SYNTHESIS OF STYRENE-DIVINYLBENZENE COPOLYMERS BY USING POLYGLUTARALDEHYDE AND THE APPLICATIONS

This invention is about the synthesis in a new form (STR-DVB-PGA co-polymers) of the styrene-diviniylbenzene (STR-DVB) co-polymer by means of polyglutaraldehyde (PGA) which allows the covalent bonding of enzymes to the matrix particularly in immobilization, and the use of this polymer in different applications. The suggested process has been named as micro porous polymeric enzyme reactors (MPPER) and can be produced easily in any size and form needed.

The polymers are large chain molecules synthesized from chemical substances named as monomers. In styrene-diviniylbenzene co-polymer synthesis, polystyrene is produced as a result of radicalic chain reaction by using diviniylbenzene as a cross-linker together with a catalyst (organic peroxide, hydroperoxides, redox starters, azo compounds etc.). There are many classical methods and processes currently used for polystyrene production in which styrene and diviniylbenzene are used.

There are many methods and technologies have been suggested for the production of styrene-diviniylbenzene co-polymer in literature. In U.S. Pat. No. 4,522,953 (Barby et al.) patent, the Lever Research Group searched this polymer by using different methods and defined the polyhipe production as such. The polymer obtained by polymerizing and drying of the oil phase prepared by mixing the styrene- diviniylbenzene monomers is with high porosity, low density, open-cell structured and rigid. Polymers with different pore sizes between a wide range from a few micrometers to a few hundred micrometers can be produced. Smaller pores allowing a greater iner surface can be obtained by increasing the mixing duration and speed.

In a study conducted by Wakeman et al. (Chem. Eng. Journal, 1998, 70, 133-141), the oil phase of polyhipe was prepared by using styrene, diviniylbenzene and span 80 providing water emulsion in oil. Water phase used as the disperse phase contains potassium persulfate (0.4% w/w) as polymerization starter.

Although there are many studies in the literature about the production and use of styrene-diviniylbenzene co-polymer in different areas, there are no studies about the polymer synthesized by using polyglutaraldehyde as a cross-linker. This invention allows the covalent bonding between various enzymes and styrene-diviniylbenzene- polyglutaraldehyde co-polymer (STR-DVB-PGA). The said polymer allows the realization of many enzymatic reactions in different reactor configurations (bead, disc, cylinder, packed colon). The immobilization of enzymes to the styrene- diviniylbenzene co-polymer can be done using two ways apart from this invention. In the first way, the co-polymer prepared with the known methods is treated with enzymes directly and the enzyme is adsorbed in the co-polymer physically. The polymers prepared with this method lose their activity after a few uses. In a study conducted by Castro et al. (Biochemical Engineering Journal, 2000, 5, 63-71) Candida rugosa lipase was immobilized to the styrene-diviniylbenzene co-polymer through adsorption method and the immobilization was realized in two different environments using buffer solution and organic solvent (heptane). When the immobilization of lipase is carried out in buffer solutions, it has been reported that the relative activity of the enzyme is zero after the third repetitive use. Another application is the treatment of prepared polymer with glutaraldehyde at first and then its interaction with enzyme. In such application, although there is a covalent bonding between the enzyme and the glutaraldehyde, the activity losses cannot be prevented as glutaraldehyde is physically adsorbed in polymer (J.P. Pat. No. 5,017,580). The most important difference of the present invention is that polyglutaraldehyde is in the chemical structure of the polymer and the enzyme is covalently attached to the polymer through the polyglutaraldehyde which is a part of the polymer. Current application increases the stabilization of enzymes, minimizes the enzyme losses, in addition to its capability of covalent bonding of enzymes.

Within the scope of this invention, the lipase and maltogenase enzymes are covalently bound to the polymers prepared in different configurations and used in fatty acid methyl esters (biodiesel) and maltose production, respectively. This invention is not limited to the said application samples.

Glutaraldehyde has various uses in immobilization including pre-activation of the amino groups on carrier, cross-linking of the enzymes to carrier matrix after the adsorption, creation of cross-linked gel with the polymers soluble in water and cross- linking of the enzymes in membranes not soluble in water or oligomers soluble in water (Applied Biochem. and Biotech., 1998, 73, 195-205; Applied Biochem. and Biotech., 2000, 89, 377-379).

