BENEDET, HD

PARK, YK

Journal of Dairy Science

Repositorio da Producao Cientifica e Intelectual da Unicamp

Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis 
of Purified Casein Fractions Treated with 
Milk-Clotting Enzymes 
HONORIO D. BENEDET and YONG K. PARK 
Universidade Estadual de Campinas 
Faculdade de Engenharia de Alimentos e Agrfcola 
(UNICAMP) 
Campinas, SP. Brazil 
ABSTRACT 
Acid casein was fractionated into as- , 
fl-, and K-casein and was treated sepa- 
rately with Mucor miebei and Endotbia 
parasitica enzymes and calf chymosin. 
The enzyme treated casein fractions were 
subjected to sodium dodecyl sulfate- 
polyacrylamide gel etectrophoresis, and 
the electrophoretic patterns were com- 
pared with those of untreated casein 
fractions. All three enzymes showed 
proteolytic activity, but Endotbia parasi- 
tica enzyme degraded as-casein most. 
I N T R O D U C T I O N  
A recently growing cheese industry and in- 
creasing scarcity of calf rennet have stimulated 
a search for milk clotting enzyme from alter- 
nate sources. Microbial clotting enzymes espe- 
cially are favored because they can be mass 
produced. Many species of filamentous fungi 
produce chymosin-like enzymes (8). Microbial 
enzymes commercially used for cheese making 
are Mucor pusillus enzyme (1), Mucor miebei 
enzyme (9), and Endotbia parasitica enzyme 
(6). Sternberg (9) pointed our similarities in 
the clotting mechanism between M. miebei 
enzyme and calf chymosin. Vanderpoorten and 
Weckx (10) also reported that microbial rennet 
from M. miebei and M. pusillus Lindt produced 
a pattern of hydrolyzed products similar to 
calf rennin. 
This paper reports the results of a compari- 
son between as- , fl-, and K-caseins treated with 
milk clotting enzymes from M. miebei, E. 
parasitica, and the calf abomasum by sodium 
Received February 9, 1981. 
dodecyl sulfate (SDS) polyacrylamide gel 
electrophoresis. 
MATERIALS A N D  METHODS 
Preparation of Acid Casein 
Acid casein was prepared from unpasteuri- 
zed fresh bovine milk as described by Hipp et 
al. (4). One liter of milk was centrifuged to 
remove fatty substance and adjusted to pH 4.5 
with 1 N HC1. The precipitated casein was 
separated by centrifugation and washed three 
times by suspending it in deionized water. The 
fresh acid casein was used for further fractiona- 
tion. 
Preparation of ~x s- and/3-Casein 
The a s- and /3-caseins were fractionated in 
aqueous urea solutions from acid casein as 
described by Hipp et al. (5). 
Preparation of ~-Casein 
K-Casein was prepared from acid casein by 
the method of Zittle (13) and McKenzie and 
Wake (7). Alternately, K-casein also was pre- 
pared from acid casein by gel filtration as de- 
scribed by Yaguchi et al. (12). 
Milk Clotting Enzymes 
Purified E. parasitica enzyme was obtained 
from Pfizer Co., SP. Brazil. M. miebei enzyme 
was prepared by incubating solid wheat bran 
medium, which was inoculated with spores of 
Mucor miebei NRRL 3420. After incubation at 
30°C for 3 days, the aqueous extract was puri- 
fied as described by Sternberg (9). Chymosin 
was extracted from calf abomasa and purified 
as described by Berridge (3). 
1982 J Dairy Sci 65:899--901 899 
900 BENEDET AND PARK 
Preparation of Enzyme-Treated Casein Fractions 
Fif ty  milligrams of  as-,/3-, and K-casein were 
dissolved in 4 ml of  deionized water with .2 
N NaOH added. After  dissolution of caseins, 
solutions were adjusted to pH 6.8 with .2 N 
HCI and diluted to 5 ml with deionized water. 
Each solution of  as-,/~-, and g-casein was mixed 
with 100 Soxhlet  units (one Soxhlet  unit  is 
defined as the enzyme activity which clots 1 ml 
of substrate in 40 min under the conditions 
described by Sternberg (9) of  clotting enzyme 
from M. rniebei, or E. parasitica, or calf chymo- 
• . . O sin. The mixtures were incubated at 35 C for 
30 rain. After  incubation, the mixtures im- 
mediately were freeze dried. 
Sodium dodecyl sulfate (SDS) polyacryla- 
mide gel electrophoresis was performed as de- 
scribed by Weber and Osborn (11) on enzyme 
treated casein fractions. One milligram was 
