Biotechnology Letters 22: 1557–1559, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
1557
Immobilization of endo-polygalacturonase from Aspergillus ustus on silica gel M. Narsimha Rao, A.A. Kembhavi & A. Pant∗ Division of Biochemical Sciences, National Chemical Laboratory, Pune 411008, India ∗ Author for correspondence (Fax: 091-20-5884032; E-mail:
[email protected]) Received 22 June 2000; Revisions requested 6 July 2000; Revisions received 2 August 2000; Accepted 3 August 2000
Key words: Aspergillus ustus, endo-polygalacturonase, immobilization, silica gel
Abstract Endo-polygalacturonase from Aspergillus ustus when immobilized on to modified silica gel retained 28% of its original activity. The immobilized enzyme could be re-used through 10 cycles of reaction with almost 90% retention of its original activity. It had increased thermostability over its soluble form: the half-life of the soluble enzyme at 40 ◦ C was less than 10 h whereas the immobilized enzyme retained 82% of its activity after 10 h at 40 ◦ C. Similarly, at 50 ◦ C the half-life of the soluble enzyme was 30 min whereas that of the immobilized enzyme was 5 h.
Introduction
Materials and methods
Pectinases are used in the food processing industry (Rombouts & Pilnik 1978, Uhlig 1990) and especially in the clarification of fruit juices. Pectinases have been immobilized on various matrices. The endo-polygalacturonase from Aspergillus niger has been immobilized by adsorption on porous polyethylene terephthalate (Rexova-Benkova et al. 1982). Omelkova et al. (1985) immobilized endopolygalacturonase on to porous poly(6-caprolactam) activated by glutaraldehyde with a relative activity of 24%. (The relative activity is the ratio of the activities of the bound and free enzyme expressed in percentage.) Other matrices which have been used for immobilization of polygalacturonases are poly(2,6dimethyl-p-phenylene oxide) (Rexova-Benkova et al. 1983), granular poultry bones (Findlay et al. 1986), porous glass (Romero et al. 1987) and nylon (Lozano et al. 1987). In the present study we report the use of silica gel, a robust and easily available matrix for immobilizing endo-polygalacturonase.
Enzyme Purified endo-polygalacturonase with a specific activity of 785 U mg−1 (Narsimha Rao et al. 1996) was used for immobilization. (One unit of enzyme activity is defined as the amount of enzyme which releases one µmol reducing sugar per min from 0.3% polygalacturonic acid in 0.1 M sodium acetate buffer, pH 4.6 at 40 ◦ C). Support Aminated silica gel was used as a support for the covalent immobilization of the enzyme. Aminated silica gel was prepared by refluxing 50 g silica gel (60–120 mesh) in 300 ml ethanolamine for 3 h. The silica gel was washed three times with 600 ml acetone, dried and activated with 250 ml 4% (w/v) glutaraldehyde in 0.1 M sodium phosphate buffer, pH 8.0 with slow shaking at 4 ◦ C for 2 h. The activated silica gel was washed with 0.1 M sodium acetate buffer, pH 4.6, to remove excess glutaraldehyde.
1558 Table 1. Effect of enzyme load on efficiency of binding on to matrix. Enzyme loaded (U g−1 silica gel)
Enzyme bound (U g−1 silica gel)
Activity expressed by bound enzyme (U g−1 silica gel)
Efficiency of binding (%)a
10 20 40 60 80 100
10 20 38 53 71 85
2.7 5.2 10.5 14.9 16.3 17.0
27 26 27.5 28 23 20
a Efficiency of binding is the ratio of the activity expressed by the bound enzyme
to the total activity actually linked to the matrix.