Polyglutaraldehyde can be obtained through different methods. In the patent obtained by Moyer et al. (U.S. Pat. No. 3,395,125), polyglutaraldehyde was obtained through intra-intra molecular polymerization of glutaraldehyde. In basic or neutral conditions at pH 7-13.5, soluble and insoluble polymers with molecular weight of between 12,000-20,000 have been obtained. Moreover, the polymers obtained contains carboxyl, hydroxyl and aldehyde groups in various amounts depending on pH. Carbon-carbon double bonds are present in proportion with the carbonyl groups. Soluble polyglutaraldehyde have more carboxyl groups than the insoluble ones. This is because of the Cannizzaro reaction formed depending on the pH of the environment. Freeman et al. in their patent (U.S. Pat. No. 4,904,592) stated that the polyglutaraldehyde gives peak in 233 and 285 nm in the aqueous solutions. Since the number of carbon-carbon double bond will increase when the polymerization is realized, optical density will increase in 233 nm. Aldehyde groups have absorbance in 285 nm. As aldole condensation reaction is realized, the amount of carbonyl group decreases.

Polyglutaraldehyde is used in enzyme immobilization and biosensor technologies. Thanks to the carbonyl groups on it, polygluteraldehyde forms a covalent bond with the compounds having amine groups by Schiff s base reaction. The polymerization of glutaraldehyde is shown in Figure 1.

polydutai aldehyde

Figure 1. Polymerization reaction of glutaraldehyde

Polyglutaraldehyde is preferred over glutaraldehyde in immobilization. In immobilization, glutaraldehyde reacts with amino groups of enzymes leading to a Schiffs base which is unstable under acidic conditions and break down to form the aldehyde and amine groups. The reverse reaction of Schiffs base compound can be prevented by reduction using NaBH ₄ , which could decrease the activity of enzymes. The reactions of polyglutaraldehde with protein involve Michael addition and Schiffs base resulting in irreversible reactions. There is no need for reduction reaction in immobilization using polyglutaraldehyde (Figure 2). (Trends in Analytical Chemistry, 1994, 13, 425-430).

poly glutar aldehyde

( n + 1 ) I Enzyme J- NH ₂

cross-linked enzyme molecules

Figure 2. Enzyme immobilization mechanism using polyglutaraldehyde

The subject of the invention, the styrene-divinylbenzene-polyglutaraldehyde (STR- DVB-PGA) co-polymer is used in immobilization of lipase and maltogenase for the production of fatty acid methyl esters (biodiesel) and maltose, respectively. There are many studies in the literature, where Upases immobilized onto various matrixes were used in fatty acid methyl esters (biodiesel) production. Lipases obtained from different sources are immobilized onto various matrixes. However, some of the matrixes used in immobilization had some drawbacks such as absorbing glycerol formed. In the study conducted by Belafi-Bako et al. (Biocatalysis and Biotransformation, 2002, vol 20 (6), 437-439), it was reported that glycerol, a byproduct, inhibits the enzyme. Matrix should be washed with organic solvents to remove glycerol. This increases the costs. The endeavors about commercializing the enzymatic production of biodiesel are still continued all over the world. Within this

scope, this patent has a potential to beused in industrial scale to production of biodiesel. This is because STR-DVB-PGA co-polymer is stable in terms of use, and resistant to microbial contamination, and can be produced in any size and form desired. The polymer in subject of the invention minimizes the possibility of inhibition of the enzyme by glycerol since it has hydrophobic properties. Generally the enzymes need special storage conditions if they are not to be used for a long time. The studies we have carried out demonstrated that enzymes immobilized onto the polymer (STR-DVB-PGA) can retain activition at room temperature without the need for special storage conditions.

