Enzymatic microfluidic fiber contactor

Abstract

The immobilized enzyme fiber contactor comprises a plurality of fibers arranged within a hollow conduit. The fibers possess an enzyme selected from oxidoreductases, transferases, hydrolases, lyases, isomerases, or ligases. The enzymes can be bound to the fibers via anchor groups and optionally bifunctional crosslinking agents. The enzymes can be applied, removed, and reapplied without disassembling the reactor or discarding the fibers. The immobilized enzyme fiber contactor can be used to carry out two-phase or single-phase enzymatic reactions.

Description

Technical Field

[0001] Related Applications This application claims the benefit of priority of U.S. Provisional Patent Application No. 63 / 499,628, "Enzymic Microchannel Fiber Contactors", the disclosure of which is hereby incorporated by reference in its entirety.

[0002] Technical Field of the Disclosure The present disclosure relates to enzymatic reaction processes facilitated by fiber contactors. More particularly, the present specification relates to immobilized enzyme fiber contactors and their use.

Background Art

[0003] A conventional commercial process that uses stirring enzymes is the enzymatic degumming process used in vegetable oil processing. An example of this process is shown in Figure 1. Jiang, X., Chang, M., Wang, X., Jin, Q., & Wang, X. (2014). A comparative study of phospholipase A1 and phospholipase C on soybean oil degumming. Journal of the American Oil Chemists'Society, 91(12), 2125-2134;Sampaio, KA, Zyaykina, N., Wozniak, B., Tsukamoto, J., Greyt, WD, & Stevens, CV (2015). Enzymatic degumming: Degumming efficiency versus yield increase. European Journal of Lipid Science and Technology, 117(1), 81-86; and Yang, B., Zhou, R., Yang, JG, Wang, YH, & Wang, WF (2008). Insight into the enzymatic degumming process of soybean oil. Journal of the American Oil See Chemists' Society, 85(5), 421-425. The above step involves adjusting the solution to the optimal pH for the enzyme, adding the enzyme dissolved in water, and then stirring for at least one hour. The reason why prolonged stirring is necessary when using additional enzyme is that the reaction between the enzyme and the oil occurs only at the oil / water interface, which is the case when hydrolase is involved in the enzymatic degumming of the oil. After enzymatic treatment, residual phospholipids are less than 10 ppm and metals are less than 5 ppm. Although a cleaner oil can be obtained by adding enzyme, the prolonged stirring under high shear conditions and continuous consumption of enzyme required to achieve this are undesirable from the standpoint of capital costs, enzyme costs, and high energy requirements.

[0004] Most commercial enzymes are used in solution in stirred-tank reactors. For example, the conversion of dextrin to glucose by amyloglucosidase is carried out in a 13-step stirred reactor with a holding time of 24–48 hours to obtain the desired conversion rate; the conversion of starch to dextrin by α-amylase is carried out in a 2–4-step stirred reactor system with a holding time of 1–4 hours to obtain the desired conversion rate; the conversion of dextrin to maltose by β-amylase is carried out in a stirred-tank reactor; phospholipase for the hydrolysis of phospholipids is carried out in a single-step stirred reactor with a reaction time of 2–6 hours; and furthermore, the production of biodiesel from vegetable oil and methanol is carried out in a stirred tank for 40–60 hours using enzymes.

[0005] Enzyme reactions in stirred tanks have drawbacks such as high cost and long reaction times. When new fluid is introduced into the stirred tank for processing, additional enzymes need to be added, and this re-introduction increases the raw material cost in operation. Furthermore, enzyme systems using stirred tank reactors often require long to extremely long incubation times. The reason for requiring long incubation times is often the high cost of the enzyme. In other words, the amount of enzyme administered is minimized by extending the reaction time in order to reduce ongoing operating costs. In many industrial applications, the substrate is in large excess relative to the enzyme, which reduces the enzyme cost, but requires very long reaction times.

[0006] Packed-bed immobilized enzyme reactors have several drawbacks, including high pressure drop or reduced surface area, inability to accommodate suspended solids, time and material costs associated with packing material replacement, and channeling problems. In particular, while a high surface area in the packed bed is achieved by smaller resin particle sizes, this results in high pressure drop in the resin-packed bed. While larger beads can be used to manage the pressure drop, this reduces the available surface area. Furthermore, packed-bed reactors do not tolerate suspended solids and are unsuitable for reactions that can produce them. These suspended solids are "filtered" by the packed bed, and even a small accumulation of suspended solids significantly impairs system performance due to increased back pressure and reduced total throughput. Additionally, if enzyme activity falls below the required minimum, the packing material must be removed and discarded, generating additional waste. The need to purchase new immobilized packing material for replacement increases operating costs. Finally, channeling within the packed bed is a problem. Effectively packing the resin into the column housing is generally a complex and time-consuming process. The task of filling the bed and forming a uniform column with few or no channels, that is, eliminating the possibility of short circuits in the fluid processing path, is not easy.

