Reversible Immobilization of Chitinase via Coupling to Reversibly Soluble Polymer

Reversible immobilization of chitinase via coupling to reversibly soluble polymer San-Lang Wang and Sau-Hwa Chio Department of Food Engineering, Da-Yeh University, Chan-Hwa, Taiwan The chitinase produced by Pseudomonas aeruginosa K-187 can be covalently immobilized on a polymeric support (hydroxypropyl methylcellulose acetate succinate, AS-L) which is soluble above pH 5.5 but insoluble below pH 4.5. Crude enzyme solution was used for the immobilization study. Efficiency of immobilization was 99%. The activation energy was 9.9 kcal g21 mol21 free enzyme system which reduced to 5.96 kcal g21 mol21 after immobilization. These values are higher than those for common insoluble solid supports. For the immobilized enzyme, the optimum pH and temperature shifted to pH 8 and 50°C. The half-life at 4°C was 9 days for free enzyme and 13 days for immobilized enzyme. Immobilized chitinase retained 70% of its original activity after 10 batches of chitinolytic reactions; meanwhile, the enzyme still showed antimicrobial (lysozyme) activity after immobilization. © 1998 Elsevier Science Inc. Keywords: Shrimp and crab shell waste; Pseudomonas aeruginosa; chitinase; reversibly soluble polymers; immobilized enzyme

Introduction Chitinases, a group of enzymes capable of degrading chitin directly to low molecular weight products, have been shown to be produced by a number of microorganisms.1–5 Pseudomonas aeruginosa K-187, which was recently isolated from the soil of Northern Taiwan, is a producer of chitinase when cultured in shrimp and crab shell powder.6,7 Two chitinases produced by strain K-187 had bifunctional chitinase/lysozyme characteristics.8 Chitooligosaccharides derived from the hydrolysis product of chitins are useful for agrochemical and medicinal purposes. Their production has attracted much attention in the related industry.9 Recovery of an enzyme after its application is a major concern in process economics. Immobilization is considered favorable in saving the enzyme for reuse. Traditional immobilization uses water-insoluble supports10 –12; however, since the chitin/chitinase is a solid-liquid heterogeneous reaction system which involves adsorption of chitinase to chitin particle and subsequent hydrolysis reaction,

Address reprint requests to Dr. S.-L. Wang, Da-Yeh University, Department of Food Engineering, Chan-Hwa, 51505, Taiwan Received 18 June 1997; revised 26 November 1997; accepted 5 December 1997

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immobilized enzyme in an insoluble form will usually result in poor performance due to diffusion limitations.13–15 A promising method of solving these problems inherent in heterogeneous reaction systems is to utilize an enzyme immobilized on a reversibly soluble-insoluble support that is able to change its solubility depending on the condition of the reaction solution (such as pH, temperature, or ionic strength).16 –20 One previous study of this type of immobilized purified chitinase has been used for chitin hydrolysis.16 Using purified chitinases for immobilization are sometimes not economical due to the high prices of commercially available purified chitinases. In this paper, we describe the immobilization of crude chitinase of P. aeruginosa K-187 to a reversibly soluble polymer. The reversible carrier used in this study was hydroxypropyl methylcellulose acetate succinate (AS-L). The original use of this carrier was for antacid encapsulation. It changes solubility with changes in pH. Furthermore, it contains active functional groups such as hydroxyl (OH2) and carboxyl (COO2). These groups are suitable for binding enzymes. Tests on the optimal operating condition for strain K-187 chitinase using AS-L immobilization were performed in this study. The conditions tested included enzyme activity, fraction immobilization, and fraction of enzyme recoverable.

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Reversible immobilization of chitinase: S.-L. Wang and S.-H. Chio

Materials and methods Materials Hydroxypropyl methylcellulose acetate succinate (AS-L), molecular weight 93,000, was manufactured by Shin-Etsu Chemical Company, Tokyo, Japan. Ethylene glycol chitin (EGC), glycol chitin (GC), and 1-ethyl-3 (3-dimethylaminopropyl)-carbondiimide hydrochloride (EDC) were obtained from Sigma (St. Louis, MO). Trichloroacetic acid (TCA), carboxymethyl cellulose (CMC), casein, chitin powder, were reagent grade (Wako Chemicals, Japan). Shrimp and crab shell powder (SCSP) was purchased locally. Microprotein assay kits were obtained from the Bio-Rad Chemical Company. Colloidal chitin was prepared from powdered chitin by the method of Jeniaux.21

The enzyme activities on GC, EGC, and CMC were assayed by the procedures as described previously.8,25

Effects of pH on solubility of AS-L and immobilization AS-L was dissolved in a 0.1 m citric acid-NaH2PO4 buffer solution pH 4 –5, NaOH-NaH2PO4 solution pH 6 – 8, or glycine-NaOH solution pH 9. The pH of the solution ranged from 4 –9. At each pH, the turbidity of the solution was measured. Under each of these conditions, a 3 ml protein solution was added and was allowed to react for 4 h. The enzyme activity and protein content of each mixture were measured based on that at pH 7 as 100%.

