Pectinolytic enzymes are a group of enzymes which hydrolyze pectin, one of the main polysaccharides in plant cell wall. The primary chain of pectin is composed of α-1,4- linked residues of D-galacturonic acid (Jayani et al., 2005). The enzymes depolymerising pectin can be divided into hydrolases and lyases. Hydrolases degrade pectin by hydrolysis of α-1,4-glycosidic linkages in the polymer chain. Lyases cause depolimerisation by transelimination which results in formation of unsaturated bond between C4 and C5 at the nonreducing end (Sakai et al., 1993). Pectinases are distributed in many higher plants and microorganisms. They play a very important role in plants since they help in cell wall extension and softening of some plant tissues (Jayani et al., 2005).
Pectinases find commercial application in production of fruit juices and wines, mainly for juice clarification and for improving pressing and extraction of the juice from fruits and vegetables (Kashyap et al., 2001). The raw fruit juice obtained after pressing is very turbid, viscous and contains a significant amount of colloidal compounds, mainly pectin which cause cloudiness. The amount of colloids present in fruit juices is in the range of 100-1000 mg L-1. Clarification involves the removal of juice haze by enzyme hydrolysis with pectolytic enzymes. After its degradation pectin- protein complexes flocculate giving a juice with lower viscosity which is easier to filter (Alvarez et al., 1998, 2000).
Microbial pectinases account for 25% of the global food enzymes sales. Most commonly used in industry are pectinases from Aspergillus niger which produces a mixture of polygalacturonase, pectinlyase and pectinesterase (Jayani et al., 2005; Almeida et al., 2005).
It is well known that immobilization of enzymes offers several advantages that
include reuse of the enzyme and its easier separation from the product. Since
in food industry it is preferably to avoid the presence of extraneous compounds
in the final products the possibility to remove the enzyme is a significant
advantage. In literature there is data about immobilization of pectinolytic
enzymes on different supports by various of methods (Demirel et al.,
2004; Vaillant et al., 2000; Rao et al., 2000; Sarioglu et
al., 2001; Sardar and Gupta, 2005).
In the present study commercial pectinase from Aspergillus niger was immobilized on polyacrylonitrile copolymer membrane via adsorption on the membrane or covalently after activation of the support with glutaraldehyde. The methods used for immobilization are simple and effective. The chosen support has suitable pore size that allows easy penetration of the enzyme; it has high mechanical, temperature and chemical stability and can be separated from the reaction mixture without contaminating the final product.
MATERIALS AND METHODS
Commercial pectinase from Aspergillus niger ( Biovet, Bulgaria), Apple pectin, 70% esterification degree, Polyacrylonitrile copolymer UF membrane (PAN) (Ekofilter, Bulgaria), 25 kDa.
The research was done in the department of Biochemistry and Molecular Biology, University of Food Technologies, Plovdiv, Bulgaria, in the period January- July, 2006.
Activation of the Membrane
The membrane was hydrolyzed with 6% NaOH for 60 min, then activated with
25% glutaraldehyde (GA) solution in water for 90 min (Godjevargova et al.,
1994). After the activation the membrane was washed thoroughly until no GA is
detected in the washings by measuring the absorbance at 235 nm.
Immobilization of Pectinase on PAN Membrane
The activated with GA and not activated membrane (20 cm2) was
immersed in 10 cm3 enzyme solution and mild stirred at room temperature
for 16 h. After the immobilization the membrane was washed thoroughly with distilled
water, 0.5 M NaCl and C - P buffer until no protein is detected in the washings.
Determination of Enzyme Activity
Pectinase activity was determined by the method of Roboz (1952) by following
the decrease in viscosity of 1% pectin solution in C-P buffer, pH 5.0, with
a capillary viscometer at 30°C.
The percent decrease of viscosity of pectin solution is calculated by the equation:
where to- flow time of the substrate solution; t - flow time after the enzymatic reaction; ts - flow time of the solvent.
RESULTS AND DISCUSSION
Commercial pectinase was immobilized on PAN membrane which is a good and used matrix for immobilization of enzymes (Godjevargova, 2004). For determining the influence of immobilization conditions only not activated membrane was used. The duration of immobilization is shown in Fig. 1.
The activity of the immobilized enzyme increased gradually till the eight hour and after that remained unchanged after 48 h immobilization time.
||Effect of duration of the immobilization process on the activity
of immobilized pectinase
||Effect of pectinase concentration on the activity of the immobilized
enzyme: (a) at pH 5.0, room temperature and (b) at pH 3.0, room temperature
The effect of pectinase concentration added for immobilization was studied.
Starting from 0.1% (w/v) the activity of the immobilized enzyme raised to a
maximal level. The optimal enzyme concentration was found to be 2% (w/v) at
pH of immobilization 5.0 and 1% (w/v) at pH of immobilization 3.0 (Fig.
2a and b). For further experiments the second concentration
of pectinase was chosen.
