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Sufang, Wang Chunyan, Zhang Yan, Liu Cuifen, Song Yongmei, Jia Jia Abstract A new method to immobilize b-galactosidase on chitosan was proposed with 2, 4, 6-trichloro-1, 3, 5-triazine as crosslinking reagent for the first time. Under the best immobilization conditions, the mean activity yield of the immobilized enzyme determined was 47%, which was much higher than that obtained from glutaraldehyde method. Besides, this new method with 2, 4, 6-trichloro-1, 3, 5-triazine as crosslinking reagent was mild, reliable and reproducible, and the coupling reagent used was much cheaper than glutaraldehyde.Keywords 2, 4, 6-trichloro-1, 3, 5- triazine, b-galactosidase, immobilization, chitosan 1. INTRODUCTION However, free b-galactosidase has many disadvantages, which largely limit its use [3], this lead to develop an immobilization procedure for this enzyme. In the previous studies, a large number of methods have been used to immobilize b-galactosidase [4-7]. One of the best carriers to immobilize enzyme is chitosan, a linear polymer of b-(1, 4)-2-amino-2-deoxy-D-glucopyranose derived from chitin by deacetylation. Chitosan has many useful features, such as hydrophilicity, biocompatibility, and biodegradability. In recent years, chitosan was generally used to immobilize various enzymes including b-galactosidase due to the presence of reactive amino function groups generally with glutaraldehyde as crosslinking reagent [7, 8]. In this paper, the activated chitosan was firstly obtained with 2, 4, 6-trichloro-1, 3, 5-triazine as crosslinking reagents in non-aqueous medium and its application to immobilize b-galactosidase was also investigated for the first time. Under the optimum conditions, the yield of the enzyme activity was determined and the results was compared with that obtained from the traditional coupling reagent—glutaraldehyde. 2. EXPERIMENTAL SECTION 2.8 Determination of Enzyme Activity The activity of the free and the immobilized enzyme were determined according to the reference [3, 4] using ONPG as a substrate. For the free enzyme activity, aliquots of it (0.1 mL) were added to the mixture of 1.4 ml 0.05M citric acid (pH 7.0) and 0.1 ml ONPG (5mM), after being incubated at 400C for 15min, the reaction of ONPG was stopped by the addition of 2 mL 1 mol/L Na2CO3 solution, and the amount of ONP was measured directly at 405nm. For the immobilized enzyme activity, 0.025 g of the immobilized enzyme was soaked in 1.5mL 0.05M citric buffer, the reaction was started by adding 0.1mL ONPG (5mM). after being carried out for 15min at 400C,the reaction was stopped and analyzed as above. One unit of activity was defined as the amount of enzyme that liberated 1mmol of product/min at 400C. 3. RESULTS AND DISCUSSION 3.2 Preparation of Chitosan Beads The pH-dependent solubility behavior of chitosan was used for the preparation of beads. The concentration of curing solution (NaOH-Methanol) and the chitosan, which govern the physical characteristics of the beads, was optimized. The results showed that the optimum concentration of chitosan in solution was 5 % ( w/v). Above this concentration, the viscosity of the solution was too high to pour easily, and, at lower concentrations, the beads showed poor mechanical properties. It was also determined from the experiments that when the curing solution was the mixture of 20 %( w/v) NaOH and 30 %( w/v) methanol in 2:3 volume ratio, the spherical beads of chitosan with good mechanical properties could be obtained and the average diameter of the beads was approximately 1.5mm as determined by the sieving method. 3.3 Preparation of the Activated Chitosan During the preparation of the activated carrier, three factors, i. e. the temperature, the reaction time and the concentration of 2, 4, 6-trichloro-1, 3, 5-triazine, were discussed. Among them, the temperature was much more important. From the structure of 2, 4, 6-trichloro-1, 3, 5-triazine, three chlorides were found. According to the alteration of the reaction temperature, the amount of chloride that could be replaced was different [11]. As seen from scheme 1, one chloride replaced was suitable during the synthesis of the activated carrier in order to get the maximum enzyme activity 