The effect of protein recovery on its activity Crude chitinase solution P. aeruginosa K-187 was cultured in a medium containing 3% shrimp and crab shell powder, 0.1% CMC, 0.1% (NH4)2SO4, 0.1% K2HPO4, 0.1% ZnSO4, 0.05% MgSO4 z 7H2O at a pH of 7 and a temperature of 37°C for 2 days. To the cell-free culture broth (850 ml), 515 g of ammonium sulfate was added. The resulting precipitate was collected by centrifugation, dissolved in a small amount of 50 mm phosphate buffer pH 7, and dialyzed using a tube of seamless cellulose (Sankyo Co., Tokyo, Japan) against the same buffer for 18 h.

Chitinase immobilization AS-L (1 g) was dissolved in 20 ml of 0.1 m phosphate buffer pH 7. This was mixed with a solution made with 150 mg EDC in 5 ml water. The mixture was agitated for 20 min before its pH was lowered to 4.5 with a 1 m phosphoric acid to precipitate AS-L. After filtration, the resulting precipitate was redissolved in 20 ml of 0.1 m phosphate buffer pH 7 and then 3 ml of crude chitinase solution was added. After incubating at 25°C for 4 h, the pH of the mixture was adjusted to 4.5 with a 1 m phosphoric acid. The resulting precipitate was collected as an immobilized enzyme by filtration. The immobilized enzyme was used after washing three times at room temperature with about 500 ml of 0.1 m citrate buffer pH 4.5.

Protein assay Protein content was estimated by the method of Bradford22 using Bio-Rad protein dye reagent concentrate. Bovine serum albumin was used as the standard.

Measurement of enzyme activity Chitinase activity was measured with colloidal chitin as a substrate.8 The amount of reducing sugar produced was measured by the method of Imoto and Yagishita23 with n-acetylglucosamine as a reference compound. To measure protease activity, an appropriately diluted enzyme solution (0.2 ml) was mixed with 2.5 ml of 1% casein in phosphate buffer pH 7 and incubated for 10 min at 37°C. The reaction was stopped by adding 5 ml of 0.19 m trichloroacetic acid followed by centrifuging for 15 min. The supernatant (2.5 ml) was added to the mixture of 5 ml 0.28 n NaOH and 2 ml phenol reagent (FolinCiocalteu phenol solution:water 5 1:2).24 After 15 min, the optical density at 578 nm was measured with a spectrophotometer (Beckman UD-70). The amount of amino acid released was determined from a calibration curve with tyrosine as a reference compound. One unit (U) of enzyme activity was defined as the amount of enzyme required to produce 1 mmol amino acid h21.

After AS-L was first prepared, its protein content was precipitated in a 1 m phosphate buffer at pH 4.5. The precipitate was redissolved in a 0.1 m phosphate buffer pH 7. This procedure was repeated 10 times and for each recovery step the activity and protein contents were measured. A value of 100% for the activity and protein content of the AS-L was based on the values obtained when it was first prepared.

Antimicrobial action of chitinase The action of chitinase against both Gram-positive and Gramnegative bacteria was examined. Cells of each organism were suspended on molten nutrient agar medium and then poured into Petri plates. Paper discs were placed on the surface of the medium and the enzyme solution to be assayed was pipetted into each disc. Buffer without enzyme was used as a blank for the control experiment. Fifteen target strains were tested. They were: Bacillus subtilis CCRC 10029, Enterococcus faecalis CCRC 10066, Escherichia coli CCRC 10239, Mycobacterium smegmatis CCRC 11565, Micrococcus leteus CCRC 10452, Rhizopus oryzae CCRC 30288, Proteus vulgaris CCRC 10486, Klebsiella pneumoniae CCRC 10692, Serratia marcescens CCRC 10768, Staphylococcus aureus CCRC 10451, Aspergillus niger CCRC 30130, Alcaligenes faecalis CCRC 10355, Penicillium turbatum CCRC 31684, Pseudomonas aeruginosa K-187, P. aeruginosa M-1001. P. aeruginosa M-1001, a lysozyme inhibitor-producing strain, was isolated from the soil in Taiwan.6 The other strains used for antimicrobial activity tests were purchased from the Culture Collection and Research Center, Hsin-Tsu, Taiwan.

