KR102468425B1 - Synthesis method of magnetic rice straw for lipase immobilization and esterification reaction using lipase immobilized on magnetic rice straw - Google Patents

Abstract

The present invention relates to a method for synthesizing magnetic rice straw for immobilizing lipase and an esterification reaction using the lipase immobilized on magnetic rice straw. , The enzyme immobilized on these magnetic rice straws showed excellent immobilization efficiency, showed stability and reusability, and had superparamagnetic potential, so it could be used repeatedly in the production of fatty acid methyl esters, so it could stably maintain its activity for a long period of time, , it can be easily separated from the substrate and has the effect of facilitating regeneration.

Description

Synthesis method of magnetic rice straw for lipase immobilization and esterification reaction using lipase immobilized on magnetic rice straw}

The present invention relates to magnetic rice straw for enzyme immobilization and a one-step hydrothermal synthesis method thereof, a method for manufacturing magnetic rice straw using the one-step hydrothermal synthesis method, a method for immobilizing lipase on magnetic rice straw prepared according to the method, and a method for immobilizing lipase produced by the method It relates to a method for producing fatty acid methyl esters using lipase immobilized on magnetic rice straw.

Enzymes have been widely used so far to convert cellulose processed from biomass into glucose, but despite the decrease in the price of enzymes, they have disadvantages such as high price and low processing speed to be applied to domestic processes. There is a need to develop hydrolytic enzymes. If the development of hydrolysis enzymes and fermentation catalysts for ethanolization, which are biofuel production catalysts, is mainly centered on the redesign of metabolic pathways, research and development in another direction is optimized transformation of input biomass or new optimal biomass discovery, and the last method can be called enzyme immobilization technology. The basic concept of enzyme immobilization is the field of enzyme utilization technology in biochemical processes using enzymes. It can be divided into the technology field of improving the biological activity of enzymes and the technology field of stabilizing and economically utilizing the activity. The former is genetic engineering. While research is progressing in a specific direction, the latter has been developed centering on the fixation method. An important purpose of immobilizing the enzyme is that the enzyme can be easily recovered and reused, so that the economic efficiency of the enzyme reaction process can be increased, and the reaction mode can be applied in a variety of batch or continuous ways. That is, since enzymes are inherently water-soluble, there is a problem in that repeated use is difficult. If the water-solubility of enzymes can be removed, problems such as mixing with by-products, difficulty in separation and purification, and weak stability can be easily solved. For this reason, depending on which protein raw material is used, hydrophilicity and hydrophobicity may appear, and due to these characteristics, the efficiency of enzyme action changes. In addition, in the case of most enzyme fixation technologies or entrapment technologies, the enzyme structure is modified, ultimately affecting the yield. Therefore, in order to effectively use natural enzymes in biochemical processes, enzymes must be immobilized, and general enzyme immobilization methods are physical adsorption methods or chemical methods.

The physical adsorption method mainly uses an ion exchange method. The ion exchange method has the advantage of being non-toxic, but has a problem in that its binding force is weak. In addition, the chemical method uses a reagent to immobilize the enzyme by forming a covalent bond through a chemical reaction. Although the cross-linking force is strong, the reagent used for immobilization of the enzyme is toxic. Or, it has a disadvantage that it is difficult to use in the pharmaceutical industry. Among these industrially used enzymes, lipase is an industrially important enzyme that catalyzes various reactions including esterification, hydrolysis, and polymerization. It has been used in the industrial manufacture of various products (Houde A, Kademi A, Leblanc D (2004). Appl Biochem Biotechnol 118:155-170.). However, because of its high price, low stability under some environmental conditions and non-reusability, large-scale industrial application of lipase is difficult.

Rice straw (RS) is the feedstock with the largest surplus availability worldwide. Rice is grown in 100 countries, producing millions of tonnes of rice straw each year. More than 50% of the rice straw produced is used for cattle feed or for the timber industry. The remaining rice straw is improperly processed, which burns to produce dangerous gases such as nitrous oxide along with carbon monoxide, volatile organic compounds and suspended particulate matter. In order to reduce serious damage to the environment, the need to efficiently manage the produced rice straw to produce value-added products is emerging.

In the past, rice straw has been used for whole cell immobilization in ethanol production. Because rice straw contains high levels of cellulose and hemicellulose materials that can be easily converted into fermentable sugars, there are several reports of rice straw being used as lignocellulosic biomass for bioethanol production. Although rice straw has been extensively utilized for bioethanol production, little or no research has been conducted on the use of lignocellulosic biomass for enzyme immobilization and biodiesel production.