Various spectrums of polymers synthesized and the micro porous polymeric enzyme reactor configurations developed using STR-DVB-PGA co-polymer realized to reach the purpose of the invention are shown in the annexed figures, and these figures demonstrate the following:

Figure 1. UV spectrum of polyglutaraldehyde,

Figure 2. FTIR spectrum of polyglutaraldehyde,

Figure 3. X-ray diffraction spectrum of polygluteraldehyde,

Figure 4. DSC anaylsis of polyglutaraldehyde,

Figure 5. FTIR spectrum of styrene-divinylbenzene co-polymer,

Figure 6. X-ray diffraction spectrum of styrene-divinyl benzene co-polymer,

Figure 7. DSC analysis of styrene-divinylbenzene co-polymer,

Figure 8. FTIR spectrum of styrene-divinylbenzene-polyglutaraldehyde co-polymer,

Figure 9. X-ray diffraction spectrum of styrene-divinyl benzene polyglutaraldehyde co-polymer,

Figure 10. DSC analysis of styrene-divinyl benzene-polyglutaraldehyde co-polymer,

Figure 11. X-ray diffraction spectrum of all polymeric structures,

Figure 12. Modification of micro porous polymer discs and the experimental system used in immobilization of enzyme to disc,

Figure 13. Micro porous polymer disc containing polyglutaraldehyde and the experimental system in which micro porous polymeric disc was used to produce biodiesel,

Figure 14. Reactor configurations prepared by powder form of micro porous polymer containing polyglutaraldehyde,

Figure 15. Mixing type reactor configuration prepared by beaded micro porous polymer containing polyglutaraldehyde,

Figure 16. Cylinder type reactor configuration of the micro porous polymer disc containing polyglutaraldehyde,

Figure 17. TLC analysis of biodiesel obtained with free and immobilized lipase after 3 -hours of reaction,

Figure 18. HPLC analysis and monitoring of triglyceride decrease in reaction against time,

Figure 19. Activity-reuse numbers graphic of immobilized lipase,

Figure 20. SEM analysis of styrene-divinylbenzene-polyglutaraldehyde co-polymer.

The parts in the figure are numbered and are as follows:

1. Feeding tank,

2. Flowmeter,

3. Peristaltic pump,

4. Holder in which the polyglutaraldehyde micro porous polymeric enzyme reactor in the form of disc is placed, 5. Reaction back cycle line,

6. Raw oil or waste oil feeding tank,

7. Glycerin collection reactor inlet line,

8. Reactor where glycerol is collected by formation of dead zone,

9. Feeding tank inlet circulation line, 10. Packed colon reactor prepared by powder form of polymer,

11. Reaction case where polymer is prepared as bead,

12. Pedal mixer,

13. Injector allowing the distilling of polymer into reaction case,

14. Complete mixing batch reactor, 15. Raw or filtered used oil inlet,

16. Reactor inlet line,

17. Pressure control valve,

18. Manometer,

19. Primary module, 20. Secondary module,

21. Biodiesel and glycerol separation tank,

22. Glycerol outlet line,

23. Biodiesel outlet line.

In this invention, the suggested micro porous polymeric enzyme reactor (MPPER) have been synthesized by using styrene, divinylbenzene, polyglutaraldehyde and potassium persulfate as a radicalic starter at 80° C, and various enzymes (lipase and maltogenase) have been immobilized and used. In order to determine the production conditions of the disc with the highest mechanical resistance and having maximum activity, an optimization study has been carried out. Table 1 and 2 show the study schedule pertaining to this optimization. 5 variables have been chosen in preparation of polymer discs. The upper, lower and the middle values demonstrating the arithmetic averages of these variables are as shown in Table 1. The values in the constant values part of the table have been taken as constant in the synthesis of each polymer.

The polymers have been prepared in 100 mL reactor in a way that the total volume is

