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Studies on Penicillium sp. ZDZ1 chitosanase immobilized on chitin by cross-linking reaction
Process Biochemistry 38 (2002) 531 /535 www.elsevier.com/locate/procbio
Studies on Penicillium sp. ZDZ1 chitosanase immobilized on chitin by cross-linking reaction Jia Zeng, Lian-Ying Zheng * Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, PR China Received 28 January 2002; received in revised form 11 May 2002; accepted 13 May 2002
Abstract Chitosanase was immobilized on chitin with glutaraldehyde by cross-linking reaction and the immobilization conditions and characterization of the immobilized enzyme were carried out. The optimal conditions for immobilization were as follows: 0.1 g chitin was treated with 5 ml 5% solution of glutaraldehyde and 2 mg chitosanase was immobilized on the carrier. Optimal temperature and pH for the immobilized enzyme were 608 C and 4.0, respectively. Kinetic parameter Km was 17.66 g/l. The immobilized enzyme showed better temperature stability than the free enzyme and its operation and storage stability were good. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Chitin; Immobilization; Chitosanase; Chitosan
1. Introduction Chitosan is one of the most abundant renewable polysaccharides prepared from chitin through chemical N -deacetylation. It is attracting more and more interest due to its potential application in medicine, industry and agriculture. Recently chito-oligosaccharides have received much attention because they are not only water soluble, but possess distinctive biological activity such as antitumour activity [1], antifungal and antibacterial activity [2,3], immuno-enhancing effects [4], enhancement of protective effects against infection with some pathogens in mice [5]. Chito-oligosaccharides can be prepared by chemical or enzymic depolymerization. Enzymic hydrolysis is a preferable method because of easy control, mild conditions of hydrolysis and low pollution to the environment. Chitin, a polysaccharide containing 2-deoxy-2-amino glucose units linked through b-1,4 linkages, is present in crustaceans, insects and marine crabs etc. It is hydrophilic, inert and nonbiodegradable. It has been reported that every five amino groups out of six in the chitin
* Corresponding author. Tel.: /86-571-8795-1192; fax: /86-5718891-3753 E-mail address: [email protected] (L.-Y. Zheng).
molecule are in the acetylated form. The presence of the amino groups in the chitin molecule provides a binding site for proteins. Thus chitin can be used as a solid support for the preparation of immobilized enzymes. Recently, a number of studies have shown that chitin is a good carrier for enzyme immobilization. Spagna et al. [6] used chitin as carrier to immobilize a-L-arabinofuranosidase by cross-linking the reaction. The immobilized enzyme activity was 150 U/g. Compared with free enzyme, the immobilized enzyme has better temperature and storage stability. Vaillant et al. [7] used chitin and nylon as carrier to immobilize pectinlyase and endocellulase respectively, the results showed that chitin was a more comfortable carrier for enzyme immobilization. It is also cheaper and the operation was very convenient. Zheng et al. [8] used chitin as carrier and glutaraldehyde as cross-linking reagent to immobilize cellulase. Compared with other carriers, chitin showed better ability for enzyme immobilization. Liu et al. [9] also chose chitin as carrier to immobilize Ficin by cross-linking reaction. The apparent Km was slightly higher than free enzyme and the pH and temperature stability were better. The optimal temperature for immobilized enzyme was lower than free enzyme, but the immobilized enzyme also showed good operation and storage stability. In a previous study, a strain was screened to produce an extracellular chitosanase with high activity hydrolyz-
0032-9592/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 1 6 3 - 2
532
J. Zeng, L.-Y. Zheng / Process Biochemistry 38 (2002) 531 /535
ing chitosan in culture. In the present work, the chitosanase was immobilized on chitin and conditions for immobilization and characterization of the immobilized enzyme were studied systematically.
2. Materials and methods 2.1. Materials Commercial chitosan, average molecular weight 290 kDa, 93% N -deacetylated; refined chitin was prepared in our laboratory. All other chemicals were of analytic grade.
2.5. Immobilization of chitosanase Refined chitin was treated with 5 ml glutaraldehyde at a definite concentration in the presence of 0.5 ml acetic acid as catalyst and stirred at room temperature for 24 h. The chitin /glutaraldehyde complex was washed several times with distilled water until the washings were free of glutaraldehyde. Chitosanase was added to chitin /glutaraldehyde complex and allowed to stand for 24 h at 4 8C occasional stirring. The supernatant was removed by centrifugation at 3000 rpm and chitin / chitosanase complex washed with distilled water until protein in the washing could not be detected. The immobilized chitosanase was then collected and stored at 4 8C.