[0007] Industrially, immobilized enzyme systems using beads in packed beds or mixing tanks offer several applications that result in significant cost savings given the scale of production. A few examples include glucose isomerase used for glucose-to-fructose conversion (this is the world's largest application for immobilized enzymes, used to convert glucose from corn starch to fructose in the production of high-fructose corn syrup); lipase used for Sn1,3 transesterification of fatty acids at the Sn-1 and Sn-3 positions of TAG (the most common commercially available lipase is derived from Thermomyces lanuginosus); β-galactosidase used for lactose-to-glucose + galactose conversion, for the production of lactose-free milk and its derivatives; pectinase used to produce pectin solutions in fruit processing facilities; and laccase derived from Pyricularia oryzae to reduce selective phenols in red and white wines and improve wine quality.

[0008] Thus, there is still a need for high-throughput and cost-effective systems for carrying out enzymatic reactions. The immobilized enzyme fiber contactor and its method of use described herein can provide superior throughput, reduced enzyme costs and / or improved reusability, and even shorter reaction times compared to agitated tanks and / or packed beds. [Brief explanation of the drawing]

[0009] The various embodiments of this disclosure will be better understood from the detailed description and accompanying drawings set forth below. In the drawings, identical or functionally similar elements may be shown with the same reference numeral. The embodiments are described in detail below with reference to the accompanying drawings.

[0010] [Figure 1] This diagram schematically illustrates the flow of the enzyme degumming process. [Figure 2]This figure schematically shows a fiber contactor according to one embodiment of the present disclosure, partially cut away. [Figure 3] This figure schematically shows a fiber contactor according to one embodiment of the present disclosure. [Figure 4] This figure schematically shows an immobilized enzyme on a fibrous substrate according to one embodiment of the present disclosure. [Figure 5] This graph shows the pressure loss results for Example 1. [Figure 6] This graph shows the results of Example 3. [Figure 7] This figure schematically illustrates the cleavage reaction in Example 6. [Figure 8] This graph shows the results of Example 6. [Figure 9] Figure 9 is a graph showing the results of Example 7. [Figure 10] Figure 10 is a graph showing the results of Example 7. [Overview of the Initiative]

[0011] The following is a description of how to prepare exemplary aerogels using the non-limiting methods of this disclosure. While the claimed subject matter is described based on specific embodiments and examples, other embodiments and examples are also included in the scope of this disclosure, including embodiments and examples that do not necessarily provide all the advantages and properties described herein. Various structural, logical, and process step modifications can be made without departing from the scope of this disclosure.

[0012] This specification discloses ranges of values. These ranges define lower and upper limits. Unless otherwise specified, such ranges encompass all values ​​in the smallest digit (whichever is smaller, the lower or upper limit) and all ranges between the values ​​listed within the range.

[0013] Referring to Figure 2, the present disclosure provides a fiber contactor 100 (hereinafter referred to as an "immobilized enzyme fiber contactor" or "IEFC") containing immobilized enzymes in internal elements 120 (e.g., fibers). The IEFC 100 comprises a hollow conduit 110 and a plurality of fibers 120 housed therein. In some embodiments, the fibers 120 are formed from steel, such as stainless steel or a steel composite. In other embodiments, the fibers 120 are formed from basalt, ceramics, glass, polymers, and / or metals. The fibers 120 may extend along the entire length or part thereof of the conduit 110, or they may extend outward from the conduit 110 as shown in Figure 2. In some embodiments, the fibers 120 fill the conduit 110 in nominal proportions of less than 40%, less than 30%, less than 25%, less than 20%, 5-30%, about 15%, or about 25%.

[0014] The conduit 110 includes one or more inlets 132,142 for introducing reactants into the conduit 110. In some embodiments, there is only one inlet, and in other embodiments, there are two, three, or more inlets. The IEFC 100 includes at least one outlet 134,144 downstream of the inlets 132,142. In some embodiments, there is only one outlet, and in other embodiments, there are two or more outlets. As shown in Figure 2, the IEFC 100 may include a separation tank or a sedimentation tank 112. In these embodiments, the separation tank 112 may include an outlet 144 for removing a higher density phase 140 (e.g., aqueous phase) and an outlet 134 for removing a lower density phase 130 (e.g., organic phase).

