Hydrolysis of K-casein in solution by chymosin, plasmin, trypsin and
Lactobacillus -proieinases

Anne Pihlanto-Leppälä, Eero Pahkala and Veijo Antila

Pihlanto-Leppälä, A., Pahkala, E. & Antila, V. 1993. Hydrolysis of K-casein in
solution by chymosin, plasmin, trypsin and Lactobacillus- proteinases. Agric. Sei.
Finl. 2: 489-496. (Agric. Res. Centre of Finland, Food Res. Inst., FIN-31600 Jokioinen,
Finland.)

The aim of this study was to examine the enzymatic hydrolysis of K-casein by isolating
and identifying the released peptides. The enzymes employed in the study were chy-
mosin, plasmin and trypsin, as well as a cell-free extract from three Lactobacillus
helveticus and nine Lactobacillus casei strains.

The findings showed that the bond most sensitive to the proteolytic activity of
chymosin was the Phe 105-Met 106. After 24 hours of hydrolysis a few other bonds in
the casein macropeptide were also cleaved. Plasmin was found to have weak proteolytic
activity under the conditions of this study. When the enzyme-substrate ratio was raised
from 1:200 to 1:50, a few peptides were released from the N-terminal region. Trypsin
was found to hydrolyze several K-casein bonds, and peptides were released from almost
all regions of the protein.

The proteases ofLactobacillus had less effect than chymosin, plasmin or trypsin. The
strains could be divided into three categories. L. helveticus strains had activity on bonds
in the mid-section and C-terminal region, L casei strains EB, P 3, P 8 and A 1 had activity
on bonds in the N- and C-terminal regions, while L. casei A 5 and M 9 had activity only
on bonds in the C-terminal region.

Key words: K-casein, enzymatic hydrolysis, peptides

Introduction

K-Casein is one of the components of bovine casein,
representing around 10% of the total casein. Its
primary structure consists of 169 amino acids, one
phosphate groupand variable amounts of N-acetyl-
galactosamine, galactose and N-acetylneuraminic
acid (Swaisgood 1982). K-Casein is heterogene-
ous with respect to its carbohydrate moiety, which
is exclusively linked to the macropeptide part of
K-casein (Mackinlay and Wake 1965).

K-Casein stabilizes casein micelles (Wheelock
and Knight 1969) and prevents as- and (f-casein

from precipitation in the presence of calcium ions.
Hydrolysis of bovineK-casein by chymosin consti-
tutes the first stage of milk clotting and has been
thoroughly investigated. It is known that this prote-
inase rapidly hydrolyzes one bond, Phe 105-Met
106, of K-casein, leading to the formation of an
N-terminal fragment (para-K-casein; residues I-
-105) and a C-terminal fragment (casein macropep-
tide, CMP; residues 106-169). In various laborat-
ories, thekinetics of this proteolytic action has been
studiedby the use of model substrates representing
parts of the amino acid sequence around the chy-
mosin-sensitive Phe 105-Met 106bond ofK-casein

489

Agric. Sei. Fin!. 2 (1993)

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(Visser et ai. 1976, 1977, 1987, Raymond and
Bricas 1979).

K-Casein is totally hydrolyzed during milk co-
agulation (Melachouris and Tuckey 1966),
whereas para-K-casein is not hydrolyzed during
cheese ripening (Green and Foster 1974, Led-
ford et al. 1966). This latter result, together with
the relatively low concentration and amino acid
composition ofK-casein, would suggest that it is not
an important source of bitter peptides in cheese.

Biologically active peptides that have been iden-
tifiedas a digestion product of K-casein include an
opioid antagonist peptide (residues 33-38) (Chiba
and Yoshikawa 1986) and a peptide (residues
106-116) which inhibits both the aggregation and
binding of fibrinogen to platelets (Jolles et al.
1986).

The aim of this study was to investigate the
hydrolysis of K-casein by proteolytic enzymes in-
volved in theripening ofcheese.

Material and methods

Substrate and enzymes

K-Casein was isolated from sodium caseinate using
ion exchange chromatography on column Mono Q
(HR 16/10, Pharmacia LKB, Sweden). The sol-
vents used were (A) 0.02 M Tris-HCI containing
0.1% mercaptoethanol and 4.5 Murea (pH 8.0), and
(B) buffer A containing 1 M of NaCl. Solvent was
delivered to the column at a flow rate of 4 ml/min
as follows: 1 % of B for 5 minutes, linear gradient
from 1 to 25% of B in 25 minutes and from 25 to
35% of B in 30 minutes. The chromatogram was
monitored at 279 nm. The K-casein fraction was
collected, dialyzed against water for 48 hours and
lyophilized. K-Casein was stored at -20°C until
used for hydrolysis.

