Indian Journal of Chemistry
Vol. 38A, November 1999, pp. 11 8 1- 11 83
Effect of solvents on the hydrolysis of
casein catalysed by pepsin-A kinetic study
A Dharmapuri & P K Saiprakas h*
Department of Chemi stry, Osmania Uni versi ty,
Hyderabad. 500007, India
.•
Received 18 Jan uary 1999; revised 14 May 1999
It is observed that pepsin shows increased catalyti c acti vity in
aquo-alcoholi c medi a compared to aqueous media. The maximum
activity of pepsi n has been recorded in the 20% to 40% range of
0.2M of aquo-alcoholic media. Kinetics of pepsin catalysed hydrolysis
of casein has been investi gated in di ffe rent alcohols vi z., methanol,
ethanol, n- propanol, isopropanol and I-butanol. At 0.2M alcohol concentrat ion thermal stability and acti vity of pepsin increases from primary to terti ary alcohols. The acti vi ty dec reases and thermal stabil ity increases with further increase in alcohol concentration.
Many e nzy mes are c ata lyti ca ll y acti ve in a wide variety of organi c so lvents 1-5 . The ir cata lyti c effici ency in
no n aqueous medi a is co mparable to that di spl ayed in
water. Ho wever, se lecti on of a suitable so lvent is impo rt ant becau se many so lve nts can inactiv ate the e nzy mes 6 The c riteri a fo r se lecting organi c so lvents for
enzy me cata lys is inc lude so lubility of reac tant s, stability o f enzy mes, tox ic ity and infl am abilit y 7 . Peps in pl ays
an important ro le as a bi ocatalyst in the produc ti on of
cheese, bre win g industry and in leathe r indu stry. Since
1970 there has been a surge of interest in casein hydro lysis kineti cs because of the first ph ase of milk coagul ati o n is hyd ro lys is of milk prote in case in by enzy mes and
the use of o rgani c sol vents in casein hyd ro lys is. Case in
is a large co ll o ida l agglo merate having a mean di a meter
g reate r th an 500 A 0 . M o lecul ar we ight of case in x is 3.28
x 109 . Hyd ro lys is of case in takes pl ace at o ne spec ific
peptide bo nd located at site Ph e l05 - M et lo6 alo ng the
mo lecul e. It was o bserved th at di gesti o n of casein w ith
pepsin , trypsin , c hymo ktrypsin and papa in y ie ld s in each
case produ cts hav ing mo lecul ar we ig ht of approxim ate ly
600 as dete rmined by freez ing po int depress ion 9 . Th e
res ult of hydro lys is is p ara-case in and mac ropeptide
products inc ludes ty ros ine w hi c h are so lubl e in tri c hl oroaceti c ac id (TC A) . The hydrol ysed products so fo rmed
cOlTespond to C te rmin a l end of case in and is re leased
during the hydro lys is of casein . An in solubl e peptide
corresponding to the N-te rmina l end is a lso form ed and
re m a in s a ss o c ia te d with th e c a se in m o lec ul e .
Nitsc hmann and Bohren lo first made an attempt to study
the kine tics of th e hydrol ys is of case in by enzy mes .
Animal protease conta ining pepsin has been tried fOf
casein hydro lys is. Be ing both a pro te in and a proteo lyti c
e nzy me, pepsin is capabl e of di gestin g itse lfll
Experimental
Procine pe psin twice crysta lli sed and casein [S isco
research lab Indi a] we re used without any furthe r puri fication . Trichloroacetic acid (M erck), sodium hyd roxide, potassium hydrogen ph osph ate, me thano l, eth ano l,
n-pro pano l, isopropan o l and t-buty l a lcoh o l and I-tyros ine were of BDH , AR g rade.