Lipases are the most studied and industrially utilized enzymes that catalyze esterification, transesterification, aminolysis and alcoholysis reactions, and most lipase substrates are insoluble in aqueous media. Therefore, these enzymes require organic solvents to carry out their reactions and can operate more efficiently in a hydrophobic environment. Lipases in free and immobilized forms are widely used in the pharmaceutical, detergent, drug and biodiesel industries because of their wide range of selectivity and specificity, as well as chemoselectivity, regioselectivity and enantioselectivity. Recent studies have reported the synthesis of various industrially important compounds such as alkyl levulinates and glycerol carbonates (Du et al., 2018; Zhou et al., 2018). Immobilized lipase has also been used to develop biosensors that can easily and rapidly detect triglycerides in food samples, juice beverages and serum (D. Sharma, 2011).

The present applicants developed an economical, powerful and simple one-step hydrothermal synthesis method and completed the present invention while studying a method of manufacturing magnetic rice straw for enzyme immobilization in order to reduce synthesis cost and prepare an effective scaffold.

An object of the present invention is to provide a magnetic rice straw for enzyme immobilization and a manufacturing method thereof.

Another object of the present invention is to provide a method for immobilizing an enzyme on magnetic rice straw and an enzyme immobilized on magnetic rice straw.

Another object of the present invention is to provide a method for producing a fatty acid alkyl ester using an enzyme.

In order to achieve the above purpose,

The present invention provides a magnetic rice straw manufacturing method comprising the steps of adding Fe ²⁺ ions to rice straw and subjecting the rice straw to hydrothermal treatment.

In addition, the present invention provides magnetic rice straw for enzyme immobilization, which is manufactured through the above manufacturing method.

Furthermore, the present invention comprises the steps of functionalizing the magnetic rice straw with glutaraldehyde; and mixing the functionalized magnetic rice straw with an enzyme and immobilizing the enzyme.

In addition, the present invention provides an enzyme immobilized on the magnetic rice straw prepared by the above method.

Furthermore, the present invention provides a method for producing a fatty acid alkyl ester comprising the step of performing an esterification reaction by mixing the enzyme immobilized on the magnetic rice straw prepared by the above method with oil and alcohol.

The present invention can produce magnetic rice straw in one-step using a hydrothermal synthesis method, and the magnetic rice straw produced according to the present invention has a structural form advantageous to the reactivity, stability and reusability of the enzyme to be fixed, which The enzyme immobilized on magnetic rice straw using a support showed excellent immobilization efficiency, stability and reusability, and has superparamagnetic potential, so it can be used repeatedly for fatty acid methyl ester production, so it can stably maintain its activity for a long period of time. It can be easily separated from the substrate and has the effect of facilitating regeneration.

1 is a schematic diagram of MRS generation and immobilization of lipase on the surface.
Figure 2 shows the magnetic properties and surface area of the RS support, A) VSM analysis of the magnetic properties of the MRS; and B) a diagram showing surface area analysis for MRS, HT@RS and RS.
Figure 3 shows the biochemical characteristics of free and immobilized lipase, as the initial lipase concentration increases (A) RS and (B) lipase loading (mg g ^-1 ) and activity recovery (%) by MRS (mg ml ^-1 ); It is a diagram showing the effect of (C) temperature and (D) pH on the activity of free lipase and lipase immobilized on MRS.
Figure 4 shows the stability and reusability of MRS-immobilized lipase, A) 4 ℃, B) 25 ℃ and C) relative activity of free lipase and MRS-immobilized lipase at 70 ℃; and D) reusability of RS-immobilized lipase and MRS-immobilized lipase in hydrolysis of p-NPP at 50° C. for 10 repeated cycles under standard assay conditions.
Figure 5 shows the esterification reaction yield and reusability of immobilized lipase, (A) FAME yield, (B) MRS-immobilized lipase and RS-immobilized lipase during FAME production by methanolysis of soybean oil at 45 ° C. It is a diagram showing the repeated use of

Hereinafter, the present invention will be described in detail.

The present invention relates to a one-step hydrothermal synthesis method of magnetic rice straw for enzyme immobilization.

The present invention provides a magnetic rice straw manufacturing method comprising the steps of adding Fe ²⁺ ions to rice straw and subjecting the rice straw to hydrothermal treatment.

In the magnetic rice straw manufacturing method of the present invention, the Fe ²⁺ ions are iron nitrate (Fe(NO ₃ ) ₂ ), iron sulfate (FeSO ₄ ), iron chloride (FeCl ₂ ), iron bromide (FeBr ₂ ) and iron iodide ( FeI ₂ ) may be added through one or more solutions selected from the group consisting of. Preferably, iron nitrate (Fe(NO ₃ ) ₂ ) may be added through a solution.

In the magnetic rice straw manufacturing method of the present invention, before the hot water treatment, the rice straw mixture to which Fe ²⁺ ions are added may be ultrasonically treated and maintained for 20 to 30 hours. The "maintenance" may mean leaving the sonicated rice straw and the Fe ²⁺ ion mixture as it is, allowing the rice straw to be sufficiently soaked with Fe ²⁺ ions.

According to one embodiment of the present invention, the ultrasonic treatment may be performed for 1 to 5 hours, preferably 1 to 3 hours.