[0015] In some embodiments, the IEFC does not include a settling tank. For example, as shown in FIG. 3, the IEFC 200 includes a conduit 210 within which a plurality of fibers 220 are disposed. The IEFC includes a single inlet 232 that communicates with a tank 250, and a solution or mixture is supplied from the tank 250 to the IEFC 200. The tank 250 may include a mixer 250a. The IEFC 200 also has a single outlet 234 for guiding the post-reaction mixture out of the IEFC 200. The post-reaction mixture discharged from the IEFC 200 may undergo further processing or be subjected to separation processes such as centrifugation or drying. Other configurations of IEFCs can also be used, such as those described in U.S. Patent No. 9,468,866 B2 by Massingill, the entire disclosure of which is incorporated herein by reference.

[0016] The IEFCs 100, 200 enable the formation of microchannels along the fibers 120 and achieve a high surface area to facilitate the enzyme reaction. As will be described in detail below, the reaction involves a two-phase chemistry utilized to treat phospholipids using a hydrophobic enzyme (Enzyme Class 3 (EC3)). The IEFCs 100, 200 enable a high interfacial two-phase diffusion rate, resulting in close contact between the immiscible phases and the fibers 120, 220 as the oil and water flow down the fibers 120, 220. In these embodiments, the fibers 120, 220 extend into a separation tank 112 where the immiscible fluids separate.

[0017] In other embodiments, the enzyme reaction includes a single-phase chemistry, and the IEFCs 100, 200 similarly provide a high surface area to facilitate the enzyme process. Hydrolases used to degum oil require the presence of water and substrate oil for hydrolysis, but most known enzyme reactions occur in a single-phase environment. Any of these enzyme classes may be equally immobilized on the IEFCs 100, 200, allowing the enzyme to be reused over multiple batch operations.

[0018] The enzyme classes used in this disclosure include, but are not limited to, EC1, EC2, EC3, EC4, EC5, and / or EC6. Oxidoreductases are classified as enzyme class 1 (EC1) and are classified based on their functional ability to transfer electrons from one substrate to another. Another group of commercially important enzymes is transferases (EC2). This enzyme class transfers functional groups from one substrate to another. These functional groups may be methyl, acyl, amino, or phosphate groups. As an example, cyclodextrin-glucosyltransferases (CGTases) have recently attracted attention due to their unique ability to produce large quantities of cyclic α-(1,4)-linked oligosaccharides (cyclodextrins) from starch. These oligomers have a wide range of applications in pharmaceutical formulations and drug delivery. Enzyme class EC3 is commonly known as hydrolase. Hydrolases add H2O to a bond in a substrate, producing two products from one substrate. These enzymes are used in a variety of applications, including amyloglucosidase, which converts dextrin to glucose; β-galactosidase, which converts lactose to glucose and galactose; and α-amylase, which converts starch to dextrin. Enzyme class EC4, commonly known as lyases, are used for the non-hydrolytic addition or removal of functional groups from substrates through modification of CC, CN, CO, or CS bonds. For example, pectinase is widely used to clarify fruit juices and wines. Isomerases, enzyme class EC5, are most commonly used in sugar production. Glucose isomerase (also called xylose isomerase) catalyzes the conversion of D-xylose and D-glucose to D-xylulose and D-fructose. Like many sugar isomerases, glucose isomerase catalyzes the interconversion of aldoses and ketoses. Ligases (EC6), which play a central role in biotechnology, have the function of joining two molecules through the synthesis of new CO, CS, CN, or CC bonds. The energy required to form these bonds is supplied by the simultaneous breakdown of ATP into ADP+P. An example of this is the DNA ligase of the T4 bacteriophage.Having the function of assembling longer DNA strands from short DNA fragments, T4 ligase can be used to assemble short fragments of reference DNA into longer segments.

[0019] In any of the embodiments, the IEFC 100, 200 provides that the formation of the emulsion is minimal, it is a sealed system isolated from the atmosphere, it has a small installation area compared to conventional systems, it has no moving parts that require energy and maintenance, and / or it can accommodate a wide range of flow rates.