An son l2 proposed a meth od fo r th e de te rminati o n of
the acti vity of pe psin . The me thod in vo lved the estim ation of hydro lysed products fo rn1ed (T C A - so lu b le materi a l).
pepsin
Casein + Hp
--7
Para k Casein + macro peptide
A mi xtgure of pepsin so luti on ( 10 mg/S.O ml , 1.0 ml )
and casein soluti o n ( 1.0 g in 100 ml 0 . 1M potassi um
hydrogen ph osph ate buffe r of pH 6.4, S.Oml ) was in cubated at 303K for 10 min . The reacti on was termin ated
by additi on of 20 % tri c hl o roacetic ac id (2. 0 ml ). The
prec ipitate fo rmed was filtoff a fte r a ll o win g it to stand
fo r 15 min at roo m te mperature . T he co ncentrati o n of
hydro lysed produc ts in the supern ata nt liquid was dete rmined by fo ll ow in g th e absorbance at 280nm . T he activity of peps in was dete rmin ed in suitabl e aliqu ots by
the repo rted procedure using tyros in e as the standard u
Under these conditi o ns, the optica l de nsity of 1.00 corresponds to 82 ~ g of tyros in e re leased pe r min , w he n
measured at 280 nm in a Hitachi spectroph otometer.
Results and discussion
Case in has been used ex tensive ly as a substrate to
in vesti gate proteo lyti c enzy me kin eti cs 14 - 16 Th e fi ndin g
th at casein is a mi xture and that the co m ponents are probabl y present in the form o f co mpl exes and are hydro lysed un equ a ll y by an enzy me suggests a re in vesti gat io n
1182
INDIAN J CHEM, SEC. A, NOVEMBER 1999
Table 1 - Effect of concentration of different alcohols on acti vity of pepsin at 303 K and at pH 6.4
104 Activity (mol dm·lmin· l )
% of
Alcohols
Methanol
Eth anol
n-propanol
Isopropan
I-Butanol
0
0.Q25
0.027
0.025
0.027
0.027
20
0.068
0.,036
0.043
0.039
0.040
40
0.041
0.063
0.054
0.073
0.081
60
0.029
0.054
0.040
0.043
0.054
80
0.024
0.034
0.032
0.027
0.044
100
0.019
0.027
0.024
0.019
0.027
of the enzymatic hydroly sis of casein in which the three
components are used as substrates. The influence of
differe nt a lcohol s like viz., methanol, e thanol , n-propanol, isopropanol and t-butyl alcohol have been utilized
to record the catalytic activity of pepsin. Activity of
pepsin (amount of casein hydrolysed per mm .) at 303K
in 0.2M alcohols at pH = 6.4 is as follows ( 104 activity,
mol dm·' mml). Aq . media 0.027, aquo-n-propanol 0.054,
aquo ethanol 0.063, aquo methanol 0.068, aquo isopropanol 0.073 and aquo t-butanol 0.081. It was observed
that the activity of pepsin gradually increased from npropanol to t-butyl alcohol compared to that in aqueous
media. Thi s enhanced activity in non aqueous solvent is
due to the presence of organic solvents around the microenvironment of the enzyme that can alter the secondary, te rtiary and quaternary structures of the protein and
can penetrate into the active sites of the enzyme and di srupt the chemical and structural balance and thereby promoting higher activity of the enzyme l7 Pepsin in 100%
alcoholic media shows no activity at all which indicates
that removal of water from the microenvironment of e nzyme drastically di storts the enzyme conformation and
inactivates the enzyme (Table I). Therefore, some degree of hydration of pepsin is necessary to maintain is
catalytic activity'x. Low concentration of alcohols enhances the activity, which may be due to restoration of
conditions more closely resembling those in cellu lar environment in which these enzymes are presumably optimall Y.
pH profile
The optimum pH of hydrolsis of casein by pepsin in
aqueous medium was 6.4. There is no a lteration in pH
optima when peps in is dis so lved in different alcoholic
media from that of aqueous medi a, a lthough slight broadening in pH curves were observed which increased with
the primary carbon to tertiary carbon of the alcohols.
Pepsin stabilised in alcohols shows catalytic activity over
a wide pH range. There is not much loss of activity at
extreme pH range.
Temp erarure profile
The optimum te mperature of pepsin at O.I M phosphate buffer of pH 6.4 is 303 K and the re is no shift in
optimum temperature when pepsin is dis so lved in different alcohols.
Thermal stability
The change in the water content of pepsin so lution
affects both the activity and the stability. Pepsin in aqueous media was de natured at 323K. Whe n incubated in
alcohols, pepsin showed increase in thermal stability.