According to one embodiment of the present invention, before the hydrothermal treatment, a process of removing loosely bound Fe ²⁺ ions by separating and washing rice straw impregnated with Fe ²⁺ ions from the mixture may be additionally performed.

According to one embodiment of the present invention, the hydrothermal treatment may be performed in an ammonium aqueous solution.

According to one embodiment of the present invention, the hydrothermal treatment may be preferably carried out at 160 to 200 ° C for 3 to 10 hours, more preferably at 170 to 190 ° C for 4 to 6 hours.

According to one embodiment of the present invention, after the step of hydrothermal treatment, the step of filtering the hydrothermal treated rice straw to separate from the reaction solution and drying it; may be further included.

According to one embodiment of the present invention, the dried magnetic rice straw can be used for fixing lipase after heating at 100 °C.

In one embodiment of the present invention, an aqueous Fe(NO ₃ ) ₂ solution was added to rice straw, and after ultrasonic treatment, the rice straw mixture solution to which Fe(NO ₃ ) ₂ was added in suspension was left as it was for 24 hours. Then, rice straw soaked with Fe ²⁺ ions from the solution was separated by filtration, and the separated rice straw was subjected to hydrothermal treatment in an ammonium aqueous solution at 180 ° C. for 5 hours, and then separated and washed to obtain magnetic rice straw.

In addition, the present invention provides a magnetic rice straw for enzyme immobilization prepared by the above method.

In the magnetic rice straw for enzyme immobilization of the present invention, the enzyme may preferably be lipase, but is not limited thereto.

In the magnetic rice straw prepared by the method according to the present invention, iron hydroxide is not only converted into magnetic nanoparticles through the hydrothermal treatment, but also affects biomass hydrolysis, so that hemicellulose and some lignin are removed from the rice straw biomass, and pores are formed on the surface. Therefore, as the surface is increased, leading to the formation of an attachment site for fixing the enzyme, the accessibility of the magnetic rice straw surface area to the enzyme protein may be improved.

Furthermore, the present invention comprises the steps of functionalizing the magnetic rice straw prepared by the above method with glutaraldehyde; and

It provides a method for immobilizing the enzyme on the magnetic rice straw, including mixing the functionalized magnetic rice straw with the enzyme and immobilizing the enzyme.

The enzyme of the present invention may be immobilized on magnetic rice straw through glutaraldehyde cross-linking using magnetic rice straw as a support.

In the method of immobilizing an enzyme on magnetic rice straw of the present invention, the enzyme may be lipase, but is not limited thereto.

In the method of immobilizing an enzyme on magnetic rice straw of the present invention, the magnetic rice straw may be functionally activated by glutaraldehyde. The functionalization may be performed by suspending and reacting magnetic rice straw in a buffer containing glutaraldehyde, and may be performed using a method generally known for glutaraldehyde functionalization. The glutaraldehyde may be contained in 0.1-5% v/v, preferably 1% v/v based on the volume of the buffer, and the buffer may be a phosphate buffer, but is not limited thereto. In addition, when the functionalization of the magnetic rice straw is completed, the functionalized magnetic rice straw may be recovered by centrifugation and washed with a phosphate buffer to remove excess glutaraldehyde.

In the method of immobilizing the enzyme on the magnetic rice straw of the present invention, the step of immobilizing the enzyme may be performed at 1 to 10 ° C for 1 to 10 hours, preferably at 3 to 5 ° C for 3 to 6 hours may be performed.

In the method of immobilizing the enzyme on the magnetic rice straw of the present invention, the step of immobilizing the enzyme may be performed at pH 5.0 to 11.0, preferably at pH 7.0 to 10.0. More preferably, it may be carried out at pH 7.5 to 9.5, and the immobilization efficiency at a pH in the above range may be better than other pH conditions. More preferably, it may be pH 8.0 to 9.0.

In the method of immobilizing the enzyme on the magnetic rice straw of the present invention, the step of immobilizing the enzyme may be performed in a buffer solution. The buffer solution is not particularly limited, and a Tris-HCL buffer solution was used in one embodiment of the present invention.

According to one embodiment of the present invention, the enzyme may be mixed at 0.5 to 1.5% by weight (w / v) based on the volume of the buffer solution, preferably 1.8 to 1.2% by weight (w / v). It may, but is not limited thereto.

In addition, the present invention provides an enzyme immobilized on the magnetic rice straw prepared by the method of immobilizing the enzyme on the magnetic rice straw.

In the enzyme immobilized on the magnetic rice straw of the present invention, the enzyme may be lipase, but is not limited thereto.

According to one embodiment of the present invention, the optimum enzyme activity temperature of the enzyme immobilized on the magnetic rice straw may be 45 to 75 ° C. In a temperature condition lower or higher than the above range, the activity of the enzyme immobilized on the magnetic rice straw may be reduced. More preferably, it may be 46 to 65 ° C., and enzyme activity may be exhibited better than other temperature conditions at a temperature in the above range, and the case of 48 to 53 ° C. is most preferable.

