An enzyme solidified carrier and its use

Abstract

The application discloses an enzyme solidification carrier and application thereof. The enzyme solidification carrier is prepared from a chitosan and coffee residue compound through the following steps: (1) mixing the chitosan and coffee residue compound with an acid to obtain solution I; (2) mixing solution I with an alkali and reacting to obtain a precursor; and (3) sequentially immersing the precursor in a polyethylene imine solution and a glutaraldehyde solution to obtain the enzyme solidification carrier. The chitosan has the characteristics of antibiosis and biodegradation, and due to the simultaneous presence of the coffee waste, the active groups increase and become stronger.

Description

Technical Field

[0001] This application relates to an enzyme immobilization carrier and its application, belonging to the field of biotechnology. Background Technology

[0002] Agricultural waste, primarily composed of lignocellulosic materials, is a sustainable and recyclable source of bioenergy and chemicals. Converting these raw materials into biofuels and fossil fuel alternatives is crucial.

[0003] Enzymatic catalysis is increasingly favored in biomass refining technologies due to its sustainability and environmental friendliness. Cellulase is a multi-enzyme system that hydrolyzes cellulose into soluble oligosaccharides, such as cellobiose and cellotriose, as well as glucose, by hydrolyzing β-bonds. This environmentally friendly method eliminates the need for toxic reagents and byproducts. The catalytic conversion of biomass produces byproducts such as sugars, ethanol, and chemicals that are essential for fuel production. Enzymes are recognized for their effectiveness and environmental safety and can be used in a variety of processes, typically immobilized on polymer materials. Enzymes can be immobilized on insoluble supports, improving their reusability and allowing for continuous operation. When properly designed, this method can withstand harsh environmental conditions, including high temperatures and extreme pH levels. The process begins with attaching the enzyme to the NH2 and C=O functional groups on the polymer surface. Enzyme immobilization is a common application for polymers, including synthetic and natural types such as alginate, carrageenan, and chitosan. Chitosan, derived from the exoskeleton of mollusks, is antibacterial, non-toxic, and biodegradable, although it may lack mechanical strength. Coffee waste was chosen for its numerous beneficial properties, including improved mechanical properties and enzyme preservation. Coffee waste offers a more efficient solution for enzyme fixation.

[0004] Coffee waste contains water, cellulose, lignin, hemicellulose, fat, ash, protein, and fatty acids, and its massive volume poses a significant threat to the environment. Globally, 18 million tons of coffee waste are generated annually, most of which end up in landfills, releasing the potent greenhouse gas methane during decomposition. Recycling programs are imperative, diverting organic waste such as coffee waste from landfills to commercial uses. This strategy reduces greenhouse gas emissions and supports a closed-loop circular economy focused on resource utilization and minimal waste.

[0005] Coffee waste is combined with chitosan to improve its mechanical properties and facilitate waste treatment. This method immobilizes cellulase and breaks down cellulose into its basic components. This energy-efficient and environmentally friendly method is characterized by its mild reaction and simple process, making it suitable for various applications. Summary of the Invention

[0006] The present invention aims to produce a new composite material from chitosan and coffee grounds, and to utilize enzyme immobilization technology to decompose cellulose materials present in environmental and agricultural waste.

[0007] According to one aspect of this application, an enzyme immobilization carrier is provided, characterized in that...

[0008] Using chitosan and coffee grounds complex as raw materials, it is obtained through the following steps:

[0009] (1) Mix the chitosan and coffee grounds complex with acid to obtain solution I;

[0010] (2) Mix solution I with the base and react to obtain the precursor;

[0011] (3) The precursor is sequentially immersed in polyethyleneimine solution and glutaraldehyde solution to obtain the enzyme-immobilized carrier.

[0012] The mass ratio of the chitosan and coffee grounds complex is 1:1 to 5:1;

[0013] The acid is selected from at least one of formic acid, acetic acid, citric acid, oxalic acid, p-toluenesulfonic acid, and citric acid.

[0014] The concentration of the acid is 1–3 wt%.

[0015] The alkaline solution is a sodium hydroxide solution;

[0016] The concentration of the alkali is 3-10 wt%;

[0017] The reaction time is 2 to 5 hours;

[0018] The reaction temperature is 25–40 degrees Celsius;

[0019] The concentration of the polyethyleneimine solution is 1–5 wt%.

