CN111454935A - An immobilized enzyme for denitrification of sewage and its preparation method and application - Google Patents

Abstract

The invention discloses an immobilized enzyme for sewage denitrification, a preparation method and application thereof, and relates to the technical field of sewage treatment. The preparation method of the immobilized enzyme includes the following steps: S1: preparing nitrifying bacteria and denitrifying bacteria S2, ultrasonically crush the cells and then centrifuge to obtain crude enzyme extract; S3, add activated carbon to the crude enzyme solution for mixed adsorption; S4 immobilize the enzyme: add the enriched enzyme solution containing polyvinyl alcohol, sodium alginate The carrier solution was then added dropwise to the boric acid saturated solution containing 2% anhydrous CaCl ₂ , and the immobilized pellets were obtained after standing, and the immobilized denitrogenase was obtained after washing with deionized water. The immobilized enzyme can be used to remove ammonia nitrogen in sewage, and has the advantages of simple preparation method, reusability, low production cost and the like.

Description

An immobilized enzyme for denitrification of sewage and its preparation method and application

technical field

The present invention relates to the technical field of sewage treatment, more particularly, it relates to an immobilized enzyme used for sewage denitrification and a preparation method and application thereof.

Background technique

At present, a major factor of surface water pollution is that the total nitrogen content in the water body is too high (total nitrogen includes ammonia nitrogen and nitrate nitrogen). The amount of dissolved oxygen in the water is low, which is not conducive to the growth of aquatic animals and plants. Secondly, the high ammonia nitrogen (NH ₃ ) in the water body not only prevents the ammonia in the organism from being excreted from the body, but also penetrates into the body from the water, which reduces or stagnates the metabolism of aquatic organisms and damages some important organs including gills. , inhibit its growth and development, and even cause death. In the existing research, one of the treatment methods for sewage denitrification is to use a combination of digesting bacteria and denitrifying bacteria to remove ammonia nitrogen and nitrate nitrogen from sewage.

The invention patent with the application number of 2019103687780 discloses a device and method for short-range denitrification-sludge fermentation coupled with anaerobic ammonia oxidation system to treat domestic sewage. of removal. The invention patent with the authorization announcement number of 105084682 discloses a method for improving the efficiency of ammonia nitrogen treatment in wastewater, and the method also uses nitrifying bacteria to perform related wastewater treatment. In the process of using the above-mentioned method for wastewater treatment, although the removal efficiency of nitrate nitrogen or ammonia nitrogen is relatively high in the wastewater treatment process, there is also the problem of long water treatment time. Generally, the qualified time for wastewater treatment for more than 3 days. This makes wastewater treatment less efficient.

SUMMARY OF THE INVENTION

In view of the deficiencies in the prior art, the first object of the present invention is to provide a method for preparing an immobilized enzyme for denitrification of sewage, which has the advantages of bacterial culture, cell disruption, and intracellular enzyme extraction and enzyme extraction. After immobilization, an immobilized enzyme with high denitrification efficiency is obtained.

The second object of the present invention is to provide an immobilized enzyme for denitrification of sewage, which has the advantage of efficiently denitrifying sewage in a short time.

The third object of the present invention is to provide an application of an immobilized enzyme for sewage denitrification, which has the advantages of simple application to sewage denitrification, high denitrification efficiency and short cycle.

In order to achieve the above-mentioned first purpose, the present invention provides the following technical scheme: a method for preparing an immobilized enzyme for denitrification of sewage, the method comprising the following steps:

S1 prepares nitrifying bacteria and denitrifying bacteria for use;

S2 acquisition of crude enzyme extract: after the bacteria resuspended in step S1, ultrasonically disrupted cells, centrifuged, and the supernatant is the enzyme crude extract;

Enrichment of S3 enzyme: add activated carbon to the above-mentioned crude enzyme extract, and obtain mixed solution A after mixed adsorption;

Immobilization of S4 enzyme: adding the mixed solution A to the carrier solution, and mixing to obtain a mixed solution B, and then dropping the mixed solution B into the cross-linking agent, immobilized and cross-linked at 4°C for 12-36 h, Washing with deionized water to obtain immobilized denitrogenase; the carrier solution is a mixed solution of polyvinyl alcohol and sodium alginate, the mass fraction of the polyvinyl alcohol is 5% to 15%, and the mass fraction of the sodium alginate is The score is 0 to 2%.

