阅读说明：本技术 一种基于疏水修饰明胶微球的脂肪酶界面固定化的方法 (Lipase interface immobilization method based on hydrophobic modified gelatin microspheres ) 是由 阮奇珺 付强 李圣男 林晨 王李平 于 2020-06-10 设计创作，主要内容包括：本发明公开了一种基于疏水修饰明胶微球的脂肪酶界面固定化的方法,采用乳液模板法制备包埋脂肪酶的明胶微球,利用脂肪醛疏水改性的交联壳聚糖纳米颗粒修饰明胶微球,增强明胶微球的界面活性,明胶微球能够稳定油水界面形成Pickering乳液,明胶微球包埋的脂肪酶在两相界面可高效催化己酸和己醇合成己酸己酯反应,解决了采用的脂肪酶的生物大分子包埋载体界面活性不足,无法稳定油水界面形成Pickering乳液的问题。(The invention discloses a lipase interface immobilization method based on a hydrophobic modified gelatin microsphere, which is characterized in that the gelatin microsphere embedded with lipase is prepared by adopting an emulsion template method, the gelatin microsphere is modified by utilizing cross-linked chitosan nanoparticles hydrophobically modified by fatty aldehyde, the interface activity of the gelatin microsphere is enhanced, the gelatin microsphere can stabilize an oil-water interface to form Pickering emulsion, the lipase embedded by the gelatin microsphere can efficiently catalyze hexanoic acid and hexanol to synthesize hexanoic acid hexyl ester at a two-phase interface, and the problems that the adopted biomacromolecule embedding carrier of the lipase has insufficient interface activity and cannot stabilize the oil-water interface to form Pickering emulsion are solved.)

1. A lipase interface immobilization method based on hydrophobic modified gelatin microspheres is characterized by comprising the following steps:

1) the fatty aldehyde hydrophobic modified cross-linked chitosan nanoparticle comprises the following components: adding the cross-linked chitosan nano-particles into a fatty aldehyde solution with the fatty aldehyde concentration of 5-15 wt.%, using normal hexane as a solvent, reacting under magnetic stirring, and centrifuging and cleaning after the reaction is finished to obtain the fatty aldehyde hydrophobic modified cross-linked chitosan nano-particles; the preparation method of the cross-linked chitosan nanoparticle comprises the following steps: dissolving chitosan powder in acetic acid aqueous solution, stirring to completely hydrate the chitosan powder, adjusting the pH value to 4-7, then dispersing and homogenizing by using a dispersion machine and a high-pressure micro-jet flow nano-homogenizer, adding 0.001-1 wt.% of genipin to covalently crosslink for 24-48h at 35-45 ℃, then homogenizing by using high-pressure micro-jet flow to obtain a uniform crosslinked chitosan nano-gel dispersion, and then spray-drying at 160 ℃ to obtain the chitosan nano-gel dispersion;

2) preparing gelatin microspheres modified by fatty aldehyde hydrophobic modified cross-linked chitosan nanoparticles and preparing corresponding Pickering emulsion thereof: the fatty aldehyde hydrophobic modified cross-linked chitosan nano-particles and span80 prepared in the step 1) are ultrasonically dispersed in mineral oil to obtain a uniformly dispersed suspended oil phase; centrifuging 5-10 wt% lipase solution to remove impurities, collecting supernatant, dissolving 10-20 wt.% type B gelatin, and adjusting pH to 6.5 to obtain water phase; placing the oil phase in a water bath at 30-40 ℃, dropwise adding the water phase into the oil phase under stirring, continuing to emulsify for 20-30min to obtain W/O type Pickering emulsion with stable aldehyde modified cross-linked chitosan nanoparticles, cooling to room temperature, and mixing the two emulsion according to a volume ratio of 1: 2, adding normal hexane into the obtained W/O type Pickering emulsion, vibrating and demulsifying, and separating out an oil phase to obtain lower-layer fatty aldehyde hydrophobic modified cross-linked chitosan nanoparticle modified gelatin microspheres; and preparing a stable Pickering emulsion by using the obtained gelatin microspheres modified by the fatty aldehyde hydrophobic modified cross-linked chitosan nano particles as a particle emulsifier, wherein the oil phase is n-hexane, and the water phase is deionized water containing the gelatin microspheres modified by the fatty aldehyde hydrophobic modified cross-linked chitosan nano particles.