[0020] The pH of the polyethyleneimine solution is 9–11;

[0021] The immersion time in the polyethyleneimine solution is 1 to 5 hours;

[0022] The concentration of the glutaraldehyde solution is 0.5–4.5 wt%.

[0023] The soaking time in glutaraldehyde solution is 1 to 5 hours.

[0024] Specifically, it includes the following steps:

[0025] a) Dissolve the chitosan-coffee waste complex in a 1% acetic acid solution at a mass ratio of 3:1 and eliminate bubbles.

[0026] b) The mixture of chitosan treated in a) and coffee waste is dripped into a 5% sodium hydroxide solution and left for 3 hours until it is completely hardened to form carrier I;

[0027] c) Immerse the carrier I in a 2% polyethyleneimine solution with a pH of 9.5 for 3 hours; after the reaction is complete, wash with deionized water.

[0028] d) The carrier I treated in c) was soaked in 1.5% glutaraldehyde for 1 hour to obtain the final product; after the reaction was completed, it was washed with deionized water.

[0029] The processing conditions for step a are as follows: coffee waste is thoroughly boiled to remove impurities. The coffee grounds are then dried at room temperature and the coffee waste is ground into small, uniform particles. 3% chitosan is dissolved in a 1% acetic acid solution and stirred until completely dissolved. Then, coffee waste is added to the solution at a concentration of 1%, resulting in a chitosan to coffee waste mass ratio of 3:1.

[0030] The treatment conditions described in b are as follows: using a 20 ml syringe, the mixture of chitosan and coffee waste is dripped from a height of 20 cm into a 5% sodium hydroxide solution and left for 3 hours until it is completely hardened to form carrier beads.

[0031] According to another aspect of this application, an enzyme immobilization method is provided, which uses the above-described enzyme immobilization carrier.

[0032] The beneficial effects that this application can produce include:

[0033] (1) This invention utilizes two types of environmental waste, chitosan and coffee waste, to prepare a novel composite (chitosan / coffee waste composite), which can be used as a carrier for enzyme immobilization technology.

[0034] (2) This invention produces a novel carrier based on chitosan and coffee waste, which combines the advantages of both components. Chitosan has antibacterial and biodegradable properties, and due to the co-existence of coffee waste, the active groups increase and become stronger.

[0035] (3) The novel carrier developed in this invention has the characteristics of uniform size distribution, good mechanical and thermal properties, and high curing efficiency. It has a wide range of applications and provides significant added value. Attached Figure Description

[0036] Figure 1 Photos of chitosan beads and chitosan / coffee waste beads.

[0037] Figure 2 This describes the reaction process of chitosan / coffee waste beads with polyethyleneimine (PEI).

[0038] Figure 3 This is the process of activating amination-treated chitosan / coffee waste beads by reacting with glutaraldehyde (GA).

[0039] Figure 4 This refers to the immobilization process of cellulase on the surface of activated chitosan / coffee waste beads, i.e., the immobilization step.

[0040] Figure 5 A bar chart showing the reusability of chitosan / coffee waste beads.

[0041] Figure 6 FT-IR spectra of coffee waste (A), chitosan (B), chitosan / coffee waste beads (C), amination beads (D), activated beads (E), and immobilized enzyme beads (F). Detailed Implementation

[0042] The present invention will now be described in more detail with reference to specific embodiments and preferred embodiments: Embodiment 1

[0043] a) Dissolve the chitosan-coffee waste complex in a 1% acetic acid solution at a mass ratio of 3:1 and eliminate bubbles.

[0044] b) The mixture of chitosan treated in a) and coffee waste is dripped into a 5% sodium hydroxide solution and left for 3 hours until it is completely hardened to form carrier I;

[0045] c) Immerse the carrier I in a 2% polyethyleneimine solution with a pH of 9.5 for 3 hours;

[0046] d) The carrier I treated in c) was soaked in 1.5% glutaraldehyde for 1 hour to obtain the final product;

[0047] Infrared spectroscopy confirmed the presence of active groups on the surface of the gel beads. The spectrum of coffee waste showed characteristic bands of lignocellulose materials, whose main components include hemicellulose, cellulose, lignin, and other small molecules, such as... Figure 6 As shown in Figure A. 3341cm -1 The broad bands at 1009 cm⁻¹ are associated with stretching vibrations of hydroxyl bonds, while the bands between 3009 and 2853 cm⁻¹ correspond to stretching vibrations of aliphatic C-H bonds. The bands at 1741, 1644, and 1532 cm⁻¹ are associated with carbonyl CO stretching vibrations in hemicellulose and chlorogenic acid, and CN stretching vibrations in caffeine. The bands at 1455 and 1375 cm⁻¹ are associated with CH₂ and CH₃ bending modes, while the bands between 1243 and 1150 cm⁻¹ originate from COC bonds in lignin, chlorogenic acid, and caffeine. Finally, the broad band between 1056 and 1029 cm⁻¹ is associated with typical COH bonds in polysaccharides.