2.2. Microorganism Penicillium sp. ZDZ1 was stored on agar slant at 4 8C. 2.3. Preparation of partial purified chitosanase The microorganism was scraped off from the agar slant and washed out with sterilized water. Then 1 ml of microorganism solution was inoculated into a 250 ml flask containing 60 ml of the medium and incubated on a rotary shaker at 180 rpm for 60 h at 308 C. The composition of the medium (pH 6.0) was (w/v), chitosan 1%, peptone 0.1%, (NH4)2SO4 0.1%, carbamide 0.1%, KH2PO4 0.06%. Finally the cells of the culture broth were removed from the medium by centrifugation at 2500 rpm for 15 min at 4 8C and the supernatant was collected. The chitosanase was salted out by adding 20/ 70% saturation ammonium sulphate, and the precipitates were collected by centrifugation at 9000 rpm for 25 min. The precipitates were dissolved in an appropriate volume of distilled water and dialyzed against water overnight at 4 8C. Finally the partial purified enzyme was used for immobilization.
3. Results and discussion 3.1. Immobilization of chitosanase on chitin 3.1.1. Effect of concentration of glutaraldehyde on chitosanase immobilization Chitin was treated with different concentrations of glutaraldehyde ranging from 1 to 6%, and chitin/ glutaraldehyde was then treated with chitosanase. The activity of the immobilized enzyme was determined as described above. As shown in Fig. 1, the optimal concentration of glutaraldehyde for immobilization was 5%, and enzyme activity increased with increasing concentration of glutaraldehyde. When the concentration of glutaraldehyde was more than 5%, enzyme activity did not increase further. 3.1.2. Effect of amount of added enzyme on chitosanase immobilization Chitin was treated with 5 ml 5% glutaraldehyde and the chitin /glutaraldehyde was then treated with different amounts of chitosanase ranging from 0.5 to 3.0 mg.
2.4. Chitosanase assay 2.4.1. Free chitosanase The activity of tree chitosanase was determined by a 3,5-dinitrosalicy acid method [10]. 2.4.2. Immobilized chitosanase Immobilized enzyme 0.1 g was added to 3 ml 1% chitosan solution and incubated at 508 C for 30 min. The reaction was stopped by adding 0.5 ml 1 M NaOH solution. The mixture was centrifuged at 3000 rpm for 15 min. The concentration of reducing sugar was determined by a 3,5-dinitrosalicy acid method. One unit was defined as the amount of enzyme that could produce l mmol reducing sugar in 1 min.
Fig. 1. Effect of concentration of glutaraldehyde on enzyme immobilization.
J. Zeng, L.-Y. Zheng / Process Biochemistry 38 (2002) 531 /535
533
Fig. 2. Effect of the amount of added enzyme on enzyme immobilization.
The activity of immobilized enzyme was assayed and the results are shown in Fig. 2. The optimal amount of added chitosanase was 2.0 mg.
Fig. 4. SEM micrograph of free carrier.
3.1.3. SEM of immobilized enzyme The immobilized enzyme and free carrier was subjected to SEM. As shown in Fig. 3 and Fig. 4, the chitosanase was immobilized on the carrier by a crosslinking reaction. 3.2. Characterization of immobilized chitosanase 3.2.1. Effect of pH on the activity of immobilized chitosanase The activity of immobilized chitosanase was determined at various pH ranging from 2.5 to 6.0 at 50 8C. The optimal pH for the immobilized enzyme was 4.0, and for the free enzyme was 5.0 (Fig. 5). 3.2.2. Effect of temperature on the activity of immobilized chitosanase The activity of immobilized chitosanase was determined at different temperatures ranging from 30 to
Fig. 3. SEM micrograph of immobilized enzyme.
Fig. 5. Effect of pH on the activity of immobilized enzyme.
80 8C at pH 4.0. As shown in Fig. 6, the optimal temperature for immobilized enzyme was 60 8C and the optimal temperature for free enzyme was 50 8C. 3.2.3. Determination of apparent Km The Km value of the immobilized enzyme was found to be 17.66 g/l by Lineweaver/Burk plots (Fig. 7). The
Fig. 6. Effect of temperature on the activity of immobilized enzyme.
