds of a S 2-casein were hydrolysed.Under the condi- tions of this study, the activity oftrypsin hydrolysed at the N and C terminals.Peptides in the sequence 25-41, which contains the cysteine residues of the protein, were not identified.
Previous findings have shown differences in the proteolytic activity of lactobacilli (Pahkala et al.  1986), but none was observed in this research.Other studies have reported weak proteolytic activ- ity of lactobacilli on ttst-and 6-caseins.L. Helveti- ans strains were shown to be more proteolytic on otsi-and 6-caseins than L. casei strains (Pahkala   et al. 1989 a, b).However, the findings of the pre- sent study indicated no differences in activity be- tween the lactobacilli, and not even the L. Helveti- ans strains differed from other Lactobacillusstrains in terms of stronger and more extensive proteolysis.
The amino acid sequence of fragment 174-181, isolated from the plasmin and trypsin hydrolysates, corresponds to that of the bitter peptide isolated by Matoba et al. (1970) from the trypsin hydrolysates of total casein.This means that bitter peptides can also be formed from as 2-casein.The identified N terminal peptide fragment (a s 2CN 1-  24), which contains a cluster of 4 phosphoserine residues, could be of special interest for studies of its functional properties.Although the concentra- tions of substrate and enzymes used in this study differed from the circumstances in milk, it may be assumed that at least some of the detected hydrolysis products are constituents of the very heterogeneous proteosepeptone fraction of milk (Andrews 1983).
Acknowledgements.The writers wish to express their thanks to the Ministry of Agriculture and Foresty, which has sup- ported the study through its biotechnical research fund.
are released primarily from its hydrophobic C terminal region (VISSER and Slangen 1977, Pahkala et al. 1989 b).