By adopting the above-mentioned technical scheme, the nitrifying and denitrifying enzymes in the cells are obtained by ultrasonically breaking the obtained nitrifying bacterial cells and denitrifying bacterial cells, and then the enzymes are immobilized to obtain relatively The immobilized enzyme with the best denitrification effect. First, the crude enzyme extract containing nitrification enzymes or denitrification enzymes in cells is obtained by ultrasonication. The ultrasonication method can be implemented in industrialization, and the ultrasonication method has less damage to the structure of the enzyme. The active substances in the cells still have good activity after the cells are disrupted by the ultrasonic method. Then, after the crude enzyme extract is adsorbed by activated carbon, a higher purity enzyme extract is obtained, and the enzyme is purified in a milder and more effective manner. Finally, the target nitrification enzyme and denitrification enzyme are further immobilized to obtain higher and more stable enzyme activity, so as to finally obtain better sewage denitrification effect, and the efficiency of sewage deamination nitrogen is high , Ammonia nitrogen removal rate is high.

Further, the conditions for ultrasonic crushing in the step S2 are as follows: 30 to 99 times of intermittent treatment, 3 to 5 s each time, 3 to 5 s in between, and a crushing power of 175 to 225 W.

By adopting the above technical scheme, the obtained immobilized enzyme has better denitrification effect under the above-mentioned cell disruption method. The above operating parameters have less damage to the structure of the enzyme mixture with denitrification effect, so that the enzyme mixture with denitrification effect has higher biological activity, and then when the enzyme mixture with denitrification effect is used for sewage denitrification. Has better denitrification effect.

Further, the centrifugation in step S2 adopts high-speed refrigerated centrifugation, and the centrifugation conditions are: centrifugation at 35000-45000×g for 25-35 min.

By adopting the above technical solution, the concentration of the enzyme mixture with denitrification effect obtained under the above centrifugal conditions is higher, so that the final enzyme mixture with denitrification effect has better sewage denitrification effect.

Further, in the step S3, the dosage of activated carbon is 2% to 6% of the mass of the crude enzyme extract, and the adsorption time is 7.5 to 12.5 minutes.

By adopting the above technical scheme, under the above-mentioned activated carbon dosage and adsorption time, the impurity substances in the crude enzyme extract are further removed, and in the remaining substances, the mass percentage of the enzyme mixture with denitrification effect is higher, so after purification In the enzyme mixture with denitrification effect, the content of biologically active substances with actual denitrification effect is higher, which finally makes the final denitrification effect of the immobilized enzyme better.

Further, the crosslinking agent in the step S4 is a saturated boric acid solution, anhydrous CaCl ₂ with a mass fraction of 2% is added to the saturated boric acid solution, and the pH of the saturated boric acid solution is adjusted to 6.5-7.0 in advance.

By adopting the above-mentioned technical scheme, the gel particles prepared by using the above-mentioned cross-linking agent have high mechanical strength, long service life and good elasticity.

Further, in the step S4, the mixed mass ratio of the mixed solution A and the carrier solution is 1:1-5.

By adopting the above technical scheme, enough target enzymes can be immobilized on the unit carrier and the state of excellent mass transfer performance can be guaranteed.

Further, the obtaining of described nitrifying bacteria and denitrifying bacteria comprises the following steps:

In S1-1, the sludge in the sewage treatment plant is inoculated into the nitrifying bacteria and denitrifying bacteria medium respectively for strain culture; S1-2, the above-mentioned culture solution is centrifuged respectively, and the supernatant is discarded to obtain concentrated nitrifying bacteria and denitrifying bacteria cells; S1-3 combines the obtained nitrifying bacteria and denitrifying bacteria cells, and then adds a buffer to resuspend the concentrated bacteria, and washes in this way for many times to obtain a bacterial suspension for later use.

By adopting the above technical scheme, the enriched nitrifying bacteria liquid and denitrifying bacteria liquid with higher concentration can be obtained.

Further, the samples inoculated into the nitrifying bacteria culture medium are cultured for 25 to 35 days under the condition that pH is 7.0-8.0; the samples inoculated into the denitrifying bacteria culture medium have a dissolved oxygen lower than 0.5 mg/L. , pH 6.5 to 7.5 and culture temperature of 25 to 35°C for 7 to 15 days.