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J. Zeng, L.-Y. Zheng / Process Biochemistry 38 (2002) 531 /535
Fig. 9. Operation stability of immobilized enzyme. Fig. 7. Lineweaver /Burk plots of immobilized enzyme and free enzyme.
Km of the free enzyme was estimated to be 2.611 g/l. The Km value of the immobilized enzyme was higher than that of the free enzyme which means the immobilized enzyme had less affinity for substrate.
3.2.4. Temperature stability of immobilized chitosanase The immobilized enzyme and free enzyme were incubated in 608 and 30 8C in water baths for 150 min respectively. During incubation, the activity of the immobilized enzyme was determined every 30 min. As shown in Fig. 8 the loss of activity of the immobilized enzyme was less than 20% but enzyme activity decreased significantly for the free enzyme after 60 min incubation and was almost all lost after 150 min incubation.
3.2.5. Operation stability of immobilized chitosanase Immobilized enzyme 0.5 g was added to 10 ml 1% chitosan solution in a water bath 50 8C for 6 h, and the reaction was repeated 10 times. After each reaction, the enzyme activity was determined. As shown in Fig. 9 the immobilized enzyme showed good operation stability,
Fig. 8. Temperature stability of immobilized enzyme.
and the enzyme activity loss was less than 20% after 10 times batch reaction. 3.2.6. Storage stability of immobilized chitosanase The immobilized enzyme was stored at 4 8C for 20 days, and enzyme activity measured every 3 days. The activity recovery was more than 80% after 20 days. The results indicate that the immobilized enzyme had good storage stability.
4. Discussion In the present work, chitosanase from Penicillium sp. ZDZ1 was partial purified and immobilized on chitin in a cross-linking reaction with 5% glutaraldehyde as crosslinking reagent. Mohamed et al. [11] reported that alkaline protease was immobilized on chitin by physical adsorption, ion-coagulate, cross-linking and entrapment methods. Among those four immobilization methods, cross-linking method was the optimal method for enzyme immobilization on chitin. Reiss et al. [12] also obtained similar results. The concentration of glutaraldehyde used for enzyme immobilization was similar to that of immobilized glucoamylase [13]. Zhou et al. [14] reported that 2% glutaraldehyde was sufficient for enzyme immobilization, and Spagna et al. [15] also reported that 0.5% was also sufficient. The more free amino groups on the chitin, the more glutaraldehyde will be needed for immobilization. Meantime, the enzyme immobilized on chitin will also increase with the increase of the amino group cross-linked by glutaraldehyde. The optimal pH for immobilized chitosanase shifted to the acidic side. This may be explained by electrostatic potential theory. Chitin is a positively charged carrier, thus the electrostatic potential within the immobilized enzyme Cim was higher than that in the bulk solution Cbulk. The concentration of [H1] within the immobilized enzyme will be lower than that in the
J. Zeng, L.-Y. Zheng / Process Biochemistry 38 (2002) 531 /535
bulk solution which causes the pH-activity curve to shift to the acidic side. The apparent Km,im of immobilized chitosanase was higher than the intrinsic Km, which means that the affinity between enzyme and substrate decreased after immobilization. This may be due to the same charge of the carrier and substrate, thus the local concentration of substrate near the carrier was lower than that in the bulk solution for the electrostatic repulsion. In general the immobilized chitosanase with chitin as carrier using glutaraldehyde as cross-linking reagent showed significant advantages over free enzyme. The temperature, operation and storage stability of the immobilized enzyme were much better than free enzyme, and the carrier is very cheap and the operation process is very simple, and it is very good for large-scale industrial application.
Acknowledgements This work was supported by Nature Science Fund of Zhejiang province, PR China.
References [1] Suzuki K, Mikami T, Okawa Y, Tokoro A, Suzuki S. Antitumor effect of hexa-N -acetylchitohexaose and chitohexase. Carbohydrate Res 1986;151:403 /8. [2] Jeon YJ, Park PJ, Kim SK. Antimicrobial effect of chitooligosaccharides produced by bioreactor. Carbohydrate Polymers 2001;44:71 /6.