In one embodiment of the present invention, as a result of evaluating the relative enzyme activity of lipase immobilized on magnetic rice straw using p-NPP as a substrate, it was confirmed that the enzyme activity was reduced to 80% or less under conditions outside the temperature range of 45 to 75 ° C. It was confirmed that 90% or more of the lipase activity was exhibited at 46 to 65 ° C, and it was confirmed that the range of 48 to 53 ° C, in particular, 50 ° C was the optimal condition for the best enzyme activity (see Experimental Example 3).

According to one embodiment of the present invention, the optimal enzyme activity pH of the enzyme immobilized on the magnetic rice straw may be 7.0 to 10.0. In pH conditions lower or higher than the above range, the activity of the enzyme immobilized on the magnetic rice straw may be reduced. More preferably, it may be pH 7.5 to 9.5, and the enzyme activity may be shown to be superior to other pH conditions at a pH in the above range, and the case of pH 8.3 to 8.8 is most preferable.

In one embodiment of the present invention, as a result of evaluating the relative enzyme activity of lipase immobilized on magnetic rice straw using p-NPP as a substrate, it was confirmed that the enzyme activity was reduced to 80% or less under conditions outside the pH range of 7.0 to 10.0. , It was confirmed that more than 90% of lipase activity was exhibited at pH 7.5 to 9.5, and it was confirmed that the range of pH 8.5-9.0, particularly pH 8.5, is the optimal condition for the best enzyme activity (see Experimental Example 3).

The enzyme immobilized on the magnetic rice straw of the present invention may be reusable repeatedly more than 10 times.

In one embodiment of the present invention, the lipase enzyme immobilized on magnetic rice straw by the method according to the present invention has higher immobilization efficiency, stability and/or reusability than lipase immobilized by other supports or immobilization methods or non-immobilized lipase (free cells). It was confirmed that it was excellent (see Example 4).

Furthermore, the present invention is a method for producing a fatty acid alkyl ester comprising the step of esterifying the enzyme immobilized on the magnetic rice straw prepared by immobilizing the enzyme on the magnetic rice straw with oil and alcohol provides

In the method for producing the fatty acid alkyl ester of the present invention, the oil may be characterized in that it contains vegetable oil, animal fat or waste oil. For example, the oil may be soybean oil, olive oil, palm oil, sunflower oil, fish oil, waste cooking oil, etc., preferably soybean oil, but is not limited thereto.

In the method for producing the fatty acid alkyl ester of the present invention, the alcohol may be selected from the group consisting of methanol, ethanol, propanol, n-butanol, iso-butanol, tert-butanol, and combinations thereof. It may be preferably methanol or ethanol, more preferably methanol.

In the method for producing the fatty acid alkyl ester of the present invention, the oil and alcohol may be mixed in a molar ratio of 1:3 to 1:10, preferably in a molar ratio of 1:5 to 1:7, , but is not limited thereto.

In the method for producing a fatty acid alkyl ester of the present invention, the enzyme may be mixed in an amount of 1 to 20% by weight based on the weight of alcohol, but is not limited thereto.

In the method for producing the fatty acid alkyl ester of the present invention, the fatty acid is oleic acid, linoleic acid, palmitic acid, stearic acid, pinolenic acid, It may be at least one selected from the group consisting of myristic acid, caprylic acid, and capric acid, but is not limited thereto.

In the method for producing a fatty acid alkyl ester of the present invention, the fatty acid alkyl ester may preferably be a fatty acid methyl ester, but is not limited thereto.

In the method for producing a fatty acid alkyl ester of the present invention, the esterification reaction may be carried out at 1 to 80 ° C, preferably at a temperature of 45 to 75 ° C. When the esterification reaction is performed under a temperature condition lower than or higher than the above range, the enzyme activity may be reduced and thus the yield of fatty acid alkyl ester may be lowered. More preferably, it may be carried out at 46 to 65 ° C., and at a temperature in the above range, enzyme activity and fatty alkyl ester yield may be better than other temperature conditions. More preferably, it may be performed at 48 to 55 ° C, and most preferably at 48 to 53 ° C.

In the method for producing the fatty acid alkyl ester of the present invention, the esterification reaction may be performed at pH 5.0 to 11.0, preferably pH 7.0 to 10.0. When the esterification reaction is performed under pH conditions lower than or higher than the above range, the enzyme activity may be reduced, resulting in a decrease in yield of fatty acid alkyl esters. More preferably, it may be carried out at pH 7.5 to 9.5, and at a pH in the above range, enzyme activity and yield of fatty alkyl esters may be superior to other pH conditions. More preferably, it may be performed at pH 8.5 to 9.0, and most preferably performed at pH 8.3 to 8.8.