The enzymes used in the study were chymosin
(Chr. Hansen, from calf), plasmin (Sigma, bovine
plasma) and TPCK trypsin (Sigma). The selection
ofLactobacilli was based on their differing pepti-
dase and caseinolytic activity (PAHKALA and AN-
TILA 1987). The following Lactobacilli were in-
cluded:

Lactobacillus helveticus LHI, LHS and LH7
Lactobacillus casei G2, S9, EB, P3, PB, AI and A5
Lactobacillus casei subsp. rhamnosus M I 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 autolyzed for 48 hours
at 42°C. The autolysis suspension was cooled to
-20°C, thawedand homogenized with an Ultra-Tur-
rax for 10 minutes in cold water. The suspension
was then centrifuged (20,000 x g, 4°C, 15min), and
the cell debris was washed with distilled water and
finally suspended in 10 ml of distilled water. This
suspension was used for the hydrolysis ofK-casein.

Hydrolysis

The enzymes (0.015 -3% in water) were added into
1.5% (w/v) solutions (0.05 M phosphate buffer, pH
6.0) ofK-casein. The ratios of enzyme to substrate
were:

E:SEnzyme
chymosin
plasmin
trypsin

1:100
1:200, 1:100, 1:50
1:10000

The cell-free extract ofLactobacillus was added
to the protein solution at a ratio of 1:10.

The mixtures were incubated at 40°C. After the
reaction period, 2, 4 and 24 hours for chymosin,
plasmin and trypsin, and 24 and 48 hours for Lacto-
bacillus-proteases, TEA to 1.1% was added to the
mixture. The mixture was filtered (0.45 pm) and the
filtrate stored at -20°C until analyzed.

Separation of peptides in protein hydrolysates

FPLC equipment (Pharmacia LKB, Sweden) was
used in the peptide analyzes. The column was Pep
RPC HR 5/5 (5 pm, 100 Å). The runs were con-
ducted at room temperature at a flow rate of

490

Agric. Sei. Fin!. 2 (1993)



1.0ml/min. Solvents and gradient were prepared as
described by Pahkala et al. (1989a). Peak detec-
tion was at 206 nm and the injection volume was
100 pi. Fractions were collected manually from a
second run according to the peptide profile ob-
tained in the first ran.

Identification of peptides in fractions

After collection, peptide fractions were evaporated
on a Waters PICO TAG Work Station and hydro-
lyzed using 6 M HCI (1% phenol) in the gas phase
for 24 hours at 110°C. Amino acids were analyzed
as phenylthiocarbamate (PTC) derivatives. Deri-
vatization and HPLC runs were performed accord-
ing to instructions issued by Millipore Corporation
(1987). The HPLC equipment consisted of the fol-
lowing parts: 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).

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Results

Hydrolysis by proteolytic enzymes

Figure 1 shows the chromatogram for the chymosin
hydrolysis of K-casein and the location of the pep-
tides identified on the basis of their amino acid
composition. It was shown that under these condi-
tions, the bond most sensitive to the activity of
chymosin was Phe 105-Met 106. The release of the
casein macropeptide was observed after 2 hours of
hydrolysis. After 24 hours, a few other bonds in the
casein macropeptide were also cleaved. Peptides
from the para-K-casein part were not identified.

Figure 2 gives theresults for the plasmin hydro-
lysis ofK-casein when the E:S ratio was 1:50 after
2, 4 and 24 hours of hydrolysis. Plasmin was found
to act on bonds in N-terminal region of K-casein.
N-terminal fragments 1-16 and 1-24were observed

already after 2 hours of hydrolysis. Plasmin cleaved
specifically at the carboxyl side of lysine. The Phe
105-Met 106 bond was found to hydrolyze slowly,
since the release of the casein macropeptide was
observed (Peak No. 10fig. 2) only after 24 hours of
hydrolysis.

Figure 3 shows the chromatograms of 2 and 4
hours of trypsin hydrolysates of K-casein. The res-
ults indicate that under the conditions used, bonds
were cleaved by trypsin all along the amino acid
chain ofK-casein.

Hydrolysis by Lactobacillus-proteases

Figures 4 and 5 present the proteolytic effect of
cell-free extracts ofL. helveticus LH7 and L. casei
subsp. rhamnosus M 9 on K-casein. The identified
fragments released from K-casein by the different

Fig. 1.Peptide profile of K-casein after 4 (A) and 24 (B) hours
of hydrolysis by chymosin (E:S = 1:100). Detection at 206
nm, injection volume 100 pi. Identified fragments: 1. 120-124
& 148-152; 2. 130-139; 3. 106-169.