535
[3] Hirano S, Nagao N. Effect of chitosan, pectic acid, lysozyme and chitinase on the growth of several photopathogens. Agric Biol Chem 1989;53:3056 /61. [4] Suzuki S. Studies on biological effect of water soluble low homologous oligosaccharides of chitin and chitosan. Fragrance J 1996;15:61 /8. [5] Yamada A, Shibuya N, Kodama O, Akatsuka T. Induction of phytolexin formation in suspension-cultured rice cells by N actylchito-oligosacchearides. Biosci Biotech Biochem 1993;57:405 /9. [6] Spagna G, Andreani F, Salatelli E, et al. Immobilization of a-Larabinofuranosidase on chitin and chitosan. Process Biochem 1998;33(1):57 /62. [7] Vaillant F, Millan A, Millan P, et al. Co-immobilized pectinlyase and endocellulase on chitin and nylon. Process Biochem 2000;35(9):989 /96. [8] Zheng LY, Zeng J, Zhu JF. Studies on hydrolysis of chitosan by chitin immobilized cellulase. Chem React Eng Technol 2001;17(4):344 /8. [9] Liu DF, Dong WY, Bai H, et al. Studies on Ficin immobilized on chitin. Food Sci 1994;15(11):3 /6. [10] Miller GL. Use of dinitrosalicitic acid reagent for determination of reducing sugar. Anal Chem 1959;31:426 /8. [11] Mohamed A, Ismail AM, Ahmed S, et al. Production and immobilization alkaline protease from Bucillus mycoides . Bioresource Technol 1998;64(3):205 /10. [12] Reiss M, Heibges A, Metzger J, et al. Determination of BODvalues of starch-containing waste water by a BOD-biosensor. Biosensors Bioelectron 1998;13(10):1083 /90. [13] Stanley WL, Watters GG, Chan BG, et al. Glucoamylase immobilized on chitin with glutaraldehyde. Biotech Bioeng 1978;20(2):135 /40. [14] Zhou H, Yang Z, Ouyang P. Study of condition of chitinimmobilized for preparation L-asparic acid by immobilized cell. J Nanjing Univ Chem Technol 1999;21(2):68 /71. [15] Spagna G, Andreani F, Salatelli S. Immobilization of a-Larabinofuranosidaseon chitin and chotsan. Process Biochem 1998;33(1):57 /62.
Studies on Penicillium sp. ZDZ1 chitosanase immobilized on chitin by cross-linking reaction Jia Zeng, Lian-Ying Zheng * Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 310027, PR China Received 28 January 2002; received in revised form 11 May 2002; accepted 13 May 2002
Abstract Chitosanase was immobilized on chitin with glutaraldehyde by cross-linking reaction and the immobilization conditions and characterization of the immobilized enzyme were carried out. The optimal conditions for immobilization were as follows: 0.1 g chitin was treated with 5 ml 5% solution of glutaraldehyde and 2 mg chitosanase was immobilized on the carrier. Optimal temperature and pH for the immobilized enzyme were 608 C and 4.0, respectively. Kinetic parameter Km was 17.66 g/l. The immobilized enzyme showed better temperature stability than the free enzyme and its operation and storage stability were good. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Chitin; Immobilization; Chitosanase; Chitosan
1. Introduction Chitosan is one of the most abundant renewable polysaccharides prepared from chitin through chemical N -deacetylation. It is attracting more and more interest due to its potential application in medicine, industry and agriculture. Recently chito-oligosaccharides have received much attention because they are not only water soluble, but possess distinctive biological activity such as antitumour activity [1], antifungal and antibacterial activity [2,3], immuno-enhancing effects [4], enhancement of protective effects against infection with some pathogens in mice [5]. Chito-oligosaccharides can be prepared by chemical or enzymic depolymerization. Enzymic hydrolysis is a preferable method because of easy control, mild conditions of hydrolysis and low pollution to the environment. Chitin, a polysaccharide containing 2-deoxy-2-amino glucose units linked through b-1,4 linkages, is present in crustaceans, insects and marine crabs etc. It is hydrophilic, inert and nonbiodegradable. It has been reported that every five amino groups out of six in the chitin
* Corresponding author. Tel.: /86-571-8795-1192; fax: /86-5718891-3753 E-mail address: [email protected] (L.-Y. Zheng).