dissolved in 1 ml of .01 M sodium phosphate 
buffer at pH 7.0, containing 1% SDS and 1% 
/3-mercaptoethanol and incubated at 37°C 
overnight• After  incubation, .1 ml of sample 
was mixed with 10/~1 of  .05% bromphenol  blue 
and .1 ml of 40% sucrose, then 50/~1 of mixture 
Figure 2. SDS-Electrophoresis of r-casein treated 
with various milk-clotting enzymes. 1) Calf rennin; 
2) Mucor miebei enzyme; 3) Endotbia parasitica 
enzyme; 4) untreated g-casein. 
were subjected to electrophroesis at a constant 
current (8 ma/gel tube) for 3 h. After  electro- 
phoresis, gels were fixed in a 50% trichloro- 
acetic acid solution overnight and for 2 h in 
.25% Coomassie blue in 20% trichloroacetic 
acid solution. 
Figure 1. SDS-Electrophoresis of as-casein treated 
with various milk-clotting enzymes. 1) Calf rennin; 
2) Mucor miebei enzyme; 3) Endotbia parasitica 
enzyme; 4) untreated as-casein. 
R ESU LTS AND D ISCUSSION 
o~s-Casein 
Effects of milk clotting enzymes on a s- 
casein are compared with untreated as-casein in 
Figure 1. All the enzymes catalyzed the degra- 
dation of themajor as-casein band. When 
treated with E. parasitica protease, the intensity 
of the major band was decreased markedly.  
/~-Casein 
Differences between electrophoretic patterns 
of  enzyme treated t3-casein and untreated con- 
trois were not  significant. 
g-Casein 
Electrophoretic patterns were run on K- 
Journal of  Dairy Science Vol. 65, No. 6, 1982 
DIGESTION OF CASEIN FRACTIONS 901 
casein p r epa red  by  two  procedures .  Bo th  g- 
casein f r ac t ions  s h o w e d  t he  same e l ec t rophore -  
t ic p a t t e r n s  w h e n  t r e a t ed  wi th  t he  same en- 
zymes  or  w h e n  used  as cont ro ls .  F igure  2 shows 
the  e l ec t rophore t i c  p a t t e r n  of  g-casein,  pre- 
pa red  by  m e t h o d  of  Zi t t le  (13) .  W h e n  t r e a t ed  
wi th  M. rniebei or E. parasitica enzymes ,  or 
chymos in ,  the  m a j o r  b a n d  was decreased  sub- 
s tant ia l ly .  E l e c t r o p h o r e t i c  p a t t e r n s  of  g-casein 
diges ted by  the  t h r ee  e n z y m e s  were similar.  
REFERENCES 
1 Arima, K., S. lwasald, and G. Tamura. 1967. Milk 
clotting enzyme from microorganisms. Agric. Biol. 
Chem. 31: 540. 
2 Beeby, R., and G. Mocquot. 1969. The sensitivity 
to rennin of an acid soluble fraction of casein. 
Biochim. Biophys. Acta 175:430. 
3 Berridge, N. J. 1955. Page 72 in Methods in enzy- 
mology. Vol. 2. Academic Press, New York, NY. 
4 Hipp, N. J., M. L. Groves, J. H. Custer, and T. L. 
McMeekin. 1950. Separation of "r-casein. J. Am. 
Chem. Soc. 72:4928. 
5 Hipp, N. J., M. L. Groves, J. H. Custer, and T. L. 
McMeeldn. 1952. Separation of ~-, a-, and K- 
casein. J. Dairy Sci. 35:272. 
6 Larson, M., and J. R. Whitaker. 1970. Endotbin 
parasitica protease. Parameters affecting stability 
of the rennin-like enzyme. J. Dairy Sci. 53:262. 
7 McKenzie, H. A., and R. G. Wake. 1961. An im- 
proved method for the isolation of g-casein. Bio- 
chim. Biophys. Acta 47:240. 
8 Prins, J., and T. L. Nielsen. 1970. Microbial rennet. 
Process Biochem. 5: 34. 
9 Sternberg, M. Z. 1971. Crystalline milk-clotting 
protease from Mucor miebei and some of its prop- 
erties. J. Dairy Sci. 54:159. 
10 Vanderpoorten, R., and M. Weckx. 1972. Nether- 
lands Milk Dairy J. 26:47. 
11 Weber, K., and M. Osborn. 1969. The reliability of 
molecular weight determination by dodecyl sul- 
fate-polyacrylamide gel electrophoresis. J. Biol. 
Chem. 244:4406. 
12 Yaguchi, M., D. T. Davies, and Y. K. Kim. 1958. 
Preparation of K-casein by gel filtration. J. Dairy 
Sci. 51:473. 
13 Zittle, C. A. 1962. Procedure for isolation of g- 
casein by use of sulfuric acid. J. Dairy Sci. 45: 
650. 
Journal of Dairy Science Vol. 65, No. 6, 1982 


SODIUM DODECYL-SULFATE POLYACRYLAMIDE-GEL ELECTROPHORESIS OF PURIFIED CASEIN FRACTIONS TREATED WITH MILK-CLOTTING ENZYMES

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SODIUM DODECYL-SULFATE POLYACRYLAMIDE-GEL ELECTROPHORESIS OF PURIFIED CASEIN FRACTIONS TREATED WITH MILK-CLOTTING ENZYMES

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