In order to achieve the above second object, the present invention provides the following technical solution: an immobilized enzyme for denitrification of sewage, the immobilized enzyme is prepared by the above method.

By adopting the above technical scheme, the prepared immobilized enzyme has high mechanical strength and long service life.

In order to achieve the above-mentioned third purpose, the present invention provides the following technical solution: the application of the above-mentioned immobilized enzyme for sewage denitrification.

By adopting the above technical scheme, the application method is simple and the ammonia nitrogen removal efficiency is high.

To sum up, the present invention has the following beneficial effects:

First, because the present invention adopts the operation of removing impurities from the intracellular extract and immobilizing the enzyme after the cells are disrupted, thereby obtaining an immobilized enzyme with high ammonia nitrogen removal efficiency.

Second, in the present invention, the nitrifying bacteria and denitrifying bacteria are preferably broken by ultrasonic fragmentation, so as to obtain better cell fragmentation results, so that the intracellular extracts are released, and then obtain more denitrification bacteria in the future. The active substance of the effect provides the basis for a higher denitrification efficiency of the resulting immobilized enzyme.

Detailed ways

The present invention will be described in further detail below in conjunction with the embodiments.

The raw materials of the present invention are all commercially available, wherein the nitrification medium is composed of (NH ₄ ) ₂ SO ₄ 500-1000 mg/L, NaHCO ₃ is 800-1200 mg/L, and trace elements such as calcium, iron and magnesium are all 0.2 mg/L. L, phosphate buffer system; the composition of denitrification medium is KNO ₃ 300-1000mg/L, CH ₃ OH 500-1500mg/L, calcium, iron, magnesium and other trace elements are 0.2mg/L, phosphate buffer The above chemical reagents are of analytical grade made in China and can be purchased from Beijing Chemical Factory; the mixed solution of trace elements can be purchased from Jiangsu Punuosheng Biotechnology Co., Ltd.; the activated carbon product is of analytical grade (AR) and can be purchased from Guangzhou Chemical Reagent Factory .

Example

Example 1

A method for preparing an immobilized enzyme for denitrification of sewage, the method comprising the following steps:

S1 prepares nitrifying bacteria and denitrifying bacteria for standby use, which specifically includes the following steps:

S1-1 Take the return sludge from the secondary sedimentation tank of the sewage plant, filter it through a 30-mesh nylon mesh to remove impurities such as larger particles, and then wash and centrifuge it with physiological saline for 2-3 times, and inoculate the concentrated activated sludge respectively. Strain culture in nitrifying bacteria and denitrifying bacteria medium;

The samples inoculated into the nitrifying bacteria medium were cultured for 30 days at a temperature of 30°C and a pH of 7.5; the samples inoculated into the denitrifying bacteria medium were incubated at a temperature of 30°C, with a dissolved oxygen of 0.3 mg/L and a pH of 7.0. 10 days under the conditions of culture;

S1-2 Centrifuge the above-mentioned culture solution respectively, and the centrifugation conditions are: 4 ℃, 6000r/min centrifugation for 5min, discard the supernatant to obtain concentrated nitrifying bacteria and denitrifying bacteria cells;

S1-3 After adding buffers to the obtained nitrifying bacteria and denitrifying bacteria, respectively, the nitrifying bacteria buffer and denitrifying bacteria buffer containing the bacterial cells were combined, and the buffer was heated at 4°C and 6000 r/min. After centrifugation for 5 min, the bacteria were washed twice with the buffer, and then resuspended with the buffer to obtain a bacterial suspension for later use.

S2 acquisition of crude enzyme extract: the bacteria re-suspension in step S1 is broken by ultrasonic breakage, and the ultrasonic breakage conditions are: 99 times of machine intermittent treatment, each time 4s, the middle interval is 4s, and the breaking power is 200W; then Centrifugation, the conditions of centrifugation are: centrifuge at 40000×g for 30min, and the supernatant is the crude enzyme extract.

Enrichment of S3 enzyme: add activated carbon to the above-mentioned crude enzyme extract, so that the dosage of activated carbon is 4% of the mass of the crude enzyme extract, mixed and adsorbed for 10min to obtain mixed solution A, which contains nitrification of enzymes, denitrification enzymes and some intracellular extracts.