Generally, enzyme stability increases when th e water
content dec reases . In the case of alco o ls, the therma l
stability increases with alcohol concentrat ion . Thermal
stability increased with the change from primary carbon
to tertiary carbon of alcohols. This unu sual stabi lity of
pe psin in alcoholic media may be du e to th e threedomens ional conformation of t he enzy me , which
stabilised low energy bonds that are broken mainly as a
result of an increase in vibration energy and collision
with water molecules as temperature is raised. Prote in
hydrogen bonds are of the same magnitude as those of
the enzyme intramolecular bond s. Compounds hav in g
low interaction with enzyme but strong interacti on with
water will stabilise the enzyme 21l • Pepsi n is more stab le
in alcohols because alcohols have greater affinity for
water.
NOTES
~
Table 2 - Kin eti c parameters of pepsin in aq ueous and 0.2M
alcohols at 303 K at p H 6.4
k
10' K m
10' V
Medi um
min-I
mol dm-)
mol dm-)I~i;r '
Aqueo us
2.78
II. I
1.86
Aquo-II-pro panol
2.6 1
11. 8
1.98
Aquo-eth anol
2.70
12.7
2_ 13
Aq uo- meth anol
2.5 1
13.5
2.26
Aq uo- isopropan ol
2.24
14_ 1
2.36
Aquo-I-butanol
1.94
14.9
2.46
Tabl e 3 - Thermod ynamic parameters in both aq ueous and 0.2 M
alco hols at p H 6.4
Med ia
~
tJ.H#
Ea
(kJmol-' ) (kJmol·' )
tJ.C#
tJ.S #(kJm ol-' ) (Jmol-' K -')
11 83
pend strongl y upon the reacti on medium. From the slopes
o f the An-h eniu s plot activati on e nergy is ca lcul ated both
in aqueous medium and pepsin stab ili sed in vari ous alcoholi c med ia. The other thermodynami c para mete rs
like ~H#, ~G # a nd ~S $ have been eva luated (Tabl e 3).
F rom the primary carbon to te rtiary carbon of alcoho ls,
activation energy, free e ne rgy of acti vati on, e nth alpy of
activation gradually dec reased , accounting for rate enhancement in alcoho lic medium . However, the e ntropy
of acti vation was found to be more negati ve indi catin g
that the activated compl ex is more ri gid in alcoholi c medi a, probabl y due to grea ter so lvat ion of the co mple x
and since ~H # va lu es are also very low. Entropy of ac tivation seems to playa domin ant rol e in the cata lysis invo lving pepsin . It was observed that w ith an in c rease in
alco hol s concentrati on above 0.2M the re is a grad ual
decrease in the hydro ly sis rates .
Aqueous
11. 87
9.35
72.7
209
References
Aquo-n-propanol
9.1 9
6.67
72.5
2 17
I.
Aquo- meth anol
6. 13
3.60
72.2
226
Aquo-eth anol
7.08
4.57
72.3
223
Aquo-isopropanol
4.2 1
1.69
72. 1
232
Aquo-I-butano l
3.64
1. 12
71.9
234
Kin etic and th ermodynamic param eters
The kinetic paramete r lik e Kill a nd V lllax were calculated from th e plots of I Iv versus I/[S] (LineweaverBurk plots) in aqueous med ia and different aquo- alcoholi c medi a. Regress ion coeffic ie nts for all the plots
we re 0 .99 ± 0.005 . There is a steady increase in V lllax '
gradual decrease in Km values from aqueous to aquoalcoholic media (Tabl e 2). Thi s is due to dec rease in the
so lvent dielectric constant. These c hanges will affec t
'substrate spec ifi city and cata lyti c effi ciency of an enzy me which initi all y de pend s on th e ability of enzy me
to utili ze the free e nergy of bindin g with the substrate 21 .
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of substrate e nzy mes and substrate so lvent interaction 22 .
Kineti c parameters describin g enzy me function such as
binding co nstant with substrate k, Mi c hae li s - Menten
co nstant K111 and catalytic turn over VIll ax therefore, de-
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22 . Fersht A Enzy_ Sl ructure & mechan ism (edited by W H Freeman) second edition ( 1985)