Sun Sufang,  Yang Gengliang, Liu Haiyan, Sun Hanwen, Liu Cuifen ^#
(College of Chemistry & Environmental Science, Hebei University, Baoding 071002; ^#Factory No.2 of Chemical Industry, Shijiazhuang 050031, China)

Received Mar. 20, 2002; Supported by the Natural Science Foundation of China (Grant No. 20075005) and the Natural Science Foundation of Hebei Province (Grant No. 200077)

Abstract A new method to immobilize enzyme on aminopropylsilica gel was proposed with cyanuric chloride as the activator, and its application to the papain was investigated for the first time. Under the optimum conditions, the immobilized enzyme had higher protein-binding capacity and yield of enzyme activity than that reported by the predecessors with silica or some kinds of synthetic polymer that are suitable to act as stationary phase in liquid chromatography as the immobilization carrier. Kinetic results showed that the immobilized enzyme had a better thermostability and pH stability than the free enzyme. Lyophilized pieces of the immobilized enzyme exhibited much better stability when stored at room temperature for 60 days. Meanwhile, the operational stability of the immobilized enzyme was also investigated, after being used 9 times repeatedly, the activity of it was kept nearly constant. Besides, the coupling reaction in this new method was mild, reliable and reproducible, the coupling reagent used was very cheap, and it was also suitable for the immobilization of other enzyme. Thus, the new method had the potentiality for industrial use.
Keywords Aminopropylsilica, Cyanuric chloride, Papain, Immobilization

Immobilized enzymes are not only widely used as biocatalyst in biotechnology^{[1, 2]}, it also has been used as stationary phase in liquid chromatography, especially as enantiomeric selective media^{[3, 4]} in recent years. Silica beads silanized with 3-aminopropyltriethoxysilane are often used as immobilized enzyme carriers. The reactive groups through which the enzyme is bonded to matrix are the amino, carboxyl and sulphur groups etc., but the amino group is mostly used, e. g. in the glutaraldehyde^{[5,6 ]}, epoxide^[6,7], and N, N-disuccinimidyl carbonate methods^[8]. However, most of above process have either complex or rigid reaction conditions. The main search trends in this area include preparation of enzyme carriers and development of immobilization method.
    Cyanuric chloride, which is an intermediate that is originally used as linker in reactive-dye synthesis^[9], has been widely used as an enzyme linker with some supports, usually through hydroxyl groups on the support ^{[10, 11]}. In this work, the s-trazine activated silica is firstly obtained from aminopropyl-silica gel with cyanuric chloride as activator in non-aqueous medium and its application to immobilize papain that has been immobilized on many supports due to its great industrial and medicinal potentials ^[12-14] is also investigated for the first time. Under the optimum conditions, the protein-binding capacity and yield of enzyme activity are obtained and the kinetic actions of the bound enzyme are also investigated in an effort to validate the applicability of the new method in many ways.

1. EXPERIMENTAL
1.1 Instrumentation and materials               
Ultraviolet spectrophotometer (UV-3000) and Vacuum Desiccator (DZF-6020) were used for the study. All the aqueous solutions were compounded by double distilled water. TCA solution was prepared by dissolving 18 g trichloroacetic acid, 30 g sodium acetate and 19 mL acetic acid glacial in double distilled water and made up 1000 mL solution. The buffer solution used to determine enzyme activity contained 2.26 mg.mL^-1 enzyme activator - cysteine hydrochloride (CHC) and 2.26 mg.mL^-1 metal inhibitor - ethylenediaminetetracetic acetate (EDTA). Solution of casein (1%) and papain (diluted to the desired concentration or pH according to the request) were prepared freshly with 0.05 M, pH 7.5 phosphate buffer before use.
    Bovine Serum Albumin (BSA) was purchased from Sino-American Biotechnology Company (SABC). Silica (about 40mm) was purchased from Marine Chemical Industry Co., Ltd.（Qingdao, China）Other reagents used were all A. R..
1.2 Synthesis of aminopropyl-silica gel
Silica (4 g) was added to 40 mL 20% HCl solution and the mixture was stirred gently at 110ºC. After 8 h, the silica was filtered and rinsed with water until pH 7.0, and then the silica was dried in a vacuum oven at 160ºC overnight. The dried silica obtained above was dispersed in 80 mL toluene under N₂, subsequently 6 mL 3-aminopropyltriethoxysilane was added and the temperature was raised to 110ºC. The reaction mixture was stirred and refluxed for 12 h under N₂ and then the toluene was filtered off. The product obtained was washed thoroughly with toluene and then acetone. After being dried in vaccum at 80ºC, the aminopropylsilica gel was stored in a desiccator to be used for the next step.
1.3 Preparation of the new carrier (s-triazine activated silica)           
4 g-aminopropylsilica gels were introduced into a slurry of 1 g cyanuric chloride dissolved in a mixture of 40 mL of dioxane and 8 ml of toluene. The temperature was kept 12-16ºC, after 4 h under stirring, the mixture was filtered and the activated silica was washed with toluene and acetone to guarantee that no excessive cyanuric chloride was absorbed on the silica gel. After being dried, the new carrier was obtained and it can be stored at desiccator or used immediately.
1.4 The application of the new carrier to immobilize papain   
The new carrier obtained above was mixed with an aqueous solution of papain dissolved in 0.05 M phosphate buffer solution (pH 8.0), with gently stirring the reaction was allowed to proceed at 35ºC, after 6 h, the immobilized enzyme was obtained and washed thoroughly with phosphate buffer solution, and it was dried and stored in the refrigerator to be used for the next step. The whole reaction process is shown as Scheme1.
　 Scheme 1