2. The method for lipase interfacial immobilization based on hydrophobically modified gelatin microspheres according to claim 1, wherein the fatty aldehyde is undecanal.

3. The application of the gelatin microsphere modified by the fatty aldehyde hydrophobic modified cross-linked chitosan nanoparticle obtained by the method for immobilizing the lipase interface based on the hydrophobic modified gelatin microsphere in the claim 1 or 2, which is characterized in that the gelatin microsphere modified by the fatty aldehyde hydrophobic modified cross-linked chitosan nanoparticle is used for enzymatic reaction.

4. The application of the gelatin microsphere modified by the fatty aldehyde hydrophobic modified cross-linked chitosan nanoparticle according to claim 3, which comprises the following steps: preparing stable Pickering emulsion by taking the obtained gelatin microspheres modified by the fatty aldehyde hydrophobic modified cross-linked chitosan nanoparticles as a particle emulsifier, wherein the oil phase is normal hexane, the water phase is deionized water containing the gelatin microspheres modified by the fatty aldehyde hydrophobic modified cross-linked chitosan nanoparticles, substrates of hexanoic acid and hexanol are added into the oil phase of the normal hexane, and the weight ratio of the hexanoic acid to the hexanoic acid in a centrifugal tube is 3: 1 shaking and emulsifying to prepare Pickering emulsion, and carrying out enzymatic reaction.

The technical field is as follows:

the invention relates to a lipase interface immobilization method, in particular to a lipase interface immobilization method based on hydrophobic modified gelatin microspheres.

Background art:

since the lipid substrate of lipases is insoluble in water, it is usually necessary to solubilize the substrate with an organic solvent. To mitigate enzyme denaturation and inactivation by solvents and polar substrates, a number of immobilization techniques have been used to improve the stability of lipases in nonaqueous environments.

The Pickering emulsion stabilized by particles is a novel lipase reaction system. Emulsions are kinetically stable two-phase systems. Theoretically, lipases are more favorable for maintaining conformation and activity in an aqueous environment. The Pickering emulsion can create a very large two-phase interface area and is very suitable for serving as a lipase catalysis platform. A simpler emulsion preparation strategy is to adopt interface active particles to construct an oil (organic solvent) water-in-oil emulsion, encapsulate lipase in a water phase, catalyze a substrate reaction in the organic phase, and consider the interface stability (maintaining a large-area two-phase interface) and the interface permeability (maintaining high substrate/product mass transfer efficiency) of the emulsion by adjusting an interface microstructure. However, in this way, the dynamic adsorption of lipase on the interface reduces the contact chance of lipase and substrate, and also has adverse effect on the conformation and activity of lipase. Another emulsion preparation strategy is to use the enzyme immobilization concept for reference, connect lipase to the interface active particles, realize the interface enrichment and immobilization of enzyme by utilizing the irreversible adsorption of the particles on an oil-water interface, and enhance the interface catalytic ability of the enzyme-carrying particles with interface activity by enlarging the contact area with a substrate by virtue of an emulsion interface platform with high specific surface area.

In the immobilization process of enzymes, the encapsulation and encapsulation provides more protection to the micro environment of the enzyme. After biological macromolecules such as protein, polysaccharide and the like embed enzyme molecules, the biological macromolecules have a cage effect (cagefect) and a crowding effect (crowdingffect) on the enzymes. The cage effect can control the leakage of the enzyme, and the crowding effect can reduce the conformational change of the enzyme. In addition, the protein has a large amount of-COOH and-NH₂The polysaccharide has a large amount of-OH and-COOH and can play a certain role in buffering environmental changesThe application is as follows. However, the biomacromolecule carrier used for immobilized enzyme at present has strong hydrophilicity and weak interfacial activity. For example, the Chinese invention patent (2014107493334) discloses a preparation method of chitosan modified biochar-based immobilized enzyme for converting high acid value kitchen waste grease into biodiesel. The biochar with a porous structure is prepared from peanut shells, the pore structure of the biochar is modified by chitosan, and the fixation of lipase molecules on a matrix carrier is promoted by electrostatic adsorption of opposite charges. The patent utilizes the electrostatic interaction between chitosan and enzyme to enhance the stability of lipase in organic solvent, but the carrier of the lipase has no interfacial activity. The Chinese patent CN1110577 uses the solidified oil phase as an inner core, and the lipase and the non-hydrophobic modified chitosan particles are adsorbed on the surface of the inner core together to form the microsphere with the core-shell structure. The lipase is positioned on the surface of the microsphere, so the lipase has low tolerance to polar substrates such as organic acid, organic alcohol and the like. At present, no patent discloses designing and controlling the interfacial activity of a biomacromolecule embedding carrier such as protein or polysaccharide and the like so that the biomacromolecule embedding carrier can be adsorbed on a two-phase interface.