molecule are in the acetylated form. The presence of the amino groups in the chitin molecule provides a binding site for proteins. Thus chitin can be used as a solid support for the preparation of immobilized enzymes. Recently, a number of studies have shown that chitin is a good carrier for enzyme immobilization. Spagna et al. [6] used chitin as carrier to immobilize a-L-arabinofuranosidase by cross-linking the reaction. The immobilized enzyme activity was 150 U/g. Compared with free enzyme, the immobilized enzyme has better temperature and storage stability. Vaillant et al. [7] used chitin and nylon as carrier to immobilize pectinlyase and endocellulase respectively, the results showed that chitin was a more comfortable carrier for enzyme immobilization. It is also cheaper and the operation was very convenient. Zheng et al. [8] used chitin as carrier and glutaraldehyde as cross-linking reagent to immobilize cellulase. Compared with other carriers, chitin showed better ability for enzyme immobilization. Liu et al. [9] also chose chitin as carrier to immobilize Ficin by cross-linking reaction. The apparent Km was slightly higher than free enzyme and the pH and temperature stability were better. The optimal temperature for immobilized enzyme was lower than free enzyme, but the immobilized enzyme also showed good operation and storage stability. In a previous study, a strain was screened to produce an extracellular chitosanase with high activity hydrolyz-
0032-9592/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 1 6 3 - 2
532
J. Zeng, L.-Y. Zheng / Process Biochemistry 38 (2002) 531 /535
ing chitosan in culture. In the present work, the chitosanase was immobilized on chitin and conditions for immobilization and characterization of the immobilized enzyme were studied systematically.
2. Materials and methods 2.1. Materials Commercial chitosan, average molecular weight 290 kDa, 93% N -deacetylated; refined chitin was prepared in our laboratory. All other chemicals were of analytic grade.
2.5. Immobilization of chitosanase Refined chitin was treated with 5 ml glutaraldehyde at a definite concentration in the presence of 0.5 ml acetic acid as catalyst and stirred at room temperature for 24 h. The chitin /glutaraldehyde complex was washed several times with distilled water until the washings were free of glutaraldehyde. Chitosanase was added to chitin /glutaraldehyde complex and allowed to stand for 24 h at 4 8C occasional stirring. The supernatant was removed by centrifugation at 3000 rpm and chitin / chitosanase complex washed with distilled water until protein in the washing could not be detected. The immobilized chitosanase was then collected and stored at 4 8C.
2.2. Microorganism Penicillium sp. ZDZ1 was stored on agar slant at 4 8C. 2.3. Preparation of partial purified chitosanase The microorganism was scraped off from the agar slant and washed out with sterilized water. Then 1 ml of microorganism solution was inoculated into a 250 ml flask containing 60 ml of the medium and incubated on a rotary shaker at 180 rpm for 60 h at 308 C. The composition of the medium (pH 6.0) was (w/v), chitosan 1%, peptone 0.1%, (NH4)2SO4 0.1%, carbamide 0.1%, KH2PO4 0.06%. Finally the cells of the culture broth were removed from the medium by centrifugation at 2500 rpm for 15 min at 4 8C and the supernatant was collected. The chitosanase was salted out by adding 20/ 70% saturation ammonium sulphate, and the precipitates were collected by centrifugation at 9000 rpm for 25 min. The precipitates were dissolved in an appropriate volume of distilled water and dialyzed against water overnight at 4 8C. Finally the partial purified enzyme was used for immobilization.
3. Results and discussion 3.1. Immobilization of chitosanase on chitin 3.1.1. Effect of concentration of glutaraldehyde on chitosanase immobilization Chitin was treated with different concentrations of glutaraldehyde ranging from 1 to 6%, and chitin/ glutaraldehyde was then treated with chitosanase. The activity of the immobilized enzyme was determined as described above. As shown in Fig. 1, the optimal concentration of glutaraldehyde for immobilization was 5%, and enzyme activity increased with increasing concentration of glutaraldehyde. When the concentration of glutaraldehyde was more than 5%, enzyme activity did not increase further. 3.1.2. Effect of amount of added enzyme on chitosanase immobilization Chitin was treated with 5 ml 5% glutaraldehyde and the chitin /glutaraldehyde was then treated with different amounts of chitosanase ranging from 0.5 to 3.0 mg.