[0048] In contrast, chitosan-related Figure 6B shows a strong absorption band in the 3000–3461 cm⁻¹ region, corresponding to the stretching of NH and OH groups and the presence of intramolecular hydrogen bonds. The absorption band near 2921 cm⁻¹ can be attributed to CH stretching. Bands at approximately 1645 cm⁻¹ (corresponding to C=O stretching of amide I) and 1325 cm⁻¹ (corresponding to CN stretching of amide III) confirm the presence of residual N-acetyl groups. The band at 1589 cm⁻¹ corresponds to the NH bending of a primary amine, while the signal at 896 cm⁻¹ corresponds to the CH bending outside the monosaccharide ring plane.

[0049] When adding coffee waste ( Figure 6 C) Most characteristic bands of chitosan and coffee waste can be observed. Specifically, the amine and hydroxyl bands in the 3000–3666 cm⁻¹ range and the band at 2925 cm⁻¹ represent symmetric or asymmetric CH stretching vibrations of fatty acids. The absorption peak at 1745 cm⁻¹ is attributed to the carbonyl bond in xanthine derivatives (e.g., caffeine). These important bands in the spectrum suggest that functional groups on the surface of chitosan / coffee waste beads may be involved in the reaction.

[0050] When chitosan / coffee waste beads react with polyethyleneimine, the characteristic spectral band in the wavelength range of 3000–3770 cm⁻¹ broadens. This is related to the addition of amine groups (NH₂) to the bead surface. Figure 6 As shown in D. In Figure 6 In E, the characteristic amine group is visible in the range of 3000–3744 cm⁻¹, and a new band appears at 1600 cm⁻¹, which is related to the carbonyl group in the surface glutaraldehyde. When the aldehyde-activated beads interact with the enzyme, the amine range (3000–3755 cm⁻¹) becomes wider due to the large number of amine groups in the enzyme, such as… Figure 6 As shown in F.

[0051] The above description is merely a few embodiments of this application and is not intended to limit this application in any way. Although this application discloses preferred embodiments as described above, it is not intended to limit this application. Any changes or modifications made by those skilled in the art without departing from the scope of the technical solution of this application using the disclosed technical content are equivalent to equivalent implementation cases and fall within the scope of the technical solution.

Claims

1. An enzyme immobilization carrier, characterized in that, Using chitosan and coffee grounds complex as raw materials, it is obtained through the following steps: (1) Mix the chitosan and coffee grounds complex with acid to obtain solution I; (2) Mix solution I with the base and react to obtain the precursor; (3) The precursor is sequentially immersed in polyethyleneimine solution and glutaraldehyde solution to obtain the enzyme-immobilized carrier.

2. The enzyme immobilization carrier according to claim 1, characterized in that, The mass ratio of the chitosan and coffee grounds complex is 1:1 to 5:1; The acid is selected from at least one of formic acid, acetic acid, citric acid, oxalic acid, p-toluenesulfonic acid, and citric acid. The concentration of the acid is 1–3 wt%.

3. The enzyme immobilization carrier according to claim 1, characterized in that, The alkaline solution is a sodium hydroxide solution; The concentration of the alkali is 3-10 wt%. The reaction time is 2 to 5 hours; The reaction temperature is 25–40 degrees Celsius.

4. The enzyme immobilization carrier according to claim 1, characterized in that, The concentration of the polyethyleneimine solution is 1–5 wt%. The pH of the polyethyleneimine solution is 9–11; The immersion time in the polyethyleneimine solution is 1 to 5 hours.

5. The enzyme immobilization carrier according to claim 1, characterized in that, The concentration of the glutaraldehyde solution is 0.5% to 4.5%. The immersion time in glutaraldehyde solution is 1 to 5 hours.

6. A method for enzyme curing, characterized in that, The enzyme-immobilized carrier described in any one of claims 1 to 5 is used.

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