50 mL. 90% of the total volume is water phase, and %10 is oil phase. Polymers

containing 15% span 80, 85% styrene (STR) and different ratios of divinylbenzene (DVB) of the oil phase have been produced. The water phase has been prepared by addition of polyglutaraldehyde (PGA) placed in the second column of Table 1 at different ratios. The polymer consists of two phases as 90% water phase and 10% oil phase. Total volume of 50 mL consists of 45 mL water phase and 5 mL oil phase. The polymer with high porosity has been synthesized in two stages. The first stage is polyglutaraldehyde synthesis. For the production of polymer block with 50 mL of outer volume, 25% of gluteraldehyde has been diluted to 50 mL in a way that it is at the range of 1-15% in water phase. To realize the aldole condensation polymerization, the medium has been added 5 mL, 0.1 M NaOH and mixed in the magnetic stirer at 600 rpm for 30 minutes. From the polyglutaraldehyde solution obtained as a result of this reaction, 45 mL has been taken and solved by adding 0.7 g of potassium persulfate which is used to start the radicalic polymerization reaction. The polymers have been prepared by feeding of the water phase into the oil phase of 5 mL consisting of 15% span 80 and with STR/DVB ratio changing at the range of 0-5 by means of a peristaltic pump whose flow is adjusted to the desired dosing time. The water and oil phase have been mixed with a mechanic stirer. The mixing time has been selected as minimum 20 minutes and maximum 60 minutes, and the mixing speed as minimum 300 rpm and maximum 600 rpm. After the polymers are produced, they are poured into tubes with 50 mL of volume and put into incubator with the adjusted temperature of 80 ⁰ C, and the polymers taken out of the tubes after 3 hours and they have been dried for 24 hours at 60 ⁰ C.

Table 2 shows the optimization distribution of 5 variables necessary for the preparation of the polymer. 19 experiments in total have been made for 5 variables. The expressions in Table 2 as (-1), (0) and (+1) represent the lower, middle and upper value in Table 1, respectively. The value of (0) in 5 ^th , 10 ^th and 15 ^th lines represent that the middle values of all variables should be taken.

Table 1: Study table about optimization of polymer preparation

Table 2: Table showing the distribution of the variables according to experiments

After the optimization, the optimum production conditions have been determined by making the mechanic test analyses and immobilization efficiency analyses of the polymer. According to the results of the analyses, it has been determined that the disc containing 59% styrene, 26% divinylbenzene, 15% span 80 and 10% polyglutaraldehyde has the optimum conditions.

Polyglutaraldehyde has been obtained in basic medium conditions (pH 9.5-10). When the polyglutaraldehyde obtained as a result of Cannizarro reaction of glutaraldehyde under these conditions is fluid, the peaks of UV spectrophotometer have been observed as 285 nm and the peaks of aldehyde groups and carbon-carbon double bond peaks as 233 nm (Figure 1).

After the polyglutaraldehyde is synthesized, solid polyglutaraldehyde has been obtained by evaporating its water for 48 hours at 50°C in a vacuumed incubator (Figure 2). In FTIR spectroscopy on the other hand, it is observed that non-conjugated aldehyde groups, conjugated aldehyde groups and carbon-carbon double bonds have absorption band at 1721, 1682 and 1641 cm ^"1 respectively and OH absorption band of water at 3428 cm ^"1 , aliphatic C-H stretching at 2946 ve 2872 cm ^"1 , aliphatic C-H in-plane bending at 1460, 1414, 1356 cm ^'1 , aliphatic C-H out- plane bending at 1120, 1044, 970 cm ^"1 have been observed.

Additionally, the X-ray diffraction of polyglutaraldehyde has been observed. According to this, it has been found that 17.720; 21.560 ; 27.320; 30.920; 31.640; 38.120; 44.280; 44.920; 45.400; 55; 59.160 and 64.360 degrees show the crystallic and semi-crystallic structures in PGA structure. The wide peak between 0 and 27 degrees represents the amorphosis of the material. Very small peaks present all along the spectrum and named as noise have been found in polymeric structures (Figure 3).

Figure 4 shows three different amorphous passage temperatures in polyglutaraldehyde (PGA). These are 62, 112 and 222 ⁰ C. The reason for this is the polymeric structure of PGA varying between 300 and 20000 Da. At 62 ° C shows the amorphous passage temperature of the PGAs with low molecule weight, 112 ⁰ C shows the amorphous passage temperature of those with greater molecule weights than the polymers having amorphous passage temperature at 62 °C, and 222 ⁰ C shows the amorphous passage temperature of the PGAs with greatest molecule weights.

