CN114561376A - Method for directionally immobilizing enzyme by photo-crosslinking short peptide - Google Patents

Abstract

Translated fromChinese

本发明公开了一种通过光交联短肽定向固定化酶的方法，属于酶工程技术领域。本发明选择蔗糖异构酶(SI)作为模型酶。利用分子模拟软件选择出远离活性位点的区域作为定向固定化区域，随后结合分子对接和分子动力学模拟方法设计与该区域有较高亲和性的光交联短肽，并在其末端引入含有光交联基团的非天然氨基酸4‑苯甲酰基‑L‑苯丙氨酸，序列命名为：VNIGGX(VG)。利用光交联短肽实现酶在树脂微球(EP)表面的定向固定。对光交联定向固定化酶进行酶活力、稳定性与重复使用性的评估。本发明赋予固定化酶良好的耐热性和可重复使用性。与传统的固定化酶相比，本发明结合定向吸附和光交联技术，提供了一种高效环保的定向固定化酶方法。

The invention discloses a method for directional immobilization of enzymes by photocrosslinking short peptides, and belongs to the technical field of enzyme engineering. The present invention selects sucrose isomerase (SI) as a model enzyme. Molecular simulation software was used to select a region far from the active site as a directional immobilization region, and then combined with molecular docking and molecular dynamics simulation methods, a short photocrosslinking peptide with high affinity to this region was designed and introduced at its end. Unnatural amino acid 4-benzoyl-L-phenylalanine containing photocrosslinking group, sequence name: VNIGGX(VG). The directional immobilization of enzymes on the surface of resin microspheres (EP) was achieved by photocrosslinking short peptides. Evaluation of enzyme activity, stability and reusability of photocrosslinking directional immobilized enzymes. The present invention imparts good heat resistance and reusability to the immobilized enzyme. Compared with the traditional immobilized enzyme, the present invention combines directional adsorption and photocrosslinking technology to provide an efficient and environmentally friendly directional immobilized enzyme method.

Description

Translated fromChinese

一种通过光交联短肽定向固定化酶的方法A method for targeted immobilization of enzymes by photocrosslinking short peptides

技术领域technical field

本申请属于酶工程领域，具体设计一种通过光交联短肽定向固定化酶的方法The application belongs to the field of enzyme engineering, and specifically designs a method for directional immobilization of enzymes by photocrosslinking short peptides

背景技术Background technique

酶是一种具有高催化效率和特异性的生物催化剂，但游离酶的低稳定性和难以回收再利用等缺点限制了其工业应用。固定化酶已被用来克服上述游离酶的缺点，但随机固定化会遮盖酶活性位点，造成酶活力的急剧下降。而定向固定化酶技术因避开酶活性位点结合于载体表面而受到广泛的关注。采用定向固定化的方法固定化酶，可使酶特定位点直接或间接与载体结合，酶活性位点位于载体外侧，天然构象基本保持不变，有利于底物进入到酶活性位点，定向固定化可以使蛋白质在载体表面的取向和构象达到统一，有利于保护酶的催化功能。近年来，定向固定化酶技术在酶工程中发挥着巨大作用，在保留酶活力，改善酶的稳定性及重复使用性等方面取得了非常有益的效果。Enzymes are biocatalysts with high catalytic efficiency and specificity, but their industrial applications are limited by their low stability and difficulty in recycling and reuse of free enzymes. Immobilized enzymes have been used to overcome the above shortcomings of free enzymes, but random immobilization will cover the active site of the enzyme, resulting in a sharp drop in enzyme activity. The technology of targeted immobilization of enzymes has received extensive attention because it avoids the active site of the enzyme and binds to the surface of the carrier. The directional immobilization method is used to immobilize the enzyme, which can directly or indirectly bind the specific site of the enzyme to the carrier. The active site of the enzyme is located on the outside of the carrier, and the natural conformation remains basically unchanged, which is conducive to the entry of the substrate into the active site of the enzyme. Immobilization can unify the orientation and conformation of the protein on the surface of the carrier, which is beneficial to protect the catalytic function of the enzyme. In recent years, directional immobilization enzyme technology has played a huge role in enzyme engineering, and has achieved very beneficial effects in retaining enzyme activity, improving enzyme stability and reusability.