In one embodiment of the present invention, using the lipase immobilized on magnetic rice straw according to the present invention, soybean oil was added as a substrate to methanol: oil (6: 1) and water (10% w / w) in a mixture of ester for 48 hours As a result of the reaction, it was confirmed that the yield of fatty acid methyl ester (FAME) was superior to that of lipase immobilized in general natural rice straw, and the reusability was remarkably maintained (see Example 5).

In the method for producing fatty acid alkyl esters of the present invention, the enzyme immobilized on the magnetic rice straw may be reusable for production of fatty acid alkyl esters, and preferably may be repeatedly reused 10 or more times.

Hereinafter, the present invention will be described in detail as an embodiment of the present invention with reference to the accompanying drawings. However, the following embodiments are presented as examples of the present invention, and if it is determined that detailed descriptions of well-known techniques or configurations may unnecessarily obscure the gist of the present invention, the detailed descriptions may be omitted. , the present invention is not limited thereby. Various modifications and applications of the present invention are possible within the scope of the claims described below and equivalents interpreted therefrom.

In addition, the terms used in this specification (terminology) are terms used to appropriately express preferred embodiments of the present invention, which may vary according to the intention of a user or operator or customs in the field to which the present invention belongs. Therefore, definitions of these terms will have to be made based on the content throughout this specification. Throughout the specification, when a certain component is said to "include", it means that it may further include other components without excluding other components unless otherwise stated.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing the present invention, the preferred materials and methods are described herein.

All technical terms used in the present invention, unless defined otherwise, are used with the same meaning as commonly understood by one of ordinary skill in the art related to the present invention. In addition, although preferred methods or samples are described in this specification, those similar or equivalent thereto are also included in the scope of the present invention. The contents of all publications incorporated herein by reference are incorporated herein by reference.

Preparation Example 1. Experiment preparation

All chemicals were of analytical grade (AG) and used without further purification.

Lipase (21.6 mg mL ^-1 ) from Thermomyces lanuginosus was purchased as Lipozyme ^® TL IM liquid formulation (Novozymes, Bagsværd, Denmark), gum acacia, n-hexane ( n -hexane), 2-propanol, p -nitrophenol, and p - nitrophenol palmitate were purchased from Sigma-Aldrich (St. Louis, USA). did Bradford reagents and protein assay kits were purchased from Bio-Rad (Hercules, USA).

Example 1. Preparation of magnetic rice straw and immobilization of enzyme

1-1. Synthesis of magnetic rice straw (MRS)

Natural rice straw (RS) (1 g) was washed with double distilled water (DW) for 2 hours. 0.25 M aqueous Fe(NO ₃ ) ₂ was added to the washed RS and the mixture was sonicated for 2 hours. After sonication, the suspension was allowed to stand for 24 hours. Subsequently, the rice straw was separated from the Fe(NO ₃ ) ₂ solution by filtering the suspension, and washed twice more with DW to remove loosely bound Fe ²⁺ ions. Rice straw soaked with Fe ²⁺ ions was immersed in an ammonium aqueous solution of pH 11, transferred to a 100 mL-Teflon reactor, and subjected to hydrothermal treatment at 180° C. for 5 hours. After the reaction, the hydrothermal treated rice straw was separated from the reaction solution by filtration, washed three times with DW and ethanol, and dried to obtain magnetic rice straw (MRS). The dried MRS was heated at 100° C. for 2 hours and used for lipase immobilization. MRS generation is schematically shown in FIG. 1 .

As shown in Fig. 1, the hydrothermal pretreatment not only converted iron hydroxide into magnetic nanoparticles but also affected biomass hydrolysis to remove hemicellulose and some lignin from RS biomass. Heating under pressure improved the accessibility of the surface area for enzyme attachment.

1-2. Preparation of rice paddy immobilized enzyme

For immobilization of the lipase enzyme, 100 mg of RS and MRS (Example 1-1) were functionalized with an optimized concentration of glutaraldehyde. Rice straw (RS) functionalized with glutaraldehyde and magnetic rice straw (MRS) were labeled as GRS and GMRS, respectively.

RS or GMRS (10 mg) was mixed with lipase (1 mg protein) in 1 mL of Tris-HCL buffer solution (50 mM, pH 8.5) and incubated for 4 hours at 4°C with agitation at 60 rpm. After the enzyme was adsorbed on the rice straw, the particles were centrifuged at 10,000 rpm for 15 minutes at 4°C. The protein concentration of the washed solution was determined by the Bradford method. Lipase immobilization is schematically shown in FIG. 1 .

Example 2. Confirmation of characteristics of magnetic rice straw and enzymes immobilized therein

2-1. Identification of magnetic properties of magnetic rice straw

The magnetic properties of the MRS of Example 1-1 were confirmed using vibrating sample magnetometry (VSM), which is shown in FIG. 2A.