491

Agric. Sei. Fin!. 2 (1993)



Lactobacillus-proteases are shown in Table 1. L.
helveticus strains released fragments from the mid-
section and the C-terminal region of K-casein. N
terminal and C-terminal peptides were identified in
hydrolysates by L. casei strains EB, P3, P 8 and Al.
In hydrolysates by L. casei strains A5, M9, G 2 and
S 9 only C-terminal peptides were identified. No
peptides could be identified in hydrolysates by
L.casei Ml. Free amino acids were released by
cell-free extracts of all L. casei and L. helveticus
strains. The strongest proteolysis, in terms of num-
ber of released peptides, was observed in L. casei
strains G2, S 9 and A5.

Discussion

The rapid and specific enzymatic cleavage of the
sensitive Phe 105-Met 106 peptide bond of K-ca-

sein by chymosin has been the subject of many
investigations (LAWRENCE and CREAMER 1969,
VISSER et al. 1976, 1977, 1987). Also in this study,
cleavage of the 105-106 bond was observed after 2
hours of incubation. After 24 hours of hydrolysis,
other bonds in the casein macropeptide were also
split by chymosin. These results are consistent with
those of Shammet et al. (1992), who observed an
increase in the number of peaks in the chroma-
togram after 30 minutes ofhydrolysis by chymosin.
This indicated that the enzyme started to cleave
other peptide bonds in the substrate.

Eigel (1977) reported that K-casein is resistant to
proteolysis by plasmin: even after 60 minutes of
incubation with plasmin no changes could be de-
tected in the electrophoretic pattern of K-casein.
The present study showed K-casein is hydrolyzed
slowly by plasmin, when the enzyme - substrate

Fig. 2. Peptide profile of K-casein after 2 (A), 4 (B) and 24 (C)
hours of hydrolysis by plasmin (E:S = 1:50). Detection at 206
nm, injection volume 100 pi. Identified fragments: 1. 22-24;
2. 17-21;3.1-16; 4.11-24; 5.17-24; 6. 1-16; 7.1-21; 8. 1-24;
9. 1-24; 10. 106-169.

Fig. 3. Peptide profile of K-casein after 2 (A) and 4 (B) hours
of hydrolysis by trypsin (E:S = 1:10000). Detection at 206
nm, injection volume 100pi. Identified fragments: 1. 1-16; 2.
1-10; 3. 1-21; 4. 87-99; 5. 17-24; 6. 98-105 ; 7. 98-112; 8.
98-111:9.69-86; 10. 106-169.

492

Agric. Sei. Fint. 2 (1993)



ratio was raised from 1:200 to 1:50. Under the
conditions of this study the N-terminal Lys-X
bonds were found to be sensitive to the proteolytic
activity of plasmin. The resistance of K-casein to
proteolysis by plasmin is fortunate considering the
importance of K-casein in stabilizing other major
caseins against precipitation by calcium (ZITTLE et
al. 1959, ZITTLE and Walter 1963).

Trypsin mainly hydrolyzed the carboxyl side of
arginine and lysine residues. This supports previous
findings concerning the specificity of trypsin
(Adler-Nissen 1986). On the basis of the hydro-
lysis products that were identified, it could be seen
that 9 of the 13 lysyl and argynyl containing bonds
of K-casein were split. Five of the cleaved bonds
were Lys-X and four were Arg-X bonds. In this
study, trypsin mainly hydrolyzed bonds in the N-
terminal and mid-section. It is presumed that se-

quence 25-68 contains three trypsin-sensitive
bonds; however, no peptides were detected from
this region. Sequence determinations have shown
that sequence 28-79 is resistant to enzymatic degra-
dation (JOLLES et al. 1970). In this sequence there
are three stretches of extended structure and an
a-helix, which may form a hydrophobic core and
render the bonds resistant to enzymatic degradation
(Raap et al. 1983).Leonil and Molle(1990) have
investigated the effect of trypsin on the casein
macropeptide. They observed three trypsin-sensit-
ive bonds, namely 111-112, 112-113 and 116-117;
bonds 111-112 and 112-113 were split much faster
than 116-117. In this study, the bond Lys 116-Thr
117 was not split, while the other two trypsin-sen-
sitive bonds (111-112, 112-113) were hydrolyzed
under the conditions used.

The bond 105-106 seems to be sensitive to the

Fig. 4. Peptide profile of K-casein after 48 hours ofhydrolysis
by cell-free extracts of L. helveticus LH7. Detection at 206
nm, injection volume 100 pi. Identified fragments: 1. Tyr
(free amino acid); 2. Phe (free amino acid); 3. 86-96; 4.
132-152; 5. 132-141.

Fig. 5. Peptide profile of K-casein after 48 hours of hydrolysis
by cell-free extracts ofL. casei subsp. rhamnosus M9. Detec-
tion at 206 nm, injection volume 100pi. Identified fragments:
1.Met (free amino acid); 2. He (free amino acid); 3. Tyr (free

amino acid); 4. Phe (free amino acid); 5.145-161;6.147-163;
7. 128-148.