[0020] Compared with a stirred tank, the IEFC 100, 200 solves the aforementioned problems such as high cost and long reaction time. In particular, the IEFC 100, 200 enables continuous reuse of the enzyme, thereby significantly reducing the operating cost of each step. Furthermore, due to the high surface area of the fibers 120, 220, the amount of enzyme that can be immobilized within the IEFC 100, 200 may be several orders of magnitude larger than the amount of enzyme that can be administered to a stirred tank system or resin beads. The reason for such extremely high enzyme activity is that after the same enzyme is immobilized on the fibers 120, 220, it continues to act efficiently for several months.

[0021] Compared to packed bed reactors, IEFC100,200 solves the aforementioned problems such as high pressure drop or reduced surface area, inability to accommodate suspended solids, time and material costs for replacing the packing material, and / or channeling issues. In particular, IEFC100,200 provides a very low pressure drop compared to resin-filled bed reactors, as shown in Example 1 below. Furthermore, IEFC100,200 has a channel that is highly tolerant of suspended particles. Small amounts of particles can pass through IEFC100,200 without accumulating in the system, which is different from the behavior seen in packed bed systems. In addition, the fibers 120,220 have high resistance to chemical treatment, can be cleaned in place (CIP) with strong treatment and relabeled with enzymes, and in extreme cases, the fibers can be removed and physically treated to remove the adhesion of large particles while retaining the enzyme-labeled fibers. Furthermore, if enzyme activity in the IEFC100,200 decreases, the enzyme can be removed from the column in situ and a new enzyme can be reapplied in situ. This eliminates the need to discard the packed bed medium and resolves the associated waste problems. Also, even in situations where high throughput causes composition deposition on the fibers, i.e., fouling, the fibers can be treated using the CIP procedure, and the function of the IEFC can be restored without the need for relabeling. Finally, the IEFC100,200 has high robustness against short-circuit channeling. There is no need for processes such as slurring the resin with the liquid and packing the medium into the column housing.

[0022] This disclosure also provides a method for immobilizing an enzyme on an internal element of a fiber contactor, as well as a method for enabling rapid enzymatic treatment using an IEFC.

[0023] Figure 4 shows a fiber 120 on which enzyme 120c is immobilized. According to one or more embodiments, the linkage between the fiber 120 and the enzyme 120c comprises a plurality of anchor groups 120a bonded to the surface of the fiber 120, and a bifunctional crosslinking agent 120b that links the anchor groups 120a to the enzyme 120c. Numerous options exist for immobilizing the enzyme. These methods include adsorption, covalent bonding, capture, copolymerization, and encapsulation. The general advantages and disadvantages of each of these methods are summarized in Table 1 below.

[0024] [Table 1]

[0025] In some embodiments, the enzyme 120c is covalently bonded to the fiber 120. In these embodiments, the process of immobilizing the enzyme on the internal element 120 of the IEFC 100 (e.g., steel fiber) comprises three steps. In the first step, an anchor group 120a is bonded to the surface of the fiber 120. In some embodiments, the anchor group 120a comprises an amino compound. For example, an aminosilane, such as aminopropyltriethoxysilane, may be circulated over a bundle of steel fibers in ethanol / aqueous solution for, for example, 5 to 10 minutes. Other aminosilanes available in this disclosure, but not limited to, include aminohexylaminomethyltriethoxysilane and aminoethylaminopropyltriethoxysilane. The ethoxysilane group is bonded to a naturally occurring hydroxyl group present on the surface via a hydrolysis-condensation reaction. A similar reaction is also applicable to any surface having naturally occurring hydroxyl groups (e.g., ceramics, metals, and / or glass). Once bonded, the amino group protrudes outward from the fiber. As another example, a dopamine hydrochloride solution may be circulated over the fiber 120 for up to 3 hours or up to 24 hours to coat the fiber 120 with polydopamine (anchor groups 120a). The dopamine hydrochloride solution may contain about 0.001 to 10 g / L or about 2 mg / L of dopamine hydrochloride and may be a buffered solution using Tris-HCl buffer (pH 7.4) or phosphate buffer (pH 8.5). In any embodiment, the first step may further include washing the fiber 120 to remove unbound anchor groups 120a (e.g., using water and / or denatured alcohol) and drying the fiber 120 to which the anchor groups 120a are attached. In some embodiments, drying is carried out at high temperature (e.g., about 40°C or about 80°C) and / or under vacuum.