The structure of styrene-divinylbenzene co-polymer has been illuminated with FTIR, X-ray and DSC. In FTIR analysis, OH absorption band of water at 3444 cm ^"1 , aromatic C-H stretching at 3061 and 3027 cm ^"1 , aliphatic C-H stretching at 2924 and 2853 cm ^"1 , aromatic C=C stretching at 1602, 1493 and 1452 cm ^"1 , aromatic C-H in- plane bending at 1026 and 905 cm ^"1 (for p-substitute benzene), aromatic out-plane bending at 798, 759 and 699 cm ^"1 (for p-substitute benzene) are observed (Figure 5).

In X-ray diffraction analysis, on the other hand, crystallic structure or non-crystallic structure has not been observed in the structure of styrene-divinylbenzene copolymer. A wide peak between 0 and 27 degrees showing the effective strength of the amorphous structure, and noise all along the spectrum has been obtained (Figure 6).

In DSC analysis of styrene-divinylbenzene polymer, amorphous passage temperature has been observed at 203 °C, and softening temperature at 287 ⁰ C. At 450 °C the copolymer was fully deformed (Figure 7).

The analysis system followed above has been applied to styrene-divinylbenzene (STR-DVB-PGA) co-polymer prepared with polyglutaraldehyde. In FTIR of STR- DVB-PGA analysis, OH absorption band of water at 3444 cm ^"1 , aromatic C-H stretching at 3061 and 3027 cm ^"1 , aliphatic C-H stretching at 2924 and 2853 cm ^"1 , aromatic C=C stretching at 1602, 1493 and 1452 cm ^"1 , aromatic C-H in-plane bending at 1026 and 905 cm ^"1 (for p-substitute benzene), aromatic out-plane bending at 798, 759 and 699 cm ^"1 (for p-substitute benzene) are observed (Figure 8). These peaks are all present in styrene-divinylbenzene co-polymer as well. But the peak seen at 1735 cm ^"1 is the peak of C=O stretching. This peak is seen in FTIR spectrum of PGA. However, there is a difference; while this peak has been seen at 1721 cm ^"1 , due to the bonds formed as a result of the radicalic reaction it makes with styrene and divinylbenzene, the peak is shifted and observed at 1735 cm ^"1 . In order to understand whether PGA is added to the structure, PGA and STR-DVB co-polymer is mixed very well in a way that polyglutaraldehyde, divinylbenzene, styrene amount is the same in the disc polymer; and when the FTIR spectrum of this mixture is observed, OH absorption band of water at 3444 cm ^"1 , aromatic C-H stretching at 3062 and

3026 cm ^'1 , aliphatic C-H stretching at 2927 and 2856 cm ^"1 , aromatic C=C stretching at 1603 cm ^"1 , aromatic C-H in-plane bending at 1126, 970 and 906 cm ^"1 (for p- substitute benzene), aromatic out-plane bending at 797, 758 and 700 cm ^"1 (for p- substitute benzene) are seen. Carbonyl stretching peak is seen at 1716 cm ^"1 (Figure 8). This situation proves the formation of the polymerization reaction.

When the X-ray diffraction spectrum of STR-DVB-PGA co-polymer is examined, it is seen that amorphous structure rate decreases between 0 and 23 degrees, and peaks representing the semi-crystallic structures are observed in this zone. Crystallic structures are observed at 23.00; 24.00; 25.240; 26.120; 27.240; 29.400; 31.00; 32.500; 37.00 degrees (Figure 9).

In DSC analysis of STR-DVB-PGA co-polymer, two amorphous passage temperature at 150.7 and 170.69 ⁰ C, softening temperature at 260 ⁰ C, and deformation temperature of the polymer at 436.21 ⁰ C are observed (Figure 10).

Figure 11 shows the X-ray diffraction spectrum of all polymeric structures.

Figure 12 shows the SEM analysis of STR-DVB-PGA co-polymer.

The process will be explained in detail in the following examples without limiting the claimed protection. The reaction products in the examples have been designated with thin layer chromatography (TLC), high pressure liquid chromatography (HPLC) and gas chromatography (GC) (Figure 18, Figure 19).

