Hidrólise controlada de proteínas de soro lático usando tripsina e quimotripsina imobilizadas em diferentes suportes.
Resumen
This work is part of a global project whose aim is the production of a cheese
whey proteins hydrolysate with controlled composition. The process is composed by
sequential hydrolysis of the whey proteins using trypsin, chymotrypsin and
carboxipeptidase A (CPA). The phenylalanine (Phe) and the other aromatic amino acids
released after action of CPA may be removed from the product, providing an adequate
source of proteins for phenylketonurics patients and a final product with more pleasant
flavor. After its separation, a final step of hydrolysis using the non-specific protease
AlcalaseÒ produces a mixture of small peptides with better properties than a mixture of
free amino acids. However, the use of enzymes in industrial processes requests its
immobilization and stabilization. We have studied in this work the first two hydrolysis
stages aiming to reach the following general objectives: to prepare derivatives of trypsin
(on sepabeads, chitosan and agarose) and chymotrypsin (just on agarose), to study the
sequential hydrolysis of the cheese whey proteins with trypsin and chymotrypsin
immobilized on glyoxyl-agarose gel and to investigate the kinetic of the hydrolysis of
these proteins with immobilized chymotrypsin.
Trypsin-sepabeads derivatives were prepared on resins modified with
iminodiacetic acid (IDA) or modified with IDA and copper. Yield of immobilization of
approximately 100% and complete recovery of the enzyme on supports were obtained.
Factors of stabilization in relation to the soluble enzyme from 90 times (Sepabeads-
IDA-Cu2+) to 138 times (Sepabeads-IDA), at 55ºC, were reached and the trypsin-
(Sepabeads-IDA) derivative showed to be more efficient in the casein hydrolysis than
the trypsin-glyoxyl-agarose one.
Trypsin-chitosan derivatives were prepared on coagulated matrices in NaOH
solution (0.1 or 1N) and activated with glutaraldehyde (pH 7 or 10) or glycydol.
Chitosan derivatives whose matrices were activated with glutaraldehyde reached 100%
of yield of immobilization, while the ones activated with glycydol reached just 60%. In
all the studied cases it was possible to recover completelly the enzyme on supports until
the enzymatic load of 20mgEnz./gGel. Derivatives prepared on coagulated matrices in
NaOH 0.1 or 1N and activated at pH 7 resulted in 100% of yield of immobilization and
complete recovery of the enzyme on gel until the enzymatic load of 40mgEnz./gGel. At
40ºC, the glutaraldehyde-chitosan derivatives were approximately 460 times more stable
than the soluble enzyme; at 70ºC, the glyoxyl-chitosan derivative showed to be
approximately 13 times more stable than the glutaraldehyde-chitosan derivative. The
trypsin-chitosan derivatives presented the highest hydrolysis activity at 50ºC and pH 9
(40ºC and pH 9 for the soluble enzyme). The best trypsin-chitosan derivative
(coagulated in NaOH 0.1M and activated at pH 7) showed similar performance to the
soluble enzyme in the hydrolysis of the cheese whey proteins (hydrolysis degree (DH)
of 12%).
Trypsin and chymotrypsin derivatives immobilized on glyoxyl-agarose gel
were prepared following a protocol available in the literature (agarose activated with
glycydol and oxidized with NaIO4 to obtain 75µmoles of aldehyde/mL of gel, at 25oC
and pH 10.05). The factors of stabilization obtained for trypsin (3920 times) and
chymotrypsin (14535 times) are according with results already published and were
confirmed through acid hydrolysis of the soluble enzymes and stabilized derivatives.
These experiments showed that 64.76% and 72.15% of the lysines present in the trypsin
and chymotrypsin, respectively, were involved in the enzyme-support attachment. In
consequence of this stabilization, the derivatives showed maximum hydrolysis activity
of synthetic substrates in temperatures and pH higher than the obtained for the soluble
enzymes (trypsin - 85ºC and pH 11; chymotrypsin - 70ºC and pH 10.5).
Hydrolysis of the cheese whey proteins assays using trypsin were developed
varing the DH from 0 to 12%. After that, the obtained mixtures were hydrolysates with
chymotrypsin and carboxipeptidase A, sequentially, using long times and high enzymes
concentrations. The results showed that removal of Phe of approximately 100% was
obtained in the following conditions: DHtrypsin of 0%, DHchymotrypsin of 12.4%
(3.05mgEnz./mL of solution - 10 hours) and 10 hours of reaction with CPA (200UHPHE/
gProtein), producing 15.5% of peptides with molecular mass (MM) lower than
1046Da.
The kinetic of the hydrolysis of the whey proteins was studied and the
apparent kinetic parameters of the Michaelis-Menten model taking into account
competitive inhibition by the substrate ( app
max V , app
m K and
app
S K ) were calculated by
initial rates method using a derivative with high enzymatic load - 40mgEnz./gGel. In
this work, the substrate concentration was defined in terms of peptides bonds that could
be cleaved by chymotrypsin. The effectiveness factor found for reactions developed in
presence of difusional effects was 0.78. The long-term assays (10 hours) were perfectly
fitted by a model where the first five minutes were described by the first order kinetic
(V=kN) and times higher than five minutes were represented by a function of P/No
(V=f(P/No)).