Sun Sufang, Wang Chunyan, Zhang Yan, Liu Cuifen, Song Yongmei, Jia Jia
(College of Chemistry and Environmental Science, Hebei University, Baoding 071002, China)

Received on Jul.7, 2007; Supported by the fund from the Natural Science Foundation of Hebei Province ( Grant No: B2007000146)

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
   Milk whey is the most important byproduct in cheese manufacturing. It represents 85-95% of processed milk by volume, after casein precipitation, and contains 55% of milk nutrients such as lactose, soluble proteins et al.. Unfortunately, over the past 50 years, half of the world’s production has not been transformed into sub-products but wasted directly in aqueous habitats. This situation creates both major environmental issues^[1]and the need of new strategies for the recovery of sub-products. It proved that bio-conversion of milk whey lactose by b-galactosidase could reduce more than 75% of water pollution and generate products of interest for animal feed, human nutrition et al ^[2]. Lactose bio-conversion by b-galactosidase could also play a primary role in a syndrome called lactose intolerance (LI). which is the inability to digest significant amounts of lactose, and it is caused by a deficiency of b-galactosidase. So, the enzymatic action of lactose by b-galactosidase is a very promising biotechnology in environmental, alimentary and health fields.
   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
   2.1 Apparatus and Reagents
   Ultraviolet Spectrophotometer (WFZ800-D3A), Vacuum Desiccator (DZ-6020), Digital pH Meter (PHS-3C) and Water Constant Temperature Oscillator (SHA-B) were used for the study. All the aqueous solutions were prepared by twice distilled water.
   Reagents: chitin was purchased from Hongxing Biochemical Factory of Zhejiang Yuhuan, b-galactosidase (3000U/g) was from Meihua Biological Technology Corporation in Haerbin, O-nitrophenyl-b-D-galactoside (ONPG) was from Sigma, 2, 4, 6-trichloro-1, 3, 5-triazine, glutaraldehyde, O-nitrophenol (ONP) and other reagents were all analytical grades.
   2.2 Preparation of Enzyme and Substrate Solution
   5g of b-galactosidase was weighed and extracted in 250mL 0.05M citric acid buffer (pH 7.0) over a period of 1-2h, after being filtered with filter paper, the enzyme solution was obtained and stored in the refrigerator for use.
   The substrate solution was prepared by dissolving 0.0751g ONPG in twice distilled water and made up 50mL solution.
   2.3 Hydrolysis of Chitin
   According to the reference ^[9], Chitin (20g) was added to 200mL 40% (w/v) aqueous NaOH solution in a flask and the reaction was allowed to proceed for different times at 100⁰C with vigorous stirring. After hydrolysis, the insoluble product was separated by filtration by using a sintered glass funnel and was washed thoroughly to remove the residual alkali by 1% NaCl solution and redistilled water successively, and then it was dried at 105⁰C. Thus the chitosan having different degrees of deacetylation was obtained.
   2.4 Determination of Deacetylation Degree
   0.1g of the above product was weighed accurately and put into 100mL flask, meanwhile, 0.1058mol/L HCl (15mL)standard solution（calibrated by standard Na₂CO₃ solution） was added into it. After the chitosan was dissolved with constant stirring over a period of 0.5-1h, the excessive HCl was titrated to the terminal point by 0.1053mol/L NaOH solution. Meanwhile, another piece of sample was taken and dried at 105⁰C, and then it was weighed to determine the water content. Thus, the deacetylation degree of the chitosan was calculated according to the following equation:
   DD％＝{[(C₁V₁-C₂V₂) ×0.016]/[G(100-W) ×0.0994]} ×100％
   Here DD％was the deacetylation degree of the chitosan, C₁ (mol/L) and V₁ (mL)were the concentration and volume of standard HCl solution added respectively, C₂ (mol/L) and V₂ (mL) were the concentration and volume of NaOH standard solution consumed separately, G(g) was the weight of the sample, W(%) was the water content of the sample, 0.016 (g/mL) was NH₂ content equaling to 1mL HCl solution(1mol/L), 0.0994（16/161）was NH₂ theoretical value in chitosan.
   2.5 Preparation of Chitosan Beads
   2g of chitosan was accurately weighed and dissolved in acetic acid (4%, w/v) to form 5% solution (w/v) ^[10], then the flesh solution was added dropwise to a NaOH-Methanol solution by using a syringe, the beads formed immediately, and the average diameter of it was approximately 1.5mm. After being washed completely with distilled water, the beads were dried at 85⁰C and stored for the next use.
   2.6 Preparation of Activated Chitosan Beads by 2, 4, 6-trichloro-1, 3, 5-triazine
   1g of the chitosan beads was taken and dipped into 0.05M citric acid buffer (pH 7.0) for 90min. After being dried with filter paper, the beads was introduced into a slurry of 1.6 g 2,4,6-trichloro-1,3,5-triazine dissolved in a mixture of 80ml of dioxane and 16 ml of toluene, the reaction was allowed to proceed 4h at 5-8⁰C with stirring. The product was filtered and washed thoroughly with toluene, acetone and then 0.05M citric buffer successively, thus the activated chitosan was obtained, and it could be stored in desiccator or used immediately.
   2.7 Immobilization of b-galactosidase on Activated Chitosan
   The activated chitosan obtained above was mixed with an aqueous solution of b-galactosidase dissolved in 0.05M citric acid (pH 7.0), with gently stirring, the reaction was allowed to proceed at 25⁰C, after 60min, the immobilized enzyme was obtained and washed thoroughly with citric buffer solution, and it was dried and stored in the refrigerator to be used for the next step, the whole reaction process was shown as Scheme 1.
   
   Scheme 1

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
   3.1 Time Selection in Chitin Hydrolysis
   Different reaction time was investigated in the hydrolysis of chitin, the results showed that the longer the reaction time, the higher the deacetylation degree. When the reaction time was 6h, the deacetylation degree of the chitosan attained to 91.32%, which would provide enough free NH₂for the immobilization ofb-galactosidase, so 6h was selected as the hydrolysis time of chitin in our experiment.
   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
   In this work,b-galactosidasewas immobilized on the chitosan beads with 2, 4, 6-trichloro-1, 3, 5-triazine which was much cheaper than glutaraldehyde as crosslinking reagent for the first time, and the reliable and reproducible results could be got. Under the optimal conditions, the mean activity yield of the immobilized enzyme could attain 47%, which was much higher than that from glutaraldehyde method. Besides, the coupling reaction in this new method was mild and easy to be controlled. Thus, the new method had the potentiality to immobilize enzyme for industrial use. At present, the work for the determination of the basic properties of the immobilized b-galactosidase and its kinetic parameters are going on in our laboratory.

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