EXAMPLE 1

In order to increase the strength of covalent bond, the micro porous polymer discs had been modified with polyglutaraldehyde using the experiment system in Figure 13 before immobilization. For this, 5 mL was taken from 25% glutaraldehyde and diluted to 50 mL. For aldole condensation polymerization, the medium had been added 5 mL of 0.1 M NaOH. The porous polymer was cut to have a 5 mm meat thickness, washed, dried and placed in the holder no 4. The prepared polyglutaraldehyde solution was put into tank no 1, and circulated for 24 hours with

flowmeter no 2 and pumps no 3, by means of the circulation line no 5 from the disc placed to the holder no 4. Then, the micro porous polymer was first washed with 300 mL distilled water, and then with buffer solution of 200 mL 25 mM, pH 6, Ca(Ac) ₂ .

After the modification process of the discs the immobilization of the lipase enzyme (Thermomyces lanuginousus) had been realized. An optimization study had been carried out for this, and the most important 4 parameters necessary for immobilization were selected. These parameters are shown in Table 3. The enzyme solution changing between the range of 1-10% in tank no 1 had been realized by using the experiment setup in Figure 13 by diluting to 30 mL with the pH 6, 25 mM calcium acetate buffer solution, and by circulation from the holder no 4 which had a disc inside within the range of 2-48 hours with peristaltic pump no 3. In order to get rid of the non-bounded enzymes the disc had been washed with 500 mL 0.5 M, pH 6 Ca(Ac) ₂ buffer solution. The prepared bioreactor has been preserved under the room conditions if not used for along time. After the analyses, it had been determined that the optimum immobilization conditions were realized in 10% enzyme solution, room temperature, 25 -hours of immobilization period and 11 mL/min. circulation speed. After the Bradford analyses, it had been found that the immobilization efficiency was 56% in optimum polymer synthesis and immobilization conditions.

Table 3: Study table about optimization of immobilized polymer

Table 4: Table showing the distribution of the variables according to experiments

EXAMPLE 2 For biodiesel production in the reactor shown in Figure 14, the soybean oil (100 mL) and methanol (24 mL) in the oil feeding tank no 6 had been circulated from the biocatalytic reactor in holder no 4 with the flow of 21 mL/min. by means of the flowmeter no 2 and pump no 3. The circulated product had been transferred to the reactor no 8 including a dead zone with the circulation line no 7 in order to separate the glycerol produced during reaction, and sent to the biocatalytic reactor with transmission line no 9 again. In order to prevent the enzyme inhibition due to methanol, it had been added to the medium at three steps with equal intervals. After the 24 hours-process, the biodiesel (fatty acid methyl esters) is separated in reactor no 6 and the glycerol is separated in reactor no 8. After the reaction 95% of the oil was turned into fatty acid methyl esters. Biocatalytic reactor maintained its activity even after 15 reuses (Figure 20).

EXAMPLE 3

The measures against the possible problems (softening, breaking, blockage, etc.) that may arise as a result of the long-use of the disc have been investigated. For this, the disc had been smashed until it had become powderized, and by placing it in the carrier column no 10, packed column reactor had been prepared. En2yme immobilization has been made as stated in example 1. Biodiesel has been produced by using the experiment setup in Figure 15. For biodiesel production, the soybean oil (100 mL) and methanol (24 mL) in the oil feeding tank no 6 had been circulated from the powderized biocatalyst reactor in holder no 4 with the flow of 21 mL/min. by means of the flowmeter no 2 and pump no 3. The circulated product had been transferred to the reactor no 8 including a dead zone in order to separate the glycerol produced during reaction, and sent to the biocatalytic reactor with transmission line no 9 again. In order to prevent the enzyme inhibition due to methanol, it had been added to the medium at three steps with equal intervals. After the reaction 98% of the oil was turned into fatty acid methyl esters.