[0026] In the second step, an aqueous solution of a bifunctional crosslinking agent (e.g., glutaraldehyde) is circulated over the fiber bundle for, for example, about 20 minutes to crosslink the aldehyde group with the primary amine of aminopropylsilane. Finally, an aqueous solution of the enzyme is circulated over the fiber bundle for, for example, about 30 minutes to allow the second aldehyde group on the glutaraldehyde to react and form a covalent bond with the amine group of the enzyme protein. The fiber 120 may be washed (e.g., with ultrapure water) to remove any unbound enzyme. All of the chemical reagents described above are inexpensive and are mass-produced industrially.

[0027] As those skilled in the art will understand, other methods can also be used to immobilize enzymes onto fibers. For example, ThermoFisher Scientific's Bioconjugation technical handbook, *Reagents for crosslinking, immobilization, modification, biotinylation, and fluorescent labeling of proteins and peptides*, contains a comprehensive list of linker molecules available for linking different functional groups. Table 2 below summarizes some linkers useful in this system, method, and apparatus. In particular, Table 2 lists 21 chemicals for amino group-amino group linking, 7 chemicals for sulfhydryl group-sulfhydryl group linking, 28 chemicals for amino group-sulfhydryl group linking, 3 chemicals for carboxyl group-amine group linking, 6 chemicals for sulfhydryl group-carboxyl group linking, 11 chemicals for photoreactive linking, and 12 chemicals for chemoselective linking.

[0028] [Table 2] TIFF2026516857000004.tif172141TIFF2026516857000005.tif94141

[0029] In some embodiments, enzyme 120c binds without the use of a bifunctional crosslinking agent 120b. For example, enzyme 120c binds to the fiber 120 using van der Waals forces. In these embodiments, the fiber 120 is first washed to remove oils or organic compounds by circulating a suitable organic solvent, such as hexane, denatured alcohol, or ethanol, over the fiber 120 for, for example, about 20 minutes. After washing the fiber 120, the solvent is rinsed off to remove the nonpolar organic solvent. A solution of ethanol (or denatured alcohol) and water in a volume ratio of 95 / 5 is acidified to about pH 4.5 using an acid such as acetic acid. This acid has the effect of promoting the hydrolysis-condensation reaction. About 1-5 volume% of trimethoxyhexadecyl (or octadecyl, or octyl)silane is added to this mixture, and the resulting mixture is circulated over the fiber 120 for, for example, about 10 minutes. The fluid is drained, and then the fiber 120 is washed with denatured alcohol to remove loosely bound alkylsilanes. Next, to complete the silane crosslinking reaction, the fibers are dried, for example, at about 110°C for about 10 minutes. Then, enzyme 120c is circulated over the alkyl-functionalized fibers for about 20-30 minutes to bind to the fibers 120. To remove unbound enzyme, the fibers 120 are washed with deionized water. In some embodiments, enzyme 120c is PLA or PLC, and the step of circulating enzyme 120c over the fibers 120 is carried out using a mixture containing 1 mL of enzyme 120c per liter of water.

[0030] In some embodiments, the enzymatic reactions described herein are carried out at room temperature or at high temperatures, such as 30–80°C, 40–70°C, 50–60°C, 50–55°C, about 50°C, about 55°C, or about 60°C. Depending on the enzyme used, the temperature is appropriately adjusted to the optimal temperature range for the enzyme. In some embodiments, the reactions are carried out under low pH conditions of less than 7, less than 6, about 4–6, about 4–5, about 4, about 4.5, or about 5. Similar to the temperature, the pH is controlled to the optimal range for the enzyme or multiple enzymes used in the IEFC.

[0031] In any embodiment, the pressure inside the IEFC is maintained at approximately 1–125 psi, approximately 1–100 psi, approximately 1–50 psi, or approximately 20 psi. In some embodiments, the cross-sectional area of ​​the IEFC (cm² 2 The total flow rate of the reactants per unit area must be at least 10 mL / min / cm³. 2 , at least 15 mL / min / cm2, at least 25 mL / min / cm2, at least 50 mL / min / cm 2 at least 75 mL / min / cm² 2 , at least 100 mL / min / cm³ 2 at least 125 mL / min / cm² 2 at least 150 mL / min / cm² 2 , or 20-250 mL / min / cm² 2 The IEFC can be easily scaled up to accommodate industrial-scale quantities. Furthermore, the contact time required to achieve the reactions described herein is significantly reduced compared to conventional methods.