光作为一种新型的引导交联反应的工具，其触发的交联反应在非物理接触下可精准控制，提供了较高的时间和空间分辨率，优化了交联反应的体系。光敏分子被分为三大类化合物：二苯甲酮类、二氮嗪类和芳香叠氮化合物。在特定波长的光照下能发生结构的变化，从而引导交联反应。波长为365nm的紫外光便能触发二苯甲酮类的光交联反应。在紫外光照射下，结构中的“酮羰基”经历n到π*的转变形成二自由基，此状态可以从附近活化的“碳氢键”中吸收氢原子并重组形成共价复合物，完成交联反应。光交联反应具有高效、无毒和普适性强的优点，作为一种快速、简单和时空可控的交联方法已广泛地应用于化学、生物、医学和材料等不同研究领域。As a novel tool for guiding the cross-linking reaction, light can trigger precise control of the cross-linking reaction under non-physical contact, providing high temporal and spatial resolution and optimizing the system of the cross-linking reaction. Photosensitive molecules are divided into three major classes of compounds: benzophenones, diazoxides, and aromatic azides. Structural changes can occur under specific wavelengths of light, which guide the cross-linking reaction. Ultraviolet light with a wavelength of 365 nm can trigger the photocrosslinking reaction of benzophenones. Under UV light irradiation, the "ketone carbonyl" in the structure undergoes the transformation from n to π* to form a diradical, which can absorb hydrogen atoms from the nearby activated "carbon-hydrogen bond" and recombine to form a covalent complex, complete cross-linking reaction. Photocrosslinking has the advantages of high efficiency, non-toxicity, and strong universality. As a fast, simple, and spatiotemporally controllable crosslinking method, it has been widely used in different research fields such as chemistry, biology, medicine, and materials.

发明内容SUMMARY OF THE INVENTION

为克服现在酶固定化方法中存在的缺陷，本发明提供了一种通过光交联短肽定向固定化酶的方法，根据酶的结构计算模拟设计出与之亲和的光交联短肽，光交联短肽在紫外光照下实现酶的定向固定化。该方法包括以下步骤：In order to overcome the defects existing in the current enzyme immobilization methods, the present invention provides a method for directional immobilization of enzymes by photocrosslinking short peptides. Photocrosslinked short peptides achieve directional immobilization of enzymes under ultraviolet light. The method includes the following steps:

(1)远离酶的活性位点进行光交联短肽的设计；(1) Design of photocrosslinking short peptides away from the active site of the enzyme;

(2)将设计好的光交联短肽连接在载体上；(2) linking the designed photocrosslinking short peptide on the carrier;

(3)酶溶液与(2)中得到的载体混合进行吸附反应；(3) the enzyme solution is mixed with the carrier obtained in (2) to carry out adsorption reaction;

(4)将(3)中得到的混合物进行紫外光照处理，产物经沉淀，洗涤后得到光交联定向固定化酶。(4) The mixture obtained in (3) is subjected to ultraviolet irradiation treatment, and the product is precipitated and washed to obtain a photocrosslinking directional immobilized enzyme.

进一步地，步骤(1)中所选择的酶分子，包括却不限于蔗糖异构酶。作为优选，蔗糖异构酶来源于Pantoea dispersa UQ68J。Further, the enzyme molecule selected in step (1) includes but is not limited to sucrose isomerase. Preferably, the sucrose isomerase is derived from Pantoea dispersa UQ68J.

进一步地，步骤(1)中所选择的模拟软件，包括单不限于Gromacs。作为优选，通过软件Gromacs和POCASA程序，避开蔗糖异构酶的活性中心，选择出蔗糖异构酶的目标固定区域。Further, the simulation software selected in step (1) includes but is not limited to Gromacs. Preferably, the target immobilization region of sucrose isomerase is selected by software Gromacs and POCASA programs, avoiding the active center of sucrose isomerase.

进一步地，步骤(1)中选择Auto Dock与MOE软件设计与蔗糖异构酶目标固定区域具有高亲和力的短肽。作为优选，短肽序列为H2N-VNIGGX-COOH，X为光敏性二苯甲酮类非天然氨基酸，包括但不限于4-L-苯甲酰基苯丙氨酸。Further, in step (1), Auto Dock and MOE software are selected to design a short peptide with high affinity to the target immobilization region of sucrose isomerase. Preferably, the short peptide sequence is H2N-VNIGGX-COOH, and X is a photosensitive benzophenone unnatural amino acid, including but not limited to 4-L-benzoylphenylalanine.

进一步地，步骤(2)中所述载体为表面带有环氧基团的材料，包括但不限于环氧树脂。作为优选，环氧树脂载体的材料为聚丙烯酸甲酯，粒径为0.2-200μm。Further, in step (2), the carrier is a material with epoxy groups on the surface, including but not limited to epoxy resin. Preferably, the material of the epoxy resin carrier is polymethyl acrylate, and the particle size is 0.2-200 μm.