It can be seen that the effect of pH was well expressed and the immobilized enzyme had maximal activity at pH 3.0 of immobilization at 10°C, (Fig. 3) room temperature and 30°C. Above this pH the activity of immobilized Pectinase decreased and at pH of immobilization 6.0 and 7.0 was below 5%.
Immobilization changed the pH optimum of the enzyme which was at pH 4.0 for
free pectinase (Fig. 4). The enzyme immobilized on not activated
membrane exhibited optimal pH at 5.0 and the enzyme immobilized on activated
with GA membrane at pH 5.5. This is in agreement with the results observed by
Pifferi et al. (1988) for immobilization of endo-polygalacturonase and
can be explained with a change of the microenvironment of the enzyme as a result
of its immobilization to the matrix although the chosen support is electrically
neutral (Chibata, 1978).
||Effect of pH and temperature of immobilization on the activity
of immobilized pectinase
||Effect of pH on the activity of free and immobilized pectinase:
(a) on not activated membrane and (b) on activated with GA membrane
A shift in the pH optimum was observed also by Rexova- Benkova et al.
(1986) in immobilization of endopolygalacturonase on ceramic materials and also
by Vaillant et al. (2000) in immobilization of another pectinolytic enzyme
pectin lyase on chitin and nylon. Except the change in the pH optimum there
was also a change in the pH profile of the immobilized enzyme compared with
the free form. Immobilized pectinase exhibited lower activity at pH below 4
than free enzyme and higher activity at pH above 5.0. The enzyme immobilized
on not activated membrane retained more than 40% of its optimal activity at
pH range 6.0-7.0 while free pectinase retained below 10% of its activity.
Free enzyme exhibited maximum activity at 50°C while the temperature optimum of immobilized on activated and not activated membrane enzyme elevated to 60°C (Fig. 5). As a result from enzyme binding to the matrix by covalent or non covalent bonds the rigidity of enzyme molecules is increased so that the protein is less affected by the denaturing effect of temperature (Spagna et al., 1997).
At 30, 40 and 50°C the stability of free pectinase was greater than that
of the immobilized one (Fig. 6). At 30°C free and immobilized
enzyme on activated with GA membrane retained its maximal activity for 6 h incubation
and the enzyme immobilized on not activated membrane only for 2 h.
||Effect of temperature on the activity of free and immobilized
pectinase: A- on not activated membrane; b- on activated with GA membrane
||Thermal stability of free and immobilized pectinase at pH
4.0: (a) -30°C; (b) -40°C; (c) -50°C; (d) -60°C
At 40°C pectinase immobilized on not activated PAN membrane almost lost
activity for 6 h. For the same time free pectinase retained 80% of its original
activity while the enzyme immobilized on activated membrane retained 43%.
||Reusability of immobilized pectinase
At 50°C pectinase immobilized on not activated membrane completely lost
activity for 30 min while free and immobilized on activated membrane enzyme
were active after 2 h incubation at this temperature. At 60°C pectinase
immobilized on not activated membrane deactivated for 10 min while the immobilized
on activated membrane retained 50% of its original activity after 20 min incubation.
It is generally considered that immobilized enzymes exhibited higher thermal
stability than that of the free ones. In this research it can be seen that free
pectinase was more stable than pectinase immobilized on PAN membrane via adsorption
on the membrane or via covalent binding. Probably this is due to weak bonds
formed between the enzyme and the support that cause easy separation of the
enzyme from the matrix. The enzyme immobilized on activated with GA membrane
exhibited higher stability than that immobilized on not activated membrane.
Obviously the covalent bonds formed between CHO- groups of the crosslinker and
NH2- groups of the enzyme retained the protein stronger to the support.
The usability of immobilized pectinase in presented in Fig. 7.
It was able to hydrolyze pectin in 22 consecutive batch cycles. After each cycle
the membrane with immobilized enzyme was washed with distilled water and incubated
with fresh portion of substrate. Activity is expressed as percentage of decrease
in viscosity of pectin solution. Three different pectin concentrations were
used for this experiment -0.5, 1 and 2%. Gradually after each new reuse the
immobilized pectinase lost activity which is expressed as lower percentage viscosity
decrease of pectin solution.
For first time Aspergillus niger pectinase was successively immobilized on polyacrylonitrile copolymer UF membrane activated with GA and not activated. The immobilization parameters were found- duration of immobilization, pectinase concentration, pH and temperature of the enzyme solution during the immobilization process. The effect of pH and temperature on the activity of free and immobilized enzyme and the stability at 30, 40, 50 and 60°C were determined. The reusability of immobilized pectinase in subsequent batch cycles was examined using apple pectin as substrate.
The immobilized pectinase preparation can be used in practice for clarifying of fruit juices and wine. The chosen support (ultrafiltration membrane) offers the opportunity the process to be held in a membrane reactor in which enzyme treatment and filtration can be done in one step.