[0032] This disclosure also provides a method for processing a solution and / or oil using an IEFC. In such a method, the solution and / or oil comprises reactants that may undergo an enzymatic reaction in the presence of an enzyme immobilized on the fibers of the IEFC. The method comprises the steps of introducing the solution and / or oil into the IEFC, bringing it into contact with the fibers and the enzyme immobilized thereon, and then receiving the product solution or product oil from the outlet of the IEFC with the reactants chemically modified in the product solution or product oil. In some embodiments, the solution or oil is a solution in which the reactants are dissolved in a solvent such as water. In some embodiments, the enzyme is an oxidoreductase, and the method comprises the step of removing one or more hydrogen atoms from the reactants. In some embodiments, the enzyme is a transferase, and the solution comprises a donor reactant and an acceptor reactant containing a functional group (e.g., a methyl group), and the method comprises the step of transferring the functional group from the donor reactant to the acceptor reactant. In some embodiments, the enzyme is a hydrolase, and the method comprises the step of reacting the reactants with water to cleave one or more bonds in the reactants. In some embodiments, the enzyme is a lyase, and the method includes the step of cleaving one or more bonds in the reactants by means other than hydrolysis or oxidation. In some embodiments, the enzyme is an isomerase, and the method includes the step of isomerizing the reactants. In some embodiments, the enzyme is a ligase, and the solution contains two reactants, which may be the same or different, and the method includes the step of forming a bond between these two reactants. [Example 1]

[0033] Example 1: Pressure drop was measured for a packed column using 50 μm silica beads and a fiber contactor with a packing density of 34% containing 50 μm fibers. The packed column and fiber contactor had the same packing density (34%), height, and fluid conditions (temperature, viscosity, and flow rate). The results are shown in Figure 2. [Example 2]

[0034] Example 2: Immobilization of enzymes onto fibers 77 g of 50 μm crimped stainless steel fibers, 1 foot in length, were washed with denatured alcohol for approximately 10 minutes to remove any oil / organic matter present on the steel fibers. A solution of 2% (3-aminopropyl)triethoxysilane (APTES), 5% water, and the remainder of denatured alcohol was circulated over the fibers for 10 minutes and then drained. The fibers were then washed again for 10 minutes with fresh denatured alcohol, drained, and then heated and cured overnight in an oven at 100°C. 500 mL of a 0.5% glutaraldehyde solution in water was circulated over the fibers for 1 hour, followed by washing with fresh water for 10 minutes. The fibers were placed in a glass column of a fiber contactor with an inner diameter of 0.5 inches and a length of 1 foot, so that the fibers occupied 25% of the void volume of the reactor column. The enzyme solution was circulated over the fibers for 2 hours, and then the column was left standing for 4 nights. Next, the enzyme solution was circulated for a further 24 hours, followed by washing with fresh water for 10 minutes to remove loosely bound enzymes. Each enzyme step has an optimal flow rate and fiber packing ratio, and parameters such as residence time, internal mixing (laminar vs. turbulent), and enzyme concentration (adjusted by the surface area of ​​the packed fibers) must be considered. For simplicity, unless otherwise specified, the following examples were carried out using a reactor packed with 25% 50 μm fibers, held for the period described before sample collection. [Example 3]

[0035] Example 3: Glucose oxidase (EC1) Following the procedure of Example 2, treatment was performed using an enzyme solution prepared by dissolving 1 g of powdered glucose oxidase enzyme in 500 mL of water. The column was maintained at 50°C, and 50 mL of a 30 mM glucose solution in a pH 5.7 buffer was circulated for 1 hour. The buffer was prepared by mixing 175 mL of 0.1 M citrate and 500 mL of 0.2 M Na2HPO4.

[0036] For comparison using a stirred tank, 1 g of powdered glucose oxidase enzyme was added to 50 mL of a 30 mM glucose solution in a pH 5.7 buffer. This was stirred at 50°C and 250 rpm for 1 hour. The results are shown in Figure 6. The immobilized contact bundle and the stirred tank were plotted for comparison with the 30 mM solution in the stirred tank (t=0) before any reaction occurred as the baseline.