The invention content is as follows:

the invention aims to provide a lipase interface immobilization method based on a hydrophobic modified gelatin microsphere, which is characterized in that the gelatin microsphere embedded with lipase is prepared by adopting an emulsion template method, the gelatin microsphere is modified by utilizing cross-linked chitosan nanoparticles hydrophobically modified by fatty aldehyde, the interface activity of the gelatin microsphere is enhanced, the gelatin microsphere becomes a particle emulsifier to stabilize an oil-water interface to form Pickering emulsion, and the lipase embedded by the gelatin microsphere can efficiently catalyze hexanoic acid and hexanol to synthesize hexanoic acid hexyl ester at a two-phase interface, so that the problems that the interface activity of a biomacromolecule embedding carrier of the adopted lipase is insufficient, and the oil-water interface cannot be stabilized to form Pickering emulsion are solved.

The invention is realized by the following technical scheme:

a lipase interface immobilization method based on a hydrophobic modified gelatin microsphere comprises the following steps:

1) the fatty aldehyde hydrophobic modified cross-linked chitosan nanoparticle comprises the following components: adding the cross-linked chitosan nanoparticles into a fatty aldehyde solution with the fatty aldehyde concentration of 5-15 wt.%, taking n-hexane as a solvent, reacting for 3-6h under magnetic stirring, and centrifuging and cleaning after the reaction is finished to obtain the fatty aldehyde hydrophobically modified cross-linked chitosan nanoparticles; the preparation method of the cross-linked chitosan nanoparticle comprises the following steps: dissolving chitosan powder in acetic acid aqueous solution, stirring to completely hydrate the chitosan powder, adjusting the pH value to 4-7, then dispersing and homogenizing by using a dispersion machine and a high-pressure micro-jet flow nano-homogenizer, adding 0.001-1 wt.% genipin (genipin) for covalent crosslinking for 24-48h at 35-45 ℃, then homogenizing by using high-pressure micro-jet flow to obtain uniform crosslinked chitosan nano-gel dispersoid, and then spray-drying at 160 ℃ to obtain the chitosan nano-gel dispersoid;

2) preparing gelatin microspheres modified by fatty aldehyde hydrophobic modified cross-linked chitosan nanoparticles and preparing corresponding Pickering emulsion thereof: the fatty aldehyde hydrophobic modified cross-linked chitosan nano-particles prepared in the step 1) and span80 (span80) are ultrasonically dispersed in mineral oil to obtain a uniformly dispersed suspended oil phase; centrifuging 5-10 wt% lipase solution to remove impurities, collecting supernatant, dissolving 10-20 wt.% type B gelatin, and adjusting pH to 6.5 to obtain water phase; placing the oil phase in a water bath at 30-40 ℃, dropwise adding the water phase into the oil phase under stirring, continuing to emulsify for 20-30min to obtain W/O type Pickering emulsion with stable aldehyde modified cross-linked chitosan nanoparticles, cooling to room temperature, and mixing the two emulsion according to a volume ratio of 1: 2, adding normal hexane into the obtained W/O type Pickering emulsion, vibrating and demulsifying, and separating out an oil phase to obtain lower-layer fatty aldehyde hydrophobic modified cross-linked chitosan nanoparticle modified gelatin microspheres; and preparing a stable Pickering emulsion by using the obtained gelatin microspheres modified by the fatty aldehyde hydrophobic modified cross-linked chitosan nano particles as a particle emulsifier, wherein the oil phase is n-hexane, and the water phase is deionized water containing the gelatin microspheres modified by the fatty aldehyde hydrophobic modified cross-linked chitosan nano particles.

The fatty aldehyde is preferably undecanal.

The invention also protects the application of the gelatin microsphere modified by the fatty aldehyde hydrophobic modified cross-linked chitosan nano particles obtained by the method, and the gelatin microsphere is used for enzymatic reaction.