As shown in FIG. 2A, it was confirmed that the modified RS exhibited superparamagnetic behavior, and the magnetic saturation value of 27.32 emu g ^-1 showed magnetic properties of the MRS. A typical magnetic hysteresis curve was obtained with zero coercive force and residual value, indicating the superparamagnetic properties of the prepared MRS. The inserted image in Fig. 2A shows photographic images of the MRS with and without an external permanent magnet, indicating that there are magnetic properties through the movement of the MRS toward the magnet. Since reusability of the immobilized enzyme is an essential requirement for economical treatment and the magnetic properties of the particles facilitate recovery, the reusability of the enzyme immobilized on the MRS was confirmed through the above results.

In addition, in order to confirm the effect of magnetic nanoparticle modification on RS, natural rice straw (MRS) and hydrothermally treated rice straw ( HT@RS) and magnetic rice straw (MRS) were confirmed for their surface area and pore size, which are shown in Table 1 and FIG. 2B below. In this case, the HT@RS means hydrothermal-treated rice straw that is not mixed with Fe(NO ₃ ) ₂ but only hydrothermal-treated.

matter BET surface area (m ² g ^-1 ) Pore volume (cm ³ g ^-1 ) Surface pore diameter (nm) MRS 103.2 0.197 21.65 HT@RS 46.72 0.171 11.03 RS 23.23 0.121 2.11

As shown in Table 1 and Fig. 2B, RS exhibited a pore size of 2.11 nm and a surface area of 23.23 m ² g ^-1 , and HT@RS without magnetic nanoparticle modification showed a pore size of 46.72 m ² with a pore diameter of 11.03 nm. The surface area of g ^-1 is shown. In MRS, the surface area increased from 23.23 m ² g ^-1 of RS to 103.2 m ² g ^-1 through magnetostriction, and the pore diameter increased from 2.11 nm to 21.65 nm.

Since high temperature and high alkalinity conditions dissolve the lignin, cellulose and hemicellulose backbone of RS to create pores on the surface, it is believed that the modified magnetic nanoparticles of the present invention increase the surface of RS to form attachment sites for enzyme fixation on MRS. do.

2-2. Confirmation of biochemical characteristics of lipase enzyme immobilized on magnetic rice straw

In order to confirm the characteristics of lipase immobilized on rice straw (RS) and magnetic rice straw (MRS) in Example 1-2, enzyme loading capacity and immobilization efficiency by RS and MRS were evaluated And the results are shown in Figures 3A and 3B.

As shown in Figures 3A and 3B, when considering initial lipase concentrations of 0.5, 0.75, 1.0, 1.25, and 1.5 mg mL ^-1 mixed with 1 mL of buffer solution for reaction with the support, the maximum enzyme loading of RS is the support (RS), respectively. ) were 15.3, 26.2, 41.6, 49.3 and 55.1 mg per g (15.3, 26.2, 41.6, 49.3, and 55.1 mg g ^-1 of support), and MRS was 0.5, 0.75, 1.0, 1.25 and 1.5 mg mL ^-1 of initial Enzyme loading capacity of 26.4, 42.2, 91.3, 105 and 110 mg (26.4, 42.2, 91.3, 105, and 110 mg g ^-1 of support) per g of support (MRS) was shown for lipase concentration, respectively. Here, it can be seen that the interaction between native RS and enzyme is weak compared to that between MRS and enzyme, because the presence of lignin imparts hydrophobicity to RS and inhibits its active interaction with soluble proteins.

The hydrothermal treatment used for the synthesis of magnetic nanoparticles under alkaline conditions dissolved some of the lignin and RS, exposing the hydrophilic components (i.e., cellulose and hemicellulose) as well as increasing the MRS surface area and allowing strong interactions with soluble lipases. . This result indicates a higher protein load in MRS compared to native RS under optimal conditions. For both materials, maximum loading was observed at an enzyme concentration of 1 mg mL ⁻¹ , with RS and MRS representing approximately 41% and 90% of the protein loading, respectively. As the enzyme concentration was further increased to 1.5 mg mL-1, the protein loading decreased to 36% for RS and 73% for MRS.

In addition, the effects of temperature and pH on the activity of free lipase and lipase immobilized on MRS were confirmed. The activities of free lipase and lipase immobilized on magnetic rice straw (MRS) prepared in 1-2 above were tested at different pH values (50 mM, pH 7.0- 11.0) and the absorbance at 410 nm at a temperature in the range of 30 to 80° C. was measured with a spectrophotometer and is shown in FIGS. 3C and 3D. At this time, the optimal pH of the buffer was determined using Phosphate-citrate buffer (pH 5.0 to 8.0) and Tris-HCl buffer (pH 8.5 to 10.5). Enzyme activity is expressed in international units (IU), where one IU (1 unit) is the enzyme involved in producing 1 μmol of p-nitrophenol (pNP) in 1 minute from the substrate p-NPP per minute under standard assay conditions. It was defined as the amount of

As shown in Fig. 3C, the optimal temperature was 50 °C under standard conditions, and lipase immobilized on MRS required the same optimal temperature. The enzyme immobilized on the MRS exhibited higher thermal stability than the free enzyme while retaining more than 70% of the initial activity at 80 °C. The high-temperature resistance of lipase immobilized on MRS may be due to the change in surface structure produced after hydrothermal treatment of MRS, which prevents lipase from being inactivated at high temperature.