2.4. Chitosanase assay 2.4.1. Free chitosanase The activity of tree chitosanase was determined by a 3,5-dinitrosalicy acid method [10]. 2.4.2. Immobilized chitosanase Immobilized enzyme 0.1 g was added to 3 ml 1% chitosan solution and incubated at 508 C for 30 min. The reaction was stopped by adding 0.5 ml 1 M NaOH solution. The mixture was centrifuged at 3000 rpm for 15 min. The concentration of reducing sugar was determined by a 3,5-dinitrosalicy acid method. One unit was defined as the amount of enzyme that could produce l mmol reducing sugar in 1 min.
Fig. 1. Effect of concentration of glutaraldehyde on enzyme immobilization.
J. Zeng, L.-Y. Zheng / Process Biochemistry 38 (2002) 531 /535
533
Fig. 2. Effect of the amount of added enzyme on enzyme immobilization.
The activity of immobilized enzyme was assayed and the results are shown in Fig. 2. The optimal amount of added chitosanase was 2.0 mg.
Fig. 4. SEM micrograph of free carrier.
3.1.3. SEM of immobilized enzyme The immobilized enzyme and free carrier was subjected to SEM. As shown in Fig. 3 and Fig. 4, the chitosanase was immobilized on the carrier by a crosslinking reaction. 3.2. Characterization of immobilized chitosanase 3.2.1. Effect of pH on the activity of immobilized chitosanase The activity of immobilized chitosanase was determined at various pH ranging from 2.5 to 6.0 at 50 8C. The optimal pH for the immobilized enzyme was 4.0, and for the free enzyme was 5.0 (Fig. 5). 3.2.2. Effect of temperature on the activity of immobilized chitosanase The activity of immobilized chitosanase was determined at different temperatures ranging from 30 to
Fig. 3. SEM micrograph of immobilized enzyme.
Fig. 5. Effect of pH on the activity of immobilized enzyme.
80 8C at pH 4.0. As shown in Fig. 6, the optimal temperature for immobilized enzyme was 60 8C and the optimal temperature for free enzyme was 50 8C. 3.2.3. Determination of apparent Km The Km value of the immobilized enzyme was found to be 17.66 g/l by Lineweaver/Burk plots (Fig. 7). The
Fig. 6. Effect of temperature on the activity of immobilized enzyme.
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J. Zeng, L.-Y. Zheng / Process Biochemistry 38 (2002) 531 /535
Fig. 9. Operation stability of immobilized enzyme. Fig. 7. Lineweaver /Burk plots of immobilized enzyme and free enzyme.
Km of the free enzyme was estimated to be 2.611 g/l. The Km value of the immobilized enzyme was higher than that of the free enzyme which means the immobilized enzyme had less affinity for substrate.
3.2.4. Temperature stability of immobilized chitosanase The immobilized enzyme and free enzyme were incubated in 608 and 30 8C in water baths for 150 min respectively. During incubation, the activity of the immobilized enzyme was determined every 30 min. As shown in Fig. 8 the loss of activity of the immobilized enzyme was less than 20% but enzyme activity decreased significantly for the free enzyme after 60 min incubation and was almost all lost after 150 min incubation.
3.2.5. Operation stability of immobilized chitosanase Immobilized enzyme 0.5 g was added to 10 ml 1% chitosan solution in a water bath 50 8C for 6 h, and the reaction was repeated 10 times. After each reaction, the enzyme activity was determined. As shown in Fig. 9 the immobilized enzyme showed good operation stability,
Fig. 8. Temperature stability of immobilized enzyme.
and the enzyme activity loss was less than 20% after 10 times batch reaction. 3.2.6. Storage stability of immobilized chitosanase The immobilized enzyme was stored at 4 8C for 20 days, and enzyme activity measured every 3 days. The activity recovery was more than 80% after 20 days. The results indicate that the immobilized enzyme had good storage stability.