Immobilization of S4 enzyme: add mixed solution A into the carrier solution, and get mixed solution B after mixing, wherein the carrier solution is a mixed solution of polyvinyl alcohol and sodium alginate, the mass fraction of polyvinyl alcohol is 10%, and the amount of sodium alginate is 10%. The mass fraction is 1%, and the mass ratio of the mixed solution A and the carrier solution is 1:2; then the mixed solution B is dropped into a saturated solution of boric acid containing 2% anhydrous CaCl ₂ , and then cured and cross-linked at 4 °C for 24 h. , and washed with deionized water for 3 to 4 times, the immobilized denitrogenase was obtained, and the immobilized enzyme was in granular form.

An immobilized enzyme for sewage denitrification prepared by the above method is used in the denitrification treatment of sewage.

Example 2

A method for preparing an immobilized enzyme for denitrification of sewage, the method comprising the following steps:

S1 prepares nitrifying bacteria and denitrifying bacteria for standby use, which specifically includes the following steps:

S1-1 Take the return sludge from the secondary sedimentation tank of the sewage plant, filter it through a 30-mesh nylon mesh to remove impurities such as larger particles, and then wash and centrifuge it with physiological saline for 2-3 times, and inoculate the concentrated activated sludge respectively. Strain culture in nitrifying bacteria and denitrifying bacteria medium;

The samples inoculated into the nitrifying bacteria medium were cultured for 25 days at a temperature of 25°C and a pH of 7.0; the samples inoculated into the denitrifying bacteria medium were incubated at a temperature of 25°C, with a dissolved oxygen of 0.3 mg/L and a pH of 6.5. The incubation time is 7 days under the conditions;

S1-2 Centrifuge the above-mentioned culture solution respectively, and the centrifugation conditions are: 4 ℃, 5500r/min centrifugation for 8min, discard the supernatant to obtain concentrated nitrifying bacteria and denitrifying bacteria cells;

S1-3 After adding buffer solution to the obtained bacterial cells of nitrifying bacteria and denitrifying bacteria, respectively, the nitrifying bacteria buffer solution and denitrifying bacteria buffer solution containing bacterial cells were combined, and then the buffer solution was heated at 4°C and 5500 r/min. After centrifugation for 8 min, the bacteria were washed twice with the buffer, and then resuspended with the buffer to obtain a bacterial suspension for later use.

The acquisition of crude enzyme extract in S2: the bacterial re-suspension in step S1 is disrupted by ultrasonic disruption, and the ultrasonic disruption conditions are as follows: 99 times of intermittent treatment, 3s each time, 3s interval, and 175W crushing power; Centrifuge at 35000×g for 35min, and the supernatant is the crude enzyme extract.

Enrichment of S3 enzyme: add activated carbon to the above-mentioned crude enzyme extract, so that the dosage of activated carbon is 2% of the mass of the crude enzyme extract, mix and adsorb and stir for 7.5min to obtain mixed solution A, in mixed solution A Contains nitrifying enzymes, denitrifying enzymes and some intracellular extracts.

Immobilization of S4 enzyme: add mixed solution A into the carrier solution, and get mixed solution B after mixing, wherein the carrier solution is a mixed solution of polyvinyl alcohol and sodium alginate, the mass fraction of polyvinyl alcohol is 5%, and the amount of sodium alginate is 5%. The mass fraction is 0.1%, and the mass ratio of mixed solution A and carrier solution is 1:1; then mixed solution B is dropped into a saturated solution of boric acid containing 2% anhydrous CaCl ₂ , and then cured and cross-linked at 4 °C for 12 h. , and washed with deionized water for 3 to 4 times, the immobilized denitrogenase was obtained, and the immobilized enzyme was in granular form.

An immobilized enzyme for sewage denitrification prepared by the above method is used in the denitrification treatment of sewage.