EXAMPLE 4

In this study, the styrene-divinylbenzene-polyglutaraldehyde co-polymer has been obtained as bead (Figure 16-a) and the immobilization and biodiesel production has been realized in a complete-mixing batch reactor (Figure 16-b). Thus, an alternative method has been developed in order that the polymer discs are used in pilot system. In the first stage, styrene-divinylbenzene HIPE had been obtained through emulsion polymerization method, and in the second stage polyHIPE beads were obtained through suspension polymerization method. For emulsion polymerization, divinylbenzene (1.2 g), styrene (2.7 g) and sorbitol monooleat (Span 80) (0.7 g) had been put into the reactor no 11 in Figure 16-a. The reaction had been realized at 350 rpm by using the pedal mixer no 12. Water phase had been prepared by solution of 0.7 g potassium persulfate (K ₂ S ₂ Os) in 45 mL polyglutaraldehyde. This prepared solution had been added to the oil phase in the reaction case no 11 by drops under constant mechanic mixing within 30 minutes. After the entire of the water phase was added, mixing had been continued for 15 minutes. The obtained homogenous emulsion phase had been put into injector no 13 and distilled into the aqueous

solution phase heated to 80 ⁰ C. The used aqueous suspension includes with high molecule weight as a stabilizer 5% poly(diallyldimethylammoniuπi chloride) (PDDAC). The mixing speed had been between 250 and 300 rpm, and after the entire of HIPE was added, it had continued for 15 minutes more. Then the mixing had been stopped, and polymerization had been realized in 6 hours at 80 ⁰ C. After cooling the reaction mixture had been filtred and the beads had been washed with water and ethanol. Finally, they had been dried for 24 hours under 60 ⁰ C vacuum. The dried beads had been sieved and the big ones had been separated. After the beads had been obtained this way, they had been put in the reactor no 14 using the experiment mechanism in Figure 16-b, and the immobilization had been realized by dilution of 3 mL lipase enzyme to 30 mL by means of pH 6, 25 mM calcium acetate buffer. After the immobilization period of 25 hours, the immobilized beads had been washed with the acetate buffer and become ready for biodiesel production. Biodiesel production had been obtained by mixing of 100 mL oil and 24 mL alcohol in the complete-mixing batch reactor no 14 for 24 hours. In order to prevent the enzyme inhibition due to methanol, it had been added to the medium at three steps with equal intervals. After the reaction 93% of the oil was turned into fatty acid methyl esters.

EXAMPLE 5

For biodiesel production with continuous process in the reactor shown in Figure 17, the soybean oil (1000 mL) and methanol (240 mL) fed by the oil inlet tank no 15 into the oil feeding tank no 6, had been sent to the primary module no 19 with the flow of 5 mL/min. by means of the flowmeter no 2 and pump no 3. The pressure in the system had been adjusted by means of pressure control valve no 17 and manometer no 18. The product had been sent to the secondary module no 20 from the primary module. The product had been sent to separation tank no 21, and glycerol had been obtained from line no 22 and biodiesel from line no 23. In order to prevent the enzyme inhibition due to methanol, it had been added in drops. After the reaction 95% of the oil was turned into fatty acid methyl esters.

EXAMPLE 6

In this study, the maltogenase enzyme has been immobilized to the disc and maltose has been obtained from maltodextrine. For this, the immobilization conditions of the enzyme to the polymer disc have been optimized. By using the experiment setup in Figure 13, 50 μL maltogenase and 50 mL 1 M, pH 5.5 sodium acetate [Na(Ac)] buffer solution had been put into the feeding tank no 1. This enzyme solution had

^• been circulated from the holder no 4 including a disc inside for 24 hours by means of peristaltic pump no 3 at 5 mL/min. flow, and covalent immobilization had been realized. In order to get rid of the non-bounded enzymes the disc had been washed with 500 mL 1 M, pH 5.5 Na(Ac) buffer solution. After the analyses, the optimum immobilization conditions had been determined as pH 5.5, 1 M Na(Ac). For maltose production in the experiment system shown in Figure 13, the maltodextrine solution in substrate feeding tank no 1 had been circulated from the biocatalytic reactor present in holder no 4 at 15 mL/min. flow by means of the pump no 3. After the 30 minutes process, maltose had been produced in substrate feeding tank no 1. As a result of the experiments, it had been determined that the chemical controlled zone starts at 15 mL/min. flow. After a 30 minutes reaction, it had been observed that 1.39 g/L of the substrate turns into product. Biocatalytic reactor maintained its activity even after 50 hours reuses.