[0037] The above example evaluates the usefulness of glucose oxidase (EC1 enzyme) in a fiber contactor and demonstrates the production of hydrogen peroxide using glucose as a starting substrate. This application can be used in the yeast fermentation process. In this process, yeast has high tolerance to hydrogen peroxide (H2O2) compared to competing bacterial contamination. In fact, when the molecule is broken down into oxygen and water, yeast often utilizes small amounts of H2O2 as a growth factor. In the fermentation process, glucose is used as the main substrate, so a large amount of low-cost glucose is available as a substrate for producing H2O2. The produced H2O2 selectively kills bacterial species, especially Lactobacillus, and as a result, yeast fermentation can proceed with higher efficiency. [Example 4]

[0038] Example 4: Glucoamylase enzyme (EC3) Glucoamylase enzyme was immobilized on a fiber contactor in the same manner as in Example 2. A commercially available maltodextrin with unknown chain length and branching degree was dissolved in 0.1 M citrate buffer at pH 4.5 to prepare a solution with a concentration of 0.17 g / mL. This solution was passed through the fiber contactor once, twice, three times, four times, five times, and six times at 50°C, and then circulated at room temperature for 16 hours. The passage time through the fiber contactor each time was less than 1 minute. The increase in glucose levels was tracked using a Brix meter and glucose test strips. The results are summarized in Table 3 below.

[0039] [Table 3]

[0040] The increase in glucose concentration was similarly confirmed using glucose test strips. [Example 5]

[0041] Example 5: Lactase enzyme (EC3) The lactase enzyme was immobilized on the fiber contactor in the same manner as in Example 2. Commercial skim milk (pH 6.8) was passed through the fiber contactor once, twice, and three times, and then circulated at room temperature for 14 hours. The passage time through the fiber contactor each time was less than 1 minute. The increase in glucose levels was tracked using a Brix meter and glucose test strips. The results are summarized in Table 4 below.

[0042] [Table 4]

[0043] The increase in glucose concentration was similarly confirmed using glucose test strips. After three passes through the solution, the glucose concentration was approximately 1% by weight / volume, and after one night, the glucose concentration approached approximately 3% by weight / volume. Since the theoretical glucose yield is 2.63% by weight / volume, it can be seen that the lactase bound to the fibers showed good activity even at room temperature. [Example 6]

[0044] Example 6: Pectinase (EC4) Using fruit pectin (available under the trademark "SURE JELL"), a 2 L solution containing 0.5 wt / vol% pectin in 50 mM citrate buffer at pH 4 was prepared. As in Example 2, pectinase was immobilized on a fiber contactor, which was heated via a jacket circulating 60°C hot water. The pectin solution was passed through the column at 25 mL / min for 2 minutes, after which the pump was stopped and the column was sealed with the pectin solution retained inside. After 1 minute, the solution was drained and recovered. The fiber column was then washed with fresh pectin solution, sealed and held for 5 minutes, and then the solution was drained and recovered. This procedure was repeated several more times to prepare multiple samples with different residence times on the fiber. The residence times of the samples were 1 minute, 5 minutes, 10 minutes, 30 minutes, and 60 minutes. All reactions were carried out at 60°C. As shown in Figure 7, when pectinase breaks down the polysaccharide bond, an unsaturated bond is formed on one ring of the product. The formation of these unsaturated uronic acid molecules was monitored by the increase in absorbance at 235 nm. The results are shown in Figure 8. At 300 nm, the bottom line represents the baseline measurement (0.5 wt / vol% pectin solution in 50 mM citrate buffer at pH 4), the line above it represents the 1-minute sample (absorbance of 0.66 at 235 nm). Further up are the 5-minute sample (absorbance of 1.18 at 238 nm), followed by the 10-minute sample (absorbance of 2.78 at 240 nm), followed by the 60-minute sample (absorbance of 2.79 at 240 nm), and the top line represents the 30-minute sample (absorbance of 3.64 at 252 nm). [Example 7]

[0045] Example 7: Glucose-6-phosphate isomerase (EC5) Following the procedure of Example 2, 0.4 mL of glucose-6-phosphate isomerase derived from Baker's yeast was dissolved in 500 mL of water as the enzyme solution and circulated over the bundle for 1 hour. After that, it was washed with fresh water for 10 minutes.

[0046] A buffer solution with a pH of 7.6 was prepared by mixing 0.2 M Na2HPO4 and 0.1 M citric acid in a ratio of approximately 14.7:1. Glucose phosphate was added to this buffer solution to prepare a solution with a concentration of 10 g / L or 38 mM. The glucose phosphate solution was held in a fiber contactor at 50°C for different residence times. The column was packed with the solution, and after starting the timer, the solution was removed and repacked with a fresh solution to start a new residence time test. The residence times tested were 1 minute, 5 minutes, 10 minutes, 30 minutes, and 60 minutes. The test results are shown in Figures 9 and 10.