Immobilization of endo-polygalacturonase The activated silica gel was used for immobilization of the purified endo-polygalacturonase. The enzyme in 0.1 M acetate buffer, pH 4.6, was mixed with 1 g activated silica gel and incubated at 4 ◦ C with slow shaking for 16 h. Enzyme assay The soluble polygalacturonase activity was assayed by measuring the reducing sugars released from 0.3% polygalacturonic acid in 0.1 M sodium acetate buffer, pH 4.6 at 40 ◦ C (Collmer et al. 1988). One unit of polygalacturonase activity is defined as the amount of enzyme which releases 1 µmol reducing sugar per min. The activity of the immobilized enzyme was assayed by incubating 0.1 g silica gel in 5 ml 0.3% polygalacturonic acid in 0.1 M acetate buffer, pH 4.6. The increase in reducing sugars expressed as equivalents of D-galacturonic acid was measured by the method of Collmer et al. (1988). The units are expressed as µmol reducing sugars released per min per g silica gel. The efficiency of binding is the ratio of the activity expressed by the bound enzyme to the total activity actually linked to the matrix. All data are means of triplicates. The standard deviation was less than 5%.
Results and discussion The free aldehyde obtained by activating ethanolaminetreated silica gel with glutaraldehyde was linked to the side-chain amino groups of the enzyme, through
Schiff’s base formation. When the enzyme was treated with trinitrobenzene sulfonate, a reagent which binds specifically to lysine residues in proteins, the enzyme failed to bind to the matrix, indicating that binding takes place through the ε-amino groups of lysine residues on the surface of the enzyme. Previous studies on chemical modification of the active site residues of the endo-polygalacturonase had shown that lysine is not essential for catalytic activity (Narsimha Rao et al. 1996). The optimum loading of the enzyme on to the silica gel (Table 1) was with about 10 to 60 units of enzyme per gram of matrix giving retentions of 27–28%. Higher binding leads to lower efficiency probably due to crowding of the enzyme molecules on the support (down to 20%). The immobilized endo-polygalacturonase could pass through 10 cycles of reaction with 90% retention of its original activity. The tight binding to the support allowed very little leaching of the enzyme. The Lineweaver–Burk plot yielded a Km value of 1.32 mg polygalacturonic acid ml−1 for the bound enzyme, as compared to 0.82 mg ml−1 for the soluble enzyme. This suggests a lower affinity of the immobilized enzyme for the substrate. The higher apparent Km of the immobilized enzyme could be due to diffusional limitations of the polymeric nature of the substrate. Immobilized enzymes often have increased thermal stability over the soluble enzymes, though this is not true for all enzymes (Woodward 1985). As seen from the comparison of Figures 1A and 1B, the immobilized endo-polygalacturonase too had an increased thermal stability over the free enzymes. At 40 ◦ C, 50% loss of activity was observed within 10 h
1559 In conclusion, a simple process for immobilization of endo-polygalacturonase on modified silica gel has been described. The immobilized enzyme would be advantageous with respect to the sturdiness of the matrix used, its reusability and its thermostability.
References
Fig. 1. Temperature stability of (A): immobilized enzyme and (B): soluble enzyme. Temperature stability was determined by incubating (A) 0.1 g aliquots of matrix (immobilized enzyme) and (B) the soluble enzyme at different temperatures [4 ◦ C ( ), 30 ◦ C (#), 40 ◦ C (4), 50 ◦ C (N) and 60 ◦ C ()] in 0.1 M sodium acetate buffer pH 4.6. Residual activities of the immobilized and soluble enzyme were determined after every 10 h as described in Materials and methods.
for the soluble enzyme whereas the immobilized enzyme retained 82% of its activity even after 10 h at 40 ◦ C. Similarly, the half-life (50% retention of activity) of the soluble enzyme at 50 ◦ C was 30 min whereas the half-life of the immobilized enzyme at 50 ◦ C was 5 h. This increased stability would be advantageous in the industrial use of the immobilized endo-polygalacturonase. This organism produces only one enzyme without isoforms (Narsimha Rao et al. 1996). The apparently biphasic behaviour of the free and immobilized enzymes at and above 40 ◦ C (Figures 1A and B) is being studied further.
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