T. Nikolic et al. / Carbohydrate Polymers 82 (2010) 976–981
977
by different methods, such as adsorption (Kotel’nikova, Mikhailova
Vlasova, 2007), entrapment (Monteiro et al., 2007) and cova-
weight loss of oxidized cotton yarn samples by applying the direct
gravimetric method (Koblyakov, 1989).
&
lent binding (Cavalcante, Carvalho, & Carneiro-da-Cunha (2006);
Kumar & Gupta, 1998). Immobilization of enzymes by covalent cou-
pling usually leads to very stable preparations compared with other
immobilization procedures. Various carriers have been used for the
immobilization of trypsin, e.g., chitosan-coated silica gel (Xi, Wu,
Jia, & Lin, 2005), polyester fleece (Nouaimi, Möschel, & Bisswanger,
2.3. Determination of aldehyde group content
The aldehyde content present in the oxidized cotton was mea-
sured according to the method described in literature (Kumar &
Yang, 2002; Praskalo et al., 2009; Saito & Isogai, 2004). The alde-
hyde groups were selectively oxidized to carboxyl groups with
sodium chlorite at pH 4–5, at room temperature for 48 h, and car-
boxyl group content was determined by calcium-acetate method
modified by Praskalo et al. (2009). Before titrations all cotton sam-
ples were ion-exchanged into acid form by suspending in 0.01 M
HCl for 1 h, followed by washing with distilled water. The aldehyde
group content was calculated by subtracting the carboxyl content
value determined in the starting cotton sample from that of chlorite
oxidized samples.
2
001), chitosan and cellulose (Zelenetskii et al., 2003). Medical
requirements considerably limit the number of carriers that can
be used in the immobilization of enzymes for therapeutic uses.
The carriers must be nontoxic, noncarcinogenic, biocompatible
and in no way injurious in the biological environment. Dialde-
hyde cellulose has been recommended as a suitable matrix for the
immobilization of drugs and hormones. Besides, it has been found
that insoluble form of the dialdehyde cellulose itself inhibits the
microbial growth, and this activity resists heat and washing (Hon,
1
996).
In this paper, we report the oxidation of cotton fibers with
2.4. Determination of fineness of oxidized cotton yarns
sodium periodate solutions under different conditions in order to
obtain a suitable carrier for subsequent immobilization of trypsin,
an enzyme with anti-inflammatory properties. Cotton, as the purest
natural form of cellulose, was chosen for periodate oxidation
because of the following: it possesses excellent properties, such as
regeneration, biodegradation, softness, affinity to skin and hygro-
scopic property, and is traditionally used as a bandaging material.
Our data demonstrated that trypsin immobilized on selective oxi-
dized cotton fibers was markedly stabilized for a long period of
storage.
Fineness in tex was determined as per standard method (SRPS
ISO 2060, 1994).
2.5. Tensile strength measurement
Cotton yarns were tested by using tensile tester Tex Test
(Switzerland), with clamps spaced at 100 mm, and with strain rate
(bottom clamp rate) of 150 mm/min, according to the usual proce-
dure described elsewhere (Koblyakov, 1989). The tensile strength
of the yarn was calculated as the mean value of 20 measurements.
2
. Experimental
2.6. Immobilization of trypsin on oxidized cotton yarn
2.1. Materials
A trypsin solution (0.8 mg/ml) was prepared by dissolving
enzyme in 100 mM Tris–HCl buffer (pH 9.2) containing 10 mM
Raw cotton ring yarn (fineness: 20.85 tex, CV = 3.4%; yarn twist:
55 t.p.m., CV = 5.5%), which is intended for gauze production, was
CaCl . The above mentioned oxidized cotton yarn was incubated
2
7
◦
with the solution of trypsin (ratio 1:25, w/v) for 18 h at 4 C. Subse-
obtained from Strumicanka (Strumica, FYR of Macedonia). Trypsin
from bovine pancreas (EC 3.4.21.4) in a powder form and N-␣-
benzoyl-dl-arginine p-nitroanilide hydrochloride (BAPNA) were
purchased from Sigma (St. Louis, MO, USA). All other chemicals used
were of analytical grade.
quent to immobilization, the yarn was washed with physiological
solution 9 times to remove unbound trypsin. The supernatants of
each washing were collected for protein measurements. The resul-
tant immobilized trypsin on cotton yarn was stored in physiological
solution. All immobilizing tests were performed in duplicate.
2.2. Oxidation of cotton yarn with sodium periodate
2.7. Protein loading assay
A sample of cotton yarn was immersed in solutions of sodium
The amount of trypsin immobilized on the cotton yarn was
periodate in 0.1 M acetic buffer (ratio 1:50, w/v), pH 4.0 at con-
centrations of 2.0 mg/ml and 4.0 mg/ml, i.e. 0.2% and 0.4% (w/v).
The mixture was then stirred in the absence of light, at room tem-
perature, for 15, 30, 45, 60, 120, 180, 240, 300 and 360 min. After
completion of the oxidation, the cotton yarn was washed with
ice-cold distilled water several times to remove the oxidant. This
oxidized cotton yarn was used for the immobilization of trypsin
without drying.
The effects of reaction time and periodate concentration on
the rate of oxidation of cotton fibers were studied. Consumption
of one periodate molecule produces two aldehyde groups. There-
fore, the rate of periodate consumption (i.e. the decrease in the
periodate content of the solution, referred to the weight of fibers
immersed in it, and expressed as molecules of periodate per 100
glucose units) may be identified with the rate of oxidation. Titrime-
try was used to calculate the periodate consumption (Nevell,
determined from a mass balance, i.e. difference between the con-
tent of protein in solution before and after immobilization, and in
the combined washings, using following equation:
Ts0 - Tsi - Tsw
Tiy
=
m
where Tiy is amount of trypsin immobilized on the cotton yarn
(mg/g of cotton); Ts0 is content of trypsin in solution before and
Tsi after immobilization (mg), and Tsw is content of trypsin in com-
bined washing solutions (mg) determined according to the method
of Bradford (Kruger, 2002) and using bovine serum albumin as the
standard, while m is the weight of absolute dry cotton yarn (g).
2.8. Trypsin activity assay
The trypsin activity in the solution and in the immobilized
state was determined by a method based on the initial rate of
hydrolysis of the substrate BAPNA, similar as described in the lit-
erature (Nouaimi et al., 2001; Ohta, Makinen, & Loesche, 1986; Xi
et al., 2005). One unit of activity was expressed as the amount of
1
957).
The formation of soluble fragments, as a result of the cellulose
destruction (i.e. cellulose chain scission caused by subsequent reac-
tion, not by oxidation itself), was determined by measuring the