[0047] While several embodiments have been disclosed in detail above, the disclosed embodiments are not limiting, and those skilled in the art will readily understand that many other modifications, changes, and substitutions are possible to the disclosed embodiments without substantially departing from the essence of the novel teachings and merits of this disclosure. Accordingly, all such modifications, changes, and substitutions are intended to fall within the scope of this disclosure as defined in the following claims. In the claims, any means-plus-function clauses are intended to encompass the structures described herein as performing the function, and are not limited to structural equivalents, but also include equivalent structures. Furthermore, the applicant expressly represents that it does not intend to apply 35 U.S. Patent Code § 112(f) to any limitation of any claim herein, except where the claim expressly uses the word “means” in conjunction with the corresponding function.

Claims

1. A step of introducing a solution containing reactants dissolved in a solvent into a microfluidic contactor, The microfluidic contactor includes a hollow conduit having a plurality of fibers arranged inside, The plurality of fibers form microchannels between them, and The plurality of fibers include an enzyme immobilized on its surface, the enzyme being selected from oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase, the introduction step, In order to chemically modify the reactant, the process involves enzymatically reacting the reactant in the microfluidic channel, and A step of recovering the solution containing the chemically modified reactant from the microfluidic contactor, A method that includes this.

2. A method according to claim 1, wherein the enzyme comprises an oxidoreductase, the reactant comprises a hydrogen atom, and the method comprises a step of removing the hydrogen atom from the reactant.

3. A method according to claim 1, wherein the enzyme comprises a transferase, the reactant comprises a donor reactant and an acceptor reactant containing a functional group, and the method comprises the step of transferring the functional group from the donor reactant to the acceptor reactant.

4. A method according to claim 1, wherein the enzyme comprises a hydrolase, and the method comprises the step of reacting the reactants with water to cleave one or more bonds in the reactants.

5. A method according to claim 1, wherein the enzyme comprises a lyase, and the method comprises a step of cleaving one or more bonds in the reactants.

6. A method according to claim 1, wherein the enzyme comprises an isomerase, and the method comprises a step of isomerizing the reactant.

7. A method according to claim 1, wherein the enzyme comprises a ligase, the reactant comprises a first reactant and a second reactant, and the method comprises the step of forming a bond between the first reactant and the second reactant.

8. The process of providing a hollow conduit, A step of arranging a plurality of fibers in the hollow conduit, wherein the fibers contain hydroxyl groups on their surface, In order to bond anchor groups to the hydroxyl groups of the aforementioned fibers, the process involves contacting the fibers with an anchor group precursor in ethanol / aqueous solution, A step of bringing an aqueous solution of a bifunctional crosslinking agent into contact with the fibers to which the anchor groups are bonded, A step of contacting an aqueous solution of the enzyme with the fiber in order to bind the enzyme to the fiber, wherein the enzyme is selected from oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase, and the step of contacting the fiber, Methods that include...

9. A method according to claim 8, wherein the enzyme comprises an oxidoreductase.

10. A method according to claim 8, wherein the enzyme comprises a transferase.

11. A method according to claim 8, wherein the enzyme comprises a hydrolase.

12. A method according to claim 8, wherein the enzyme comprises a lyase.

13. A method according to claim 8, wherein the enzyme comprises isomerase.

14. A method according to claim 8, wherein the enzyme comprises a ligase.

15. The method according to claim 8, wherein the anchor group precursor is aminopropyltriethoxysilane, aminohexylaminomethyltriethoxysilane, aminoethylaminopropyltriethoxysilane, or dopamine hydrochloride, and the bifunctional crosslinking agent is glutaraldehyde.

16. In the method according to claim 8, a strong base and an alcohol, or H ２ O ２ A method further comprising the step of removing the enzyme from the fibers by simultaneously introducing a sulfuric acid solution into the hollow conduit.

17. A method according to claim 16, further comprising the step of reapplying the enzyme by repeating the steps of contacting the anchor group precursor, contacting the difunctional crosslinking agent, and contacting the enzyme with an aqueous solution.

18. A device including an inlet, an outlet, and a hollow conduit having multiple fibers arranged inside therein, The plurality of fibers form microchannels between them, The plurality of fibers include enzymes immobilized on their surfaces, the enzymes being selected from oxidoreductases, transferases, hydrolases, lyases, isomerases, or ligases. The apparatus wherein the enzyme is immobilized on the plurality of fibers via a silane compound.

19. The apparatus according to claim 18, further comprising a mixing tank including a mixing means, wherein the mixing tank is fluidly connected to the inlet.

20. The apparatus according to claim 18, wherein the silane compound is aminosilane or polydopamine, and the enzyme is covalently bonded to the silane compound via glutaraldehyde.

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