Results and discussion Optimum immobilization conditions Conditions that affect immobilization include: immobilization curing time, protein amount, and EDC amount. The effects of these factors on immobilization are shown in Figures 1–3. Under room temperature, 150 mg EDC, and an immobilization time of 20 h, the amount of protein immobilized increased with the amount of enzyme added (Figure 1). Activity of chitinase was the highest when 5 ml enzyme solution was used; activity at this concentration was 4.2 U g21 polymer. From this point on, enzyme activity leveled out. Specific activities were 194 and 197 U g21 protein, respectively; however, specific enzyme activity was higher when 3 ml enzyme solution was used, amounting to 3.5 U g21 polymer; therefore, a 3-ml dosage was considered optimum. Enzyme Microb. Technol., 1998, vol. 22, May 15

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Figure 1 The amount of protein and chitinase activity immobilized at varying chitinase dosages. EDC, 150 mg and reaction time, 20 h

Under a 3-ml enzyme dosage, 150 mg of EDC, the amount of protein immobilized was proportional to immobilization time (Figure 2). Enzyme activity fell after 8 h because the long immobilized process may cause denaturation of the enzyme. A 4-h immobilization time was optimum, judging by the protein activity and specific activity which were 5.4 U g21 polymer and 346 U g21 protein, respectively. When the enzyme dosage was 3 ml, immobilization time was 4 h, EDC dosage of 150 mg, immobilization was optimal in producing chitinase activity of 5.4 U g21 polymer, and a specific activity of 346 U g21 protein (Figure 3). These activity values were 69 and 99%, respectively, when compared to those of free enzyme. In summary, when a crude chitinase of 7.4 mg ml21,

Figure 2 Effect of reaction time on the coupling yield of protein and chitinase activity. EDC, 150 mg and chitinase dosage, 3 ml

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activity of 2.6 U ml21, was the immobilizing enzyme, the immobilization conditions were optimal with 150 mg EDC, 3 ml enzyme, and 4 h of immobilization time. Under these conditions, 99% protein was immobilized. This percentage of immobilization was high compared to most other immobilization techniques.16 –20 The reasons for the higher percentage of immobilization found in this study may be because the chitinase used are thermally stabile.8

Effects of pH on immobilization Figure 4 shows the solubility of AS-L and protein immobilization when pH was varied. When pH , 4.5, AS-L was totally insoluble. The high turbidity at this time was

Figure 3 Effect of EDC dosage on the coupling yield of protein and chitinase activity. Reaction time, 4 h and chitinase dosage, 3 ml

Reversible immobilization of chitinase: S.-L. Wang and S.-H. Chio to the stability of enzyme at varying pH. When free enzyme was examined, it showed a 100% activity and was stable at pH values between 6.0 – 8.0. The loss of activity when it was immobilized at pH 5.5 was significant. The enzyme suspension was nonhomogeneous at pH 5.5, the fraction of immobilization was lowered when pH was lowered, reaching 20% when pH reached 4.0. These results showed that AS-L changes its property at different pH values and its solubility variation resulted in different fraction of immobilization. In summary, the most stable immobilization occurred at pH values between 6.0 – 8.0. Under this condition, the solution was homogeneous and the resulting enzyme was stable and high in activity.

Effects of pH and temperature on chitinase activity and stability

Figure 4 Effect of pH on solubility of AS-L and immobilization. Reaction condition included: room temperature; reaction time, 4 h; chitinase, 3 ml. The enzyme activity and protein content of each mixture were measured based on that at pH 7 as 100%

designated 100%. When pH . 5.5, AS-L was totally soluble. The low turbidity at this time was designated 0%. Between the pH of 4.5 and 5.5, the turbidity decreased linearly with the rise in pH. When pH . 6.0, 100% protein binding was achieved; however, when pH was between 5.5–9.0, the activity of chitinase decreased as pH was lowered. This corresponded

Hydrolysis of substrate chitin using free and immobilized chitinases is shown in Figure 5A as a function of pH. The optimum pH for free and immobilized chitinase hydrolysis of chitin was 6.0 and 8.0, respectively. The pH range for free and immobilized chitinase to remain stable was between 6.0 – 8.0 (Figure 5B). Hydrolysis of substrate chitin using free and immobilized chitinase is shown in Figure 6A as a function of temperature. The optimum temperature for free and immobilized chitinase hydrolysis of chitin was 40 and 50°C, respectively. The free and immobilized chitinase remained stable for at least 30 min under these temperatures (Figure 6B). Based on the Arrhenius equation, the activation energy for free chitinase was 9.94 kcal g21 mol21 while that for immobilized chitinase was 5.96 kcal g21 mol21. Activation

Figure 5 Effects of pH on the activity and stability of free and immobilized chitinase. Chitinase activities were measured at various pHs at 37°C for 10 min (A). Enzyme solutions were incubated at various pHs at 37°C for 30 min and residual activities were assayed at pH 7

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Figure 6 Effects of temperature on activity and stability of free and immobilized chitinase. Chitinase activities were measured at various temperatures at pH 7 (A). Enzyme solutions were incubated at pH 7 for 10 min, and remaining activities were measured at 37°C (B)

energy was lowered after immobilization, making the hydrolysis reaction more favorable.