4. Discussion In the present work, chitosanase from Penicillium sp. ZDZ1 was partial purified and immobilized on chitin in a cross-linking reaction with 5% glutaraldehyde as crosslinking reagent. Mohamed et al. [11] reported that alkaline protease was immobilized on chitin by physical adsorption, ion-coagulate, cross-linking and entrapment methods. Among those four immobilization methods, cross-linking method was the optimal method for enzyme immobilization on chitin. Reiss et al. [12] also obtained similar results. The concentration of glutaraldehyde used for enzyme immobilization was similar to that of immobilized glucoamylase [13]. Zhou et al. [14] reported that 2% glutaraldehyde was sufficient for enzyme immobilization, and Spagna et al. [15] also reported that 0.5% was also sufficient. The more free amino groups on the chitin, the more glutaraldehyde will be needed for immobilization. Meantime, the enzyme immobilized on chitin will also increase with the increase of the amino group cross-linked by glutaraldehyde. The optimal pH for immobilized chitosanase shifted to the acidic side. This may be explained by electrostatic potential theory. Chitin is a positively charged carrier, thus the electrostatic potential within the immobilized enzyme Cim was higher than that in the bulk solution Cbulk. The concentration of [H1] within the immobilized enzyme will be lower than that in the
J. Zeng, L.-Y. Zheng / Process Biochemistry 38 (2002) 531 /535
bulk solution which causes the pH-activity curve to shift to the acidic side. The apparent Km,im of immobilized chitosanase was higher than the intrinsic Km, which means that the affinity between enzyme and substrate decreased after immobilization. This may be due to the same charge of the carrier and substrate, thus the local concentration of substrate near the carrier was lower than that in the bulk solution for the electrostatic repulsion. In general the immobilized chitosanase with chitin as carrier using glutaraldehyde as cross-linking reagent showed significant advantages over free enzyme. The temperature, operation and storage stability of the immobilized enzyme were much better than free enzyme, and the carrier is very cheap and the operation process is very simple, and it is very good for large-scale industrial application.
Acknowledgements This work was supported by Nature Science Fund of Zhejiang province, PR China.
References [1] Suzuki K, Mikami T, Okawa Y, Tokoro A, Suzuki S. Antitumor effect of hexa-N -acetylchitohexaose and chitohexase. Carbohydrate Res 1986;151:403 /8. [2] Jeon YJ, Park PJ, Kim SK. Antimicrobial effect of chitooligosaccharides produced by bioreactor. Carbohydrate Polymers 2001;44:71 /6.
535
[3] Hirano S, Nagao N. Effect of chitosan, pectic acid, lysozyme and chitinase on the growth of several photopathogens. Agric Biol Chem 1989;53:3056 /61. [4] Suzuki S. Studies on biological effect of water soluble low homologous oligosaccharides of chitin and chitosan. Fragrance J 1996;15:61 /8. [5] Yamada A, Shibuya N, Kodama O, Akatsuka T. Induction of phytolexin formation in suspension-cultured rice cells by N actylchito-oligosacchearides. Biosci Biotech Biochem 1993;57:405 /9. [6] Spagna G, Andreani F, Salatelli E, et al. Immobilization of a-Larabinofuranosidase on chitin and chitosan. Process Biochem 1998;33(1):57 /62. [7] Vaillant F, Millan A, Millan P, et al. Co-immobilized pectinlyase and endocellulase on chitin and nylon. Process Biochem 2000;35(9):989 /96. [8] Zheng LY, Zeng J, Zhu JF. Studies on hydrolysis of chitosan by chitin immobilized cellulase. Chem React Eng Technol 2001;17(4):344 /8. [9] Liu DF, Dong WY, Bai H, et al. Studies on Ficin immobilized on chitin. Food Sci 1994;15(11):3 /6. [10] Miller GL. Use of dinitrosalicitic acid reagent for determination of reducing sugar. Anal Chem 1959;31:426 /8. [11] Mohamed A, Ismail AM, Ahmed S, et al. Production and immobilization alkaline protease from Bucillus mycoides . Bioresource Technol 1998;64(3):205 /10. [12] Reiss M, Heibges A, Metzger J, et al. Determination of BODvalues of starch-containing waste water by a BOD-biosensor. Biosensors Bioelectron 1998;13(10):1083 /90. [13] Stanley WL, Watters GG, Chan BG, et al. Glucoamylase immobilized on chitin with glutaraldehyde. Biotech Bioeng 1978;20(2):135 /40. [14] Zhou H, Yang Z, Ouyang P. Study of condition of chitinimmobilized for preparation L-asparic acid by immobilized cell. J Nanjing Univ Chem Technol 1999;21(2):68 /71. [15] Spagna G, Andreani F, Salatelli S. Immobilization of a-Larabinofuranosidaseon chitin and chotsan. Process Biochem 1998;33(1):57 /62.