1.5 Enzyme activity assay   
The activity of the free and the immobilized papain were determined according to the published method ^[15] using casein as a substrate. For the soluble enzyme activity, aliquots of it (0.2ml) were added to the mixture of 1.4 mL 0.05 M phosphate buffer (pH 7.5, containing CHC and EDTA) and 1.0 mL 1% casein solution, after being incubated at 37ºC for 10min, the digestion of casein was stopped by the addition of 3 mL TCA solution, and the amount of tyrosine was measured directly at 275 nm. For the immobilized enzyme activity, 0.02 g of the immobilized enzyme was soaked in the solution containing 1.6 mL 0.05 M phosphate buffer (pH 7.5, containing CHC and EDTA), the reaction was started by adding 1.0 mL casein solution. It was carried out for 10 min at 37ºC, the ended product was analyzed as above. One unit of activity was defined as the amount of enzyme that liberated 1 mmol of product/min at 37ºC.

2. RESULTS AND DISCUSSION
2.1 Factors affecting the synthesis of the activated carrier
During the preparation of the activated carrier, three factors, i.e. the temperature, the reaction time and the concentration of cyanuric chloride, were discussed. Among them, the temperature was much more important. From the structure of cyanuric chloride, three chlorides were found. According to the alteration of the reaction temperature, the amount of chloride that could be replaced was different^[9]. As seen from Scheme1, one chloride replaced was suitable during the synthesis of the activated carrier in order to get the maximum enzyme activity yield and avoid the self-coupling of the carrier. Thus, the control of temperature in this process was very important. If the reaction temperature was too high, two or three chlorides would be replaced, the reaction chance of the enzyme with the carrier would decrease greatly. If the temperature was too low, the reaction rate was so slow that a lot of time would be wasted. The optimum conditions of preparing the activated carrier was investigated in the experiment and the results were described in section 2.3.
2.2 The results of the immobilization          
Papain was immobilized on the carrier activated by cyanuric chloride under the optimum conditions obtained from the experiments and three parallel runs were performed. The immobilization results could be seen in Table 1. The amount of protein attached was obtained from the difference in absorbance at 275 nm between the papain solution added and that present in the washings with BSA as standard reagent. The activity yield was defined as the ratio of the activity of the amount of the enzyme known to be coupled to the carrier (from the measurement of the amount of immobilized enzyme) to the activity that would be shown by the same amount of the enzyme in free solution.

Table 1. Immobilization results of papain obtained for three parallel runs

As shown in Table 1, for every content listed in it, the bias for the three runs was very small, so the reproducibility of this immobilization method was very good. Meanwhile, it also could be seen that the mean activity yield of the immobilized enzyme obtained from cyanuric chloride was 49.1%. The activity yield reported here seemed lower than those immobilized on membrane or sepharose^[12,14], but it was the highest value with silica or some kinds of synthetic polymer that are suitable to act as the stationary phase in liquid chromatography as immobilization support (casein as substrate to determine enzyme activity) to our knowledage^[16-18], Results above indicated that cyanuric chloride was successful to immobilize papain on aminopropylsilica.
2.3 The optimum temperature and the thermostability                             
As shown in Fig.1, the optimum temperature of the immobilized enzyme was 80ºC, which was 15ºC higher than that of the free enzyme. Enzyme activity was determined with casein as substrate at various temperature (30-90ºC), pH 7.5 for 10 min.