进一步地，步骤(2)中的连接反应包括：将0.5～5mg的短肽溶解于磷酸盐缓冲溶液中，缓冲液浓度为10～100mM，pH为7.5～10.0；短肽溶液与环氧树脂在管中混合，树脂表面环氧基团与短肽的摩尔质量比为5～50。作为优选，混合后的反应时间为10-20h，反应温度为20～30℃。连接反应完成后收集得到经短肽修饰的载体。Further, the ligation reaction in step (2) includes: dissolving 0.5-5 mg of the short peptide in a phosphate buffer solution, the buffer concentration is 10-100 mM, and the pH is 7.5-10.0; Mix in a tube, and the molar mass ratio of epoxy groups on the resin surface to short peptides is 5-50. Preferably, the reaction time after mixing is 10-20h, and the reaction temperature is 20-30°C. After the ligation reaction is completed, the short peptide-modified vector is collected.

进一步地，步骤(3)中吸附反应的缓冲溶液为磷酸盐缓冲溶液，浓度为10～100mM，pH为5.0～6.5。作为优选，酶溶液初始浓度为0.5～10mg/mL，载体的质量为5～20mg，吸附反应时间为2～10h。Further, the buffer solution for the adsorption reaction in step (3) is a phosphate buffer solution with a concentration of 10-100 mM and a pH of 5.0-6.5. Preferably, the initial concentration of the enzyme solution is 0.5-10 mg/mL, the mass of the carrier is 5-20 mg, and the adsorption reaction time is 2-10 h.

进一步地，步骤(4)中紫外光照的波长为350～365nm，功率为25w～120w，照射时间为1～30min。反应结束后，收集固定化酶并洗涤，得到光交联定向固定化酶。Further, in step (4), the wavelength of the ultraviolet light is 350-365 nm, the power is 25w-120w, and the irradiation time is 1-30min. After the reaction, the immobilized enzyme was collected and washed to obtain a photocrosslinking directional immobilized enzyme.

本发明的方法具有下列优点和积极效果：The method of the present invention has the following advantages and positive effects:

1)可定向控制固定化酶在载体表面的方向1) The orientation of the immobilized enzyme on the surface of the carrier can be directionally controlled

2)固定化酶能保留相比于游离酶的高酶活力2) The immobilized enzyme can retain the high enzyme activity compared to the free enzyme

3)固定化酶的热稳定性有极大的提高3) The thermal stability of the immobilized enzyme is greatly improved

4)固定化酶的重复使用性能高4) High reusability of immobilized enzymes

5)固定化工艺流程简单易操作，环保高效。5) The immobilization process is simple and easy to operate, environmentally friendly and efficient.

附图说明Description of drawings

图1为载体连接光交联短肽的HPLC分析；Fig. 1 is the HPLC analysis of carrier-linked photocross-linked short peptides;

图2为本发明的光交联反应示意图；Fig. 2 is the photocrosslinking reaction schematic diagram of the present invention;

图3为固定化酶的FTIR检测光谱；Fig. 3 is the FTIR detection spectrum of immobilized enzyme;

图4为固定化酶的热稳定性；Fig. 4 is the thermostability of immobilized enzyme;

图5固定化酶的重复使用性。Figure 5 Reusability of immobilized enzymes.

具体实施方式Detailed ways

下面结合实施例对本发明做详细说明，但并不构成对本发明的限制。The present invention will be described in detail below in conjunction with the embodiments, but it does not constitute a limitation of the present invention.

实施例1：蔗糖异构酶(SI)的目标固定区域的选择及光交联短肽的设计Example 1: Selection of target immobilization region for sucrose isomerase (SI) and design of short photocrosslinking peptides

蔗糖异构酶的三维结构从Protein Data Bank(PDB)(https：//www.rcsb.org/)数据库中获得，PDB ID为4G18。使用POCASA程序搜索蛋白质表面的空洞和口袋，考虑到短肽结合位点应该远离活性中心并具有正确的空间位置，由残基R339-G359及残基P456-L557形成的区域被选为亲和短肽配基的目标结合区域。使用Auto Dock与MOE软件设计与蔗糖异构酶具有高亲和力的短肽。应用Auto Dock软件将20个常见氨基酸与蔗糖异构酶目标结合区域进行对接操作。格点间距被设为

同时格点中心为x＝-37.788，y＝46.928，z＝45.369。格点参数设为

对接所得构象数目为300。随后，以蔗糖异构酶整个蛋白结构为目标区域，使用MOE软件进行备选亲和短肽配基的对接筛选。最终得到合适的光交联短肽VNIGGX(VG)。The three-dimensional structure of sucrose isomerase was obtained from the Protein Data Bank (PDB) (https://www.rcsb.org/) database with the PDB ID 4G18. Using the POCASA program to search for holes and pockets on the surface of the protein, considering that the short peptide binding site should be far from the active center and have the correct spatial position, the region formed by residues R339-G359 and residues P456-L557 was selected as the affinity short. The target binding region of the peptide ligand. Short peptides with high affinity for sucrose isomerase were designed using Auto Dock and MOE software. The Auto Dock software was used to dock 20 common amino acids with the target binding region of sucrose isomerase. The grid spacing is set to

At the same time, the center of the grid point is x=-37.788, y=46.928, and z=45.369. The grid parameter is set to

The number of conformations obtained by docking was 300. Subsequently, the whole protein structure of sucrose isomerase was used as the target region, and the MOE software was used for docking screening of candidate affinity short peptide ligands. Finally, a suitable photocrosslinking short peptide VNIGGX (VG) was obtained.