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Agric. Sei. Finl. 2 (1993)



Table 1. Peptide sequences identified in hydrolysates of k-
casein produced by cell-free extracts of Lactobacillus-strains.

Strain N-terminal Mid- C-terminal
peptides section peptides

peptides

L. heiveticus LHI 87-94 126-140
L. heiveticus LHS 87-95 154-168
L. heiveticus LH7 86-96 132-141,

132-152
L. casei ES 1-8 126-151,

147-161
L. casei P 3 1-8 147-161
L. casei P 8 1-8 147-152
L. casei M 1-8 112-138,

147-161
L. casei G 2 112-118,

114-118
L. casei S 9 114-120
L. casei A 5 114-120
L. casei 128-148,
(rhamnosus) M 9 145-161,

147-163

action of proteinases other than chymosin, since the
casein macropeptide was released also by plasmin
and trypsin.

K-Casein was observed to be more susceptible to
the proteolytic action of the different Lactobacil-
/w.v-cultures used in this study than the other caseins
studied earlier (Pahkala et al. 1989a, 1989b,
Pihlanto-Leppälä et al. 1993). The differences in
activity observed earlier between differentLacto-
bacillus-strains (Pahkala et al. 1986) were not
apparent in this study. Instead, the Lactobacillus-
strains could be divided into three categories ac-
cording to the identified fragments; those that hy-
drolyzed bonds in the C-terminal region and mid-
section, only in the C-terminal region, or in the bl-
and C-terminal regions of K-casein. No clear sub-
strate specificity was observed among the peptides
identified. The results of this study would indicate
that para-K-casein is hydrolyzed during cheese rip-
ening. Harper et al. (1971) have obtained similar
results which showed extensive degradation of
para-K-casein during the ripening of Cheddar
cheese slurries. However, other previous studies
(Green and Foster 1974, Ledford et al. 1966)
have reported nohydrolysis ofpara-K-casein during
cheese ripening.

Acknowledgements. The writers wish to express their thanks
to the Ministry of Agriculture and Forestry ofFinland, which
has supported the study through its biotechnical research
fund.

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Manuscript received April 1993

Anne Pihlanto-Leppälä
Eero Pahkala
Veijo Antila
Agricultural Research Centre of Finland
Food Research Institute
FIN-31600 Jokioinen,Finland

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Agric. Sei. Fint. 2 (1993)



SELOSTUS

K-kaseiinin hydrolysoituminen kymosiinilla, plasmiinilla, trypsiinillä ja
Lactobacillus-proteinaaseilla

Anne Pihlanto-Leppälä, Eero Pahkala ja Veijo Antila

Maatalouden tutkimuskeskus

Tässä tutkimuksessa selvitettiin K-kaseiinin entsymaattista
hydrolyysiä eristämällä ja identifioimalla vapautuvia peptide-
jä. Entsyymeinä käytettiin kymosiinia, plasmiinia, trypsiiniä
sekä kolmesta Lactobacillus helveticus- ja yhdeksästä Lacto-
bacillus casei -kannasta eristettyä preparaattia.

K-kaseiinin Phe 105-Met 106 sidos oli herkkä kymosiinin
proteolyyttiselle aktiivisuudelle. Havaittiin, että 24 tunnin
hydrolyysin jälkeen myös muutamia muita kaseiinimakro-
peptidin sidoksia oli hydrolysoitunut. Plasmiinin proteolyytti-
nen aktiivisuus hydrolysoida käytettyä substraattia oli heikko,
sillä muutamia N-terminaalisia peptidejä vapautui kun ent-
syymi-proteiini suhde nostettiin l:200:sta l:so:neen. Trypsii-

ni hydrolysoi useita K-kaseiinin sidoksiapa peptidejä vapautui
lähes koko aminohappoketjun matkalta.

Lactobacillus-proteimasien vaikutus oli vähäisempi kuin
käytetyillä proteolyyttisillä entsyymeillä. K-kaseiinin hydro-
lysoituminen laktobasiHei 11a oli voimakkaampaa kuin muilla
aikaisemmin tutkituilla kaseiini-komponenteilla. Tunnistettu-
jen peptidien perusteella Lactobacillus- kannat voitiin jakaa
kolmeen luokkaan: L. helveticus kannat hydrolysoivat sidok-
sia aminohappoketjun keskialueelta ja C-terminaalista, L.
casei EB, P 3, P 8 ja AI kannat hydrolysoivat sidoksia N- ja
C-terminaalista ja L casei A 5 ja M 9 hydrolysoivat sidoksia
C-terminaalista.

496

Agric. Sei. Fin!. 2(1993)