Substrate specificity of enzyme From the previous study,8 the enzyme produced by strain K-187 possessed the activity of chitinase and lysozyme. Protease and cellulase activities were also discovered in the present study. Table 1 lists the activity of free and immobilized K-187 enzymes on various substrates: colloidal chitin (CC; chitinase); glycol chitin (GC; chitinase); ethylene glycol chitin (EGC; lysozyme); carboxymethyl cellulose (CMC; cellulase); and casein (protease). The hydrolysis reactions were performed in 50 mm phosphate buffer pH 7 at 37°C. Using the activity of free enzyme at the 100% level, the immobilized enzyme showed 99, 104, 80, 74, and Table 1 Enzyme activities with various substratesa Activityb on:

Enzyme Free enzyme Immobilized enzyme

Colloidal chitin

GC

EGC

CMC

Casein

351

379

227

290

545

346

394

182

215

109

a

Two enzymes were assayed on five different substrates at a constant ionic strength (50 mM phosphate buffer) and temperature (37°C) at pH 7 (10 min) for colloidal chitin and pH 7 (30 min) for GC or EGC and pH 7 (10 min) for CMC or casein. The amount of enzyme used was controlled to a concentration that showed an optical density (l 5 420 nm) range of 0.05– 0.20. The assay conditions are described in MATERIALS AND METHODS b Expressed as U g21 protein

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20% activities to the respective substrates listed above. The enzyme had a higher activity on the water-soluble GC than to CC, indicating hydrolysis was favored in a homogeneous reaction system. For the protease reaction with casein, a 20% activity remained after immobilization, indicating that these immobilization conditions were suitable for chitinase but not for protease. Whether AS-L was a suitable carrier for protease or not requires further studies.

Half-life of immobilized enzyme The free and immobilized enzymes were tested for their activity after different times in cold storage (4°C). A relative activity of 100% was designated for enzyme activity at day 0. The times required for a 50% reduction in activity were 9 and 13 days for free and immobilized enzyme, respectively. Stability of the enzyme was enhanced after immobilization. This result was similar to that reported above. The reason for this enhancement was probably due to the covalent bond between AS-L and the protein protecting it from being denatured. Chen and Chang16 have reported the immobilized chitinase lost 30% of its enzyme activity during the first cycle due to protein release and enzyme denaturation. As for other reports,16 –20 it is difficult to compare because the differences in the immobilized methods, enzyme substrates, and calculation approach would influence the similarity of the results.

Effect of redissolution on the activity of enzyme To use the reversible soluble/insoluble property of the AS-L carrier, recovery of enzyme took place when the pH of the solution was lowered and raised between 4.5–7.0 several times. The recovery process also involved filtration such that the carrier, protein, and enzyme may be lost. Figure 7

Reversible immobilization of chitinase: S.-L. Wang and S.-H. Chio

Acknowledgments This work was supported by a grant from the National Science Council, Republic of China (NSC 87-2313-B-212-003).

References 1. 2. 3. 4. 5.

6. Figure 7 Effect of recovery on the chitinase activity 7.

shows protease activity, amount of immobilized protein, and specific activity of immobilized enzyme after a number of recovery times. The loss of protein and enzyme activity was obvious; however, the specific activity was not lowered significantly with 90 and 70% remaining after 6 and 10 times of recovery, respectively. The relative value of activity recovery after the first pH change was 95%. This percentage of immobilization was high compared to 70% reported by the other report.16 The loss of specific activity was due to deactivation exerted by the fluctuating pH. The loss of only 3– 4% at each recovery rendered AS-L immobilization a feasible process.

8.

9. 10. 11. 12. 13.

Antimicrobial activity of immobilized chitinase

14.

The two purified chitinases produced by strain K-187 was found to be antimicrobial.8 The action of immobilized chitinase against Gram-positive and Gram-negative bacteria was examined. After 3 days of incubation at 37°C, the susceptible cells grew uniformly in the medium except for the area where antibiotic had diffused into the medium. This was indicated by the formation of clear zones of inhibition; as immobilized chitinase inhibited growth, zones of microbial inhibition were visible. In a test of 15 target strains, the immobilized chitinase was fatal to all strains except strain K-187 itself. The effect was similar to the free purified chitinases as described in our previous paper.8 In this study, we found that the culture supernatant of strain P. aeruginosa K-187 antagonistic to the fungal plant pathogen (Fusarium oxysporum) (data not shown). The antifungal activity was also found when immobilized enzyme was used. Although there are reports on chitinolytic microorganisms antagonistic to fungal plant pathogens,26 –28 the chitinases produced by these microorganisms are not known to have antibacterial effects similar to those of the K-187 chitinases.

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