As shown in Figure 3D, the optimal pH of lipase immobilized in MRS for p-NPP at 50 °C was 8.5. MRS-immobilized lipase (Lipase@MRS) showed higher stability than RS-immobilized lipase under more alkaline conditions.

Example 3. Confirmation of stability and reusability of lipase immobilized on magnetic rice straw

Enzyme stability was investigated by incubating free lipase and MRS-immobilized lipase at various temperatures (25, 50 and 70° C.) under standard assay conditions. Samples were collected regularly to determine residual activity. The storage stability of lipase was evaluated at 4°C, 25°C and 70°C for 15 days, and the results are shown in FIG. 4 .

As shown in Fig. 4A, after 30 days, the relative activities of MRS-immobilized lipase and free lipase were 81% and 42%, respectively, at 4°C. indicates that the

As shown in Figure 4B, as a result of studying the stability of free lipase and MRS-immobilized lipase at 25 ° C for 15 days, MRS-immobilized lipase showed much higher stability than free lipase, and at 15 days of storage 85% of the initial activity was retained. Because oil hydrolysis is performed at high temperatures in biodiesel production, the thermal stability of immobilized lipase is essential.

As shown in Fig. 4C, lipase immobilized on MRS at 70 °C retained 53% of its initial activity after 210 min, whereas free lipase completely lost activity after 150 min. The high stability of the lipase immobilized on MRS may be due to the fibrous nature of the scaffold and the strong interaction between the scaffold and enzyme allowing for tight binding, reduced leaching and protection from harsh environmental conditions. Therefore, stability studies performed at various temperatures can support the potential use of MRS-immobilized lipases in various industrial processes.

In addition, one of the most important advantages of immobilized biocatalysts is their reusability, free enzymes are not reusable. The reusability of the immobilized lipase was evaluated by p-NPP oxidation assay under standard conditions using 10 repeated cycles, and the results are shown in Table 1 and FIG. 4D.

As shown in Figure 4D, the lipase immobilized on MRS retained 81% of its activity, whereas the lipase immobilized on RS showed 45% of its original activity. MRS-immobilized lipase is much more stable than RS-immobilized lipase and can be reused efficiently over repeated cycles.

Through the above experiments, it was demonstrated that the interaction of RS with lipase without self-transformation was very weak due to the presence of lignin in the cellulosic material, which hindered the strong interaction because lignin increased RS hydrophobicity.

Example 4. Comparison of Lipase Immobilization Characteristics of Various Supports and Magnetic Rice Straw

Enzyme loading and immobilization efficiency for immobilization of lipase according to the immobilization method and the use of various supports were compared with those described in the existing literature, and the results are shown in Table 2.

support material Enzyme loading (mg g ^-1 ) Immobilization efficiency (%) immobilization method Reuse rate after 10 times (%) References Chitosan/Poly(vinyl alcohol) (PVA) 63.6 49.8 Covalent bonding 46 (Huang et al., 2007) Poly(acrylonitrile-co-maleic acid) ~20 49.7 Glutaraldehyde cross-linking 55 (Ye et al., 2006) CNC@Bacterial Cellulose (BC) 7.8 35 EDC-NHS cross-linking - (Kim et al., 2015) SiO ₂ @Fe ₃ O ₄ nanoflower 93 65 Glutaraldehyde cross-linking 40 (Gao et al., 2017) Chitosan/Nanocellulose 81 76.3 Schiff's base 44 (Elias et al., 2018) Rice straw (RS) 41.6 85.3 Glutaraldehyde cross-linking 45 the present invention Magnetic Rice Straw (MRS) 91.3 94.3 Glutaraldehyde cross-linking 81 the present invention

As shown in Table 2, it can be seen that the present invention provides a solid structure of a support material for immobilizing lipase with high enzyme loading capacity and immobilization efficiency. In addition, it was confirmed that the reusability rate was significantly better when using the magnetic rice straw of the present invention than when using other supports.

On the other hand, the amine-functionalized multi-walled carbon nanotube (CNT) binding lipase in the previous literature maintained 55% of its original activity after 10 repeated uses (Verma et al., 2013), and bound to egg white after 7 repeated uses. Lipase was maintained at 45% (Karimpil et al., 2011). T. lanuginosus lipase immobilized on the Fe ₃ O ₄ immobilized chitosan magnetic particle complex showed 70% of the initial activity after being reused 10 times (Wang et al., 2015). The present invention exhibits better performance in reusability when compared to previously presented techniques.