In particular, the following steps are included: preparing stable Pickering emulsion by taking the obtained gelatin microspheres modified by the fatty aldehyde hydrophobic modified cross-linked chitosan nanoparticles as a particle emulsifier, wherein the oil phase is normal hexane, the water phase is deionized water containing the gelatin microspheres modified by the fatty aldehyde hydrophobic modified cross-linked chitosan nanoparticles, substrates of hexanoic acid and hexanol are added into the oil phase of the normal hexane, and the weight ratio of the hexanoic acid to the hexanoic acid in a centrifugal tube is 3: 1 shaking and emulsifying to prepare Pickering emulsion, and carrying out enzymatic reaction.

The invention has the following beneficial effects:

1) the invention constructs gelatin microspheres embedded with lipase and Pickering emulsion stabilized by the gelatin microspheres, embeds the lipase into gelatin matrix, makes hydrophobically modified cross-linked chitosan particles adsorbed on the surface of the gelatin microspheres, enhances the interfacial activity of the gelatin microspheres, thereby making the gelatin microspheres capable of being used as particle emulsifier to stabilize the Pickering emulsion, the lipase in the gelatin spheres has enhanced tolerance to polar substrates such as organic acid, organic alcohol and the like, can be used for catalyzing esterification reaction of hexanoic acid and hexanol under the Pickering system, is used for two-phase catalytic reaction of lipase, and the emulsion system of the gelatin microspheres adsorbed with the embedded lipase at the two-phase interface has obviously higher catalytic activity, and can efficiently catalyze esterification of hexanoic acid and hexanol to generate hexanoic acid hexyl ester due to the mild embedding condition of the gelatin microspheres embedded with lipase and biocompatible microenvironment, and generates larger oil-water interfacial area and short diffusion distance by the Pickering emulsion stabilized by the gelatin microspheres modified by the cross-linked chitosan particles. Furthermore, the product/substrate containing the organic phase can be easily separated by gravity settling, so that the Pickering emulsion can be recovered and reused several times.

2) The invention adjusts the length of the carbon chain of the cross-linked chitosan nano-particle grafted by the fatty aldehyde to adjust the hydrophilic gelatin microsphere to have proper wettability, thereby stabilizing the water-hexane Pickering emulsion.

Description of the drawings:

FIG. 1 is an SEM photograph of the cross-linked chitosan nanoparticle obtained in example 1;

FIG. 2 is a graph showing a potential comparison of crosslinked chitosan nanoparticles before and after modification;

FIG. 3 is a graph comparing contact angles of fatty aldehyde-modified cross-linked chitosan nanoparticles of different carbon chain lengths;

in fig. 2 and 3, CS _ hep is n-heptaldehyde hydrophobically modified cross-linked chitosan nanoparticle, CS _ non is n-nonanal hydrophobically modified cross-linked chitosan nanoparticle, CS _ und is undecanal hydrophobically modified cross-linked chitosan nanoparticle, and CS _0C is cross-linked chitosan particle before aldehyde modification;

FIG. 4 is a contact angle comparison graph of gelatin microspheres modified by fatty aldehyde hydrophobically modified cross-linked chitosan nanoparticles with different carbon chain lengths;

wherein CS _ hep is gelatin microsphere modified by n-heptanal hydrophobically modified cross-linked chitosan nanoparticle, CS _ non is gelatin microsphere modified by n-nonanal hydrophobically modified cross-linked chitosan nanoparticle, CS _ und is gelatin microsphere modified by undecanal hydrophobically modified cross-linked chitosan nanoparticle, and CS _0C is gelatin microsphere modified by cross-linked chitosan particle powder before aldehyde modification;

FIG. 5 is an appearance diagram and a white light microscope diagram of a Pickering emulsion stabilized by undecylenic aldehyde hydrophobically modified cross-linked chitosan nanoparticle modified gelatin microspheres;

FIG. 6 is a CLSM diagram of Pickering latex stabilized by undecylenic aldehyde hydrophobically modified cross-linked chitosan nanoparticle modified gelatin microspheres;

FIG. 7 is a graph of the enzymatic change rate of Pickering latex stabilized by undecylenic aldehyde hydrophobically modified cross-linked chitosan nanoparticles modified gelatin microspheres;

FIG. 8 is a graph showing the effect of batch reactions on the conversion of enzymatic reactions.

The specific implementation mode is as follows:

the following is a further description of the invention and is not intended to be limiting.