Example 3

A method for preparing an immobilized enzyme for denitrification of sewage, the method comprising the following steps:

S1 prepares nitrifying bacteria and denitrifying bacteria for standby use, which specifically includes the following steps:

S1-1 Take the return sludge from the secondary sedimentation tank of the sewage plant, filter it through a 30-mesh nylon mesh to remove impurities such as larger particles, and then wash and centrifuge it with physiological saline for 2-3 times, and inoculate the concentrated activated sludge respectively. Strain culture in nitrifying bacteria and denitrifying bacteria medium;

The samples inoculated into the nitrifying bacteria medium were cultured for 35 days at a temperature of 35°C and a pH of 8.0; the samples inoculated into the denitrifying bacteria medium were incubated at a temperature of 35°C, with a dissolved oxygen of 0.48 mg/L and a pH of 7.5. 15 days under the conditions of culture;

S1-2 Centrifuge the above-mentioned culture solution respectively, and the centrifugation conditions are: 4 ℃, 6500r/min centrifugation for 4min, discard the supernatant to obtain concentrated nitrifying bacteria and denitrifying bacteria cells;

S1-3 After adding buffers to the obtained nitrifying bacteria and denitrifying bacteria, respectively, the nitrifying bacteria buffer and denitrifying bacteria buffer containing the bacteria were combined, and then the buffer was heated at 4°C and 6500 r/min. After centrifugation for 4 min, the bacteria were washed twice with the buffer, and then resuspended with the buffer to obtain a bacterial suspension for later use.

S2 acquisition of crude enzyme extract: the bacterial re-suspension in step S1 is broken by ultrasonic crushing, and the conditions of ultrasonic crushing are: intermittent treatment for 30 times, each time 5s, the middle interval is 5s, and the crushing power is 225W; then Centrifuge at 45000×g for 25min, and the supernatant is the crude enzyme extract.

Enrichment of S3 enzyme: add activated carbon to the above-mentioned crude enzyme extract, so that the dosage of activated carbon is 6% of the mass of the crude enzyme extract, mixed and adsorbed and stirred for 12.5min to obtain mixed solution A. In mixed solution A Contains nitrifying enzymes, denitrifying enzymes and some intracellular extracts.

Immobilization of S4 enzyme: add mixed solution A into the carrier solution, and get mixed solution B after mixing, wherein the carrier solution is a mixed solution of polyvinyl alcohol and sodium alginate, the mass fraction of polyvinyl alcohol is 15%, and the amount of sodium alginate is 15%. The mass fraction is 2%, and the mass ratio of the mixed solution A and the carrier solution is 1:4; then the mixed solution B is dropped into a saturated solution of boric acid containing 2% anhydrous CaCl ₂ , and then cured and cross-linked at 4 °C for 36 h. , and washed with deionized water for 3 to 4 times, the immobilized denitrogenase was obtained, and the immobilized enzyme was in granular form.

An immobilized enzyme for sewage denitrification prepared by the above method is used in the denitrification treatment of sewage.

Examples 4-16

The difference between Examples 4-16 and Example 1 is that some process parameters in the preparation process of the immobilized enzyme are different, as shown in Table 1 for details.

The preparation technology of the immobilized enzyme of table 1 embodiment 1-16

Comparative Examples 1-7

The difference between Comparative Examples 1-7 and Example 9 is that some process parameters in the preparation process of the immobilized enzyme are different, as shown in Table 2 for details.

The preparation technology of the immobilized enzyme of table 2 comparative examples 1-7

The immobilized enzyme of the invention is used for the removal of ammonia nitrogen from wastewater, and 4% of the immobilized denitrification enzyme is added according to the quality of the wastewater, and the ammonia nitrogen removal rate reaches 75% after 48 hours.

The immobilized enzymes prepared in Examples 1-15 and Comparative Examples 1-3 were used in the simulated organic nitrogen wastewater, and the simulated organic nitrogen wastewater was composed of: NH ₄ Cl was 30 mg/L, NaHCO ₃ was 35 mg/L, and glucose was 30 mg/L. 40mg/L, CH ₃ COONa·3H ₂ O is 40mg/L, MgSO ₄ ·7H ₂ O is 1.5mg/L, FeSO ₄ ·7H ₂ O is 1.5mg/L, a small amount of trace elements (MnSO ₄ , CuSO ₄ are both 0.5mg/L), the ammonia nitrogen content is 9-10mg/L, and the COD is 50-60mg/L. The amount of immobilized enzyme added in the simulated organic nitrogen wastewater was 4% (wt, %). After 48 hours of wastewater treatment, the ammonia nitrogen content in the wastewater was detected. Among them, the detection of ammonia nitrogen is detected by Nessler's reagent colorimetric method.