Example 5. Esterification reaction yield and reusability of immobilized lipase

Biodiesel is being considered as an alternative fuel to fossil fuels for use in industry and vehicles. Therefore, in order to evaluate the possibility of using rice straw immobilized lipase in biodiesel production, esterification was performed in a mixture of methanol:oil (6:1) and water (10% w/w) for 48 hours.

Specifically, for the esterification reaction, 100 mg of lipase immobilized on rice straw (RS or MRS) and 0.5 g of soybean oil (10 wt% of oil) were added to a mixture of water and methanol at various concentrations at 45 °C. Methanol was added at three intervals: the initial stage (starting point) of the esterification reaction, 12 hours and 24 hours, so that the final concentration was 150 μM. Samples (reactants) were collected periodically for 48 hours and mixed with n-hexane. The sample was treated in a high-speed vacuum concentrator at 85 °C, 2000 rpm and -0.1 MPa to remove methanol and then washed with water. The washed samples were mixed in a rotary shaker, and 1 μL of each sample was used for GC analysis to measure the yield (% FAME) of fatty acid methyl ester (FAME), and the results are shown in FIG. 5A .

The reusability of MRS-immobilized lipase (lipase@MRS) and RS-immobilized lipase (lipase@RS) was confirmed in Example 2 above, under the optimized conditions (temperature and pH), in the esterification reaction using soybean oil. It was irradiated with 10 repeated cycles. All data were averaged over three experiments, and the results are shown in Figure 5B.

As shown in Figures 5A and 5B, lipase immobilized on RS and lipase immobilized on MRS showed 38% and 80% of the initial % FAME yields, respectively, after 10 repetitions. These results indicate that the transformation of RS into magnetic nanoparticles MRS provides a wider surface for lipase attachment, thereby reducing lipase leaching during esterification, and therefore, lipase immobilized on MRS remains intact even after repeated use of 10 cycles of esterification. It can be seen that reusability is maintained. The low yield in repeated use of lipase immobilized on RS was due to the weak interaction between lipase and RS.

As described above, through the experiment, it was confirmed that the magnetic rice straw immobilized with the enzyme showed excellent immobilization efficiency, showed stability and reusability, and could be used repeatedly for fatty acid methyl ester production, furthermore, industrial-scale biodiesel It was confirmed that there is a possibility of using it for production.

Claims (20)

Fe ²⁺ ions were added to rice straw through an iron nitrate (Fe(NO ₃ ) ₂ ) solution, and the rice straw mixture to which the Fe ²⁺ ions were added was sonicated and maintained for 20 to 30 hours, followed by hot water A method for manufacturing magnetic rice straw for immobilizing lipase, comprising the step of hydrothermal treatment. delete delete According to claim 1,
Characterized in that the hydrothermal treatment is carried out at 170 to 190 ℃ for 4 to 6 hours, magnetic rice straw manufacturing method. Magnetic rice straw for immobilizing lipase, prepared by the method of claim 1. delete Functionalizing the magnetic rice straw prepared by the method of claim 1 with glutaraldehyde; and
A method for immobilizing lipase on magnetic rice straw, comprising: mixing the functionalized magnetic rice straw with lipase and immobilizing the lipase. delete According to claim 7,
The method of immobilizing lipase on magnetic rice straw, characterized in that the step of immobilizing the lipase is performed at 3 to 5 ° C. for 3 to 6 hours. According to claim 7,
The method of immobilizing lipase on magnetic rice straw, characterized in that the step of immobilizing the lipase is performed at pH 7.0 to 10.0. Lipase immobilized on magnetic rice straw prepared by the method of claim 7. According to claim 11,
Lipase immobilized on magnetic rice straw, characterized in that the optimum enzyme activity temperature of the lipase immobilized on the magnetic rice straw is 45 to 70 ° C. According to claim 11,
Lipase immobilized on magnetic rice straw, characterized in that the optimal enzyme activity pH of the lipase immobilized on the magnetic rice straw is 7.0 to 10.0. Production of fatty acid alkyl esters comprising the step of esterifying lipase immobilized on magnetic rice straw prepared by the method of claim 7 with soybean oil and methanol at a temperature of 40 to 80 ° C. and pH 7.0 to 10.0 How to. delete delete According to claim 14,
The fatty acids include oleic acid, linoleic acid, palmitic acid, stearic acid, pinolenic acid, myristic acid, and caprylic acid. ) And capric acid (capric acid) characterized in that at least one selected from the group consisting of, a method for producing a fatty acid alkyl ester. According to claim 14,
The method for producing a fatty acid alkyl ester, characterized in that the esterification reaction proceeds at a temperature of 48 to 53 ℃. According to claim 14,
The esterification reaction is a method for producing a fatty acid alkyl ester, characterized in that proceeding at pH 8.3 to 8.8. According to claim 14,
The method for producing fatty acid alkyl esters, characterized in that the lipase immobilized on the magnetic rice straw can be reused for fatty acid alkyl ester production.

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