    After all the enzyme preparations were incubated at indicated temperature (30-80ºC) for 2 h, the enzyme activity was determined in aliquots of enzyme (0.2 mL free enzyme or 0.02 g immobilized enzyme) at 37ºC, pH 7.5 for 10 min, with casein as substrate. The results showed that the immobilized enzyme was much more stable than the free enzyme, and this could be seen in Fig.2. As shown in it, with the temperature being increased, the activity of the free enzyme decreased quickly, but that of the immobilized enzyme firstly increased and then decreased slowly, after being incubated at 70ºC, the relative activity of the bound enzyme and the free papain were 95.6% and 31.6% respectively, when the incubated temperature was 80ºC, the residual activity of the bound enzyme was 83%, and that was 17.2% for the free papain.

Fig.2 Thermal stability of papain ( a: the free enzyme, b: the immobilized enzyme)

Fig.3 pH dependence of papain, a: the free enzyme, b: the immobilized enzyme

2.4 pH optima and pH stability                    
The pH profiles of both the immobilized and the free enzyme were determined. The optimum pH of the immobilized enzyme was 7.5, which was the same as that of the free enzyme, the results are shown in Fig.3 and the enzyme activity was determined with casein as substrate, at 37ºC in various phosphate buffer (pH 6-9, 0.05 M) for 10 min.
    After all of the enzymes were exposed to different pH values (2.0-9.0) at room temperature for 1h, enzyme activity was determined with casein as substrate at pH 7.5. The results showed that the activity of the immobilized enzyme remained high in the range pH 4.5-9, and the stability of the free enzyme was very good when pH ranged from 6.0-9.0. This indicated that the immobilized enzyme from the new method had wider pH stability than that of the free enzyme.


Fig.4 Storage stability of the enzyme, A: the enzyme was stored at 0-4ºC. B: the enzyme was stored at room temperature (about 17-25ºC), a: the free enzyme, b: the immobilized enzyme

2.5 Storage stability of enzyme
After all the enzyme preparations were stored at 0-4ºC and at room temperature (about 17-25ºC), the enzyme activity was determined. As seen in Fig.4 (A), a 23.3% loss of activity was observed for the free enzyme after being kept one month at 4ºC, which might be caused by the self-dissolution of the papain, almost no loss in activity of the immobilized enzyme was observed after 4 months at the same temperature. While at room temperature, as shown in Fig.4 (B), a 45.4% loss was seen only after 5 days for the free enzyme, whereas the immobilized enzyme could retain its activity (95.4%) without serious loss (4.6%) for 60 days. The results mentioned above indicated that the immobilized enzyme had a much better storage stability than the free enzyme, and it supplied a piece of good information for us to use it in many ways.
2.6 Operational stability     
The experimental was repeated nine times according to the procedure described above, and the enzyme activity was determined. It was interesting to note that the activities of the immobilized enzyme which had been used at different times were almost the same, which indicated that no enzyme was dissociated from the surface of the carrier in the course of reaction, this could be explained by the structure of s-trazine carrier, i.e. because of the induction and resonance effects on trazine ring, the carbon-nitrogen bonding was very stable under normal conditions. The results described above showed that the operational stability of the immobilized enzyme obtained from cyanuric chloride was excellent and it had potential to be used in industry.

3. CONCLUSIONS                 
In this work, the s-trazine activated carrier was synthesized in non-aqueous solution at the first time and it was applied to immobilize papain. Under the optimum conditions, the protein-binding capacity and yield of enzyme activity were obtained for three runs and the reliable and reproducible results could be got, the mean yield of enzyme activity could be attained 49.1%, which was much higher than that reported by predecessors with silica or some kinds of synthetic polymer that are suitable to be used as stationary phase in liquid chromatography as immobilization carrier. Meanwhile, the thermostability, storage stability and operational stability of the immobilized enzyme were excellent. In addition, cyanuric chloride, which was used as activator, was very cheap. All the results described above indicated that the method reported here was a reliable and easy procedure for the immobilization of enzyme, which might not only be useful to covalently bound other enzyme, but also it would have a promising use in industry. At present, the work that used the immobilized enzyme obtained by the new method as stationary phase in liquid chromatography to separate chiral medicine was going on.

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