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 be decreased greatly. If the temperature was too low, the reaction velocity was so slow that a lot of time would be wasted. In our work, the optimal reaction temperature was obtained and it was described in section 2.6. Meanwhile, the effect of 2,4,6-trichloro-1,3,5-triazine concentration to the activated carrier was also studied, the results showed that the immobilized enzyme activity increased firstly then decreased with the increase of the concentration of the crosslinking regent, when the concentration of the crosslinking regent was 0.67g/m L (per g chitosan), the immobilized enzyme activity attained to the maximum, the phenomena above can be explained by the following facts: With the increase of the concentration of the crosslinking reagent, the activated group was increased, which benefited the immobilization of the enzyme and the activity of the immobilized enzyme was increased also. But if the concentration of the crosslinking reagent was high enough, a large number of activated groups obtained might make the enzyme combine with the activated carrier in multipoint way and the space obstruction appeared, thus the activity of the immobilized enzyme was decreased again. Finally, the reaction time was also investigated in our experiments, the results showed that 15min was the best choice in the preparation of activated carrier. 3.4 Immobilization of b-galactosidase Three factors (the temperature, enzyme quantity and reaction time) were investigated during the immobilization of b-galactosidase on the chitosan with 2, 4, 6-trichloro-1, 3, 5-triazine as crosslinking reagent. Firstly, b-galacotosidase was immobilized on the activated chitosan at different temperatures (from 200C to 400C), and the activity of the immobilized enzyme determined showed that 250C was the optimal temperature. When the temperature was lower than 250C, almost no enzyme activity was determined, this was decided by the reaction characteristics of 2, 4, 6-trichloro-1, 3, 5-triazine [11], when the temperature was higher than 250C, the enzyme activity was also less, which might be caused by the enzyme thermal lost. Secondly, the reaction time was studied in our experiments, the results showed that the highest enzyme activity could be obtained when the reaction time was 60 min. Finally, different enzyme quantity added was discussed, and the immobilized enzyme activity was determined also. The results showed that the enzyme activity increased firstly then decreased with the increase of enzyme quantity, when the enzyme quantity was 16mL per g activated chitosan, the activity of the immobilized enzyme was the best. This kind of phenomenon might be explained by the following facts, with the increase of the enzyme quantity, the enzyme immobilized on the chitosan was raised, and the activity of the immobilized enzyme was increased as a result. But when the enzyme quantity was high enough, a great quantity of enzyme was immobilized and arranged closely on the chitosan, which would make the enzyme overlap in space and affect the active center of the enzyme, thus the activity of the immobilized enzyme was decreased consequently. 3.5 The Results of the Immobilization Under the optimum conditions obtained from the experiments, b-galactosidase was immobilized on the chitosan with 2, 4, 6-trichloro-1, 3, 5-triazine as crosslinking reagent for three parallel runs and the yield of the enzyme activity was determined. From the experiments, it could be seen that the reproducibility of this immobilization method was very good and the mean activity yield determined was 47%. Meanwhile, b-galactosidase was also immobilized on the chitosan with glutaraldehyde as coupling reagent according to the reference [7], the mean activity yield from our experiments was 37%, which was lower than that obtained from 2, 4, 6-trichloro-1, 3, 5-triazine method. The above results indicated that 2, 4, 6-trichloro-1, 3, 5-triazine was potential to be used to immobilize enzyme in industrial. 4. CONCLUSION REFERENCES 三氯均三嗪法固定化β-半乳糖苷酶的研究 孙素芳,王春燕,张燕,刘翠芬,宋咏梅,贾嘉 (河北大学化学与环境科学学院 保定071002) 摘要 本文首次以三氯均三嗪为交联剂,实现了b-半乳糖苷酶在壳聚糖载体上的固定化过程。在最优化制备条件下,测定了固定化酶的活性回收率为47%,此值高于传统交联剂--戊二醛法固定化结果(37%)。另外,以三氯均三嗪作为交联剂制备固定化β-半乳糖苷酶,反应条件温和,操作简单,耗时短。 关键词 三氯均三嗪, β-半乳糖苷酶, 固定化,壳聚糖 |