Table 3 Removal rate of ammonia nitrogen in wastewater by immobilized enzymes of Examples 1-16 and Comparative Examples 1-10

The data in Table 3 show that Example 9 is the best embodiment. Among them, in Comparative Example 6, when the mass ratio of solution A and carrier liquid was 1:0.5, the gel concentration was low, and the dropped gel floated on the surface of the cross-linking agent and formed oil droplets, which could not be formed into balls, and could not form a stable fixation in the later stage. enzyme, so this scheme is not used.

This specific embodiment is only an explanation of the present invention, and it does not limit the present invention. Those skilled in the art can make modifications without creative contribution to the present embodiment as required after reading this specification, but as long as the rights of the present invention are used All claims are protected by patent law.

Claims (10)

1. a preparation method for the immobilized enzyme of sewage denitrification, is characterized in that, described method comprises the following steps: S1 prepares nitrifying bacteria and denitrifying bacteria for use; S2 acquisition of crude enzyme extract: take the bacterial re-suspension in step S1, after ultrasonically disrupting the cells, centrifuge, and the supernatant is the enzyme crude extract; Enrichment of S3 enzyme: add activated carbon to the above-mentioned crude enzyme extract, and obtain mixed solution A after mixed adsorption; Immobilization of S4 enzyme: add the mixed solution A into the carrier solution, and get mixed solution B after mixing, then drop the mixed solution B into the cross-linking agent solution, and immobilize and cross-link at 4°C for 12-36 h Then, wash with deionized water to obtain immobilized denitrogenase; the carrier solution is a mixed solution of polyvinyl alcohol and sodium alginate, the mass fraction of polyvinyl alcohol is 5% to 15%, and the sodium alginate The quality fraction is 0~2%. 2. the preparation method of a kind of immobilized enzyme for sewage denitrification according to claim 1, is characterized in that, the condition of ultrasonic crushing in described step S2 is: machine batch treatment 30～99 times, each time 3 ~5 s, the middle interval is 3~5 s, and the crushing power is 175~225 W. 3. the preparation method of a kind of immobilized enzyme for sewage denitrification according to claim 1, is characterized in that, the centrifugation of described step S2 adopts high-speed refrigerated centrifugation, and described centrifugal condition is: at 35000～45000×g Centrifuge for 25-35 min. 4. the preparation method of a kind of immobilized enzyme for sewage denitrification according to claim 1, is characterized in that, in described step S3, the dosage of activated carbon is 2%～6% of crude enzyme extract quality , the adsorption time was 7.5-12.5 min. 5. the preparation method of a kind of immobilized enzyme for sewage denitrification according to claim 1, is characterized in that, the crosslinking agent in described step S4 is saturated boric acid solution, and described saturated boric acid solution is added with The mass fraction of anhydrous CaCl ₂ is 2%, and the pH of the saturated boric acid solution is adjusted to 6.5-7.0 in advance. 6. the preparation method of a kind of immobilized enzyme for sewage denitrification according to claim 1, is characterized in that, in described step S4, the mixed mass ratio of described mixed solution A and described carrier solution is 1 : 1~5. 7. the preparation method of a kind of immobilized enzyme for sewage denitrification according to claim 1, is characterized in that, the acquisition of described nitrifying bacteria and denitrifying bacteria comprises the following steps: S1-1 takes the sludge in the sewage treatment plant and inoculates it into the nitrifying bacteria and denitrifying bacteria medium respectively for bacterial culture; S1-2 separates the above-mentioned culture solution by centrifugation, discards the supernatant to obtain concentrated nitrifying bacteria and denitrifying bacteria cells; S1-3 combines the obtained nitrifying bacteria and denitrifying bacteria cells, and then adds a buffer to resuspend the concentrated bacteria, and washes in this way for many times to obtain a bacterial suspension for later use. 8. the preparation method of a kind of immobilized enzyme for sewage denitrification according to claim 1, is characterized in that, the sample inoculated to described nitrifying bacteria medium is cultivated under the condition that pH is 7.0～8.0 25 ~35 days; the samples inoculated into the denitrifying bacteria medium are cultured for 7-15 days at a culture temperature of dissolved oxygen lower than 0.5 mg/L, pH of 6.5 to 7.5 and 25 to 35°C. 9 . An immobilized enzyme for sewage denitrification, characterized in that, the immobilized enzyme is prepared by the method described in any one of claims 1-8. 10 . 10. An application of the immobilized enzyme for denitrification of sewage according to claim 9.