实施例2：环氧树脂表面修饰光交联短肽Example 2: Epoxy resin surface modification photocrosslinking short peptide

将2mg的短肽溶解于2mL磷酸盐缓冲溶液(pH 8.0，10mM)中，随后与10mg环氧树脂(EP)在管中混合(EP表面环氧基团与VG的摩尔质量比为10)。混合物在25℃的水浴恒温振荡器中连续反应12h，避光处理。反应完成后静置使载体沉淀分离，并用缓冲溶液反复洗涤3次，收集沉淀得到经VG修饰的载体。随后利用盐酸溶液(1M)对上述载体和EP的表面剩余环氧基进行开环处理，得到EP-VG和EP*。采用高效液相色谱法(HPLC)检测环氧树脂表面修饰的VG含量。结果见图1。从HPLC分析可以得出，EP表面成功修饰光交联短肽。VG的连接率达到80±5％，连接量为0.14±0.02mmol/g。2 mg of the short peptide was dissolved in 2 mL of phosphate buffer solution (pH 8.0, 10 mM) and then mixed with 10 mg of epoxy resin (EP) in a tube (the molar mass ratio of EP surface epoxy groups to VG was 10). The mixture was continuously reacted for 12 h in a water bath thermostatic oscillator at 25°C, and treated in the dark. After the completion of the reaction, stand still to separate the carrier precipitate, and repeatedly wash with abuffer solution 3 times, and collect the precipitate to obtain a VG-modified carrier. The remaining epoxy groups on the surface of the above support and EP were then subjected to ring-opening treatment with hydrochloric acid solution (1M) to obtain EP-VG and EP*. The VG content of epoxy resin surface modification was detected by high performance liquid chromatography (HPLC). The results are shown in Figure 1. From HPLC analysis, it can be concluded that the surface of EP was successfully modified with photocrosslinked short peptides. The linking rate of VG reached 80±5%, and the linking amount was 0.14±0.02 mmol/g.

实施例3：光交联定向固定化酶的制备Example 3: Preparation of photocrosslinking directional immobilized enzymes

将SI溶解于磷酸盐缓冲溶液(pH 6.0，10mM)中，配制成5mg/mL初始酶溶液。分别取10mg的EP-VG(2份)、EP*分散于5mL酶溶液中，在0℃、70rpm条件下振荡吸附反应10h，分别得到非定向吸附固定酶(EP*-SI)和定向吸附固定酶(EP-VG-SI)。随后将其中一份EP-VG-SI在波长365nm、功率120w的紫外光条件下照射3min，得到定向光交联固定化酶(EP-VG-SI hv)。光交联示意图见图2。反应结束后，收集固定化酶并用缓冲液反复洗涤3次，于4℃保存待用。使用傅立叶变换红外光谱(FTIR)检测载体和固定化酶的表面化学结构的变化。将冷冻干燥的样品与溴化钾粉末混合研磨，进行压片处理后，利用FTIR进行测定，扫描范围为4000-400cm^-1。结果见图3。VG的特征峰为3060cm^-1、1670cm^-1和1545cm^-1。由图3可知VG连接在EP表面后其-C＝O吸收峰从1670cm^-1蓝移到1722cm^-1。光交联定向固定化酶EP-VG-SI hv的-C＝O吸收峰峰值降低，且其1670cm^-1处峰值也随之降低，表明-C＝O与C-H在光照后发生反应而减少，VG与SI光交联成功使得SI固定于载体表面。SI was dissolved in phosphate buffered solution (pH 6.0, 10 mM) to make a 5 mg/mL initial enzyme solution. Disperse 10 mg of EP-VG (2 parts) and EP* in 5 mL of enzyme solution, respectively, and shake the adsorption reaction at 0 °C and 70 rpm for 10 h to obtain non-directional adsorption immobilized enzyme (EP*-SI) and directional adsorption immobilized enzyme, respectively. Enzyme (EP-VG-SI). Then, one part of EP-VG-SI was irradiated for 3 min under the condition of ultraviolet light with a wavelength of 365 nm and a power of 120 w to obtain a directional photocrosslinking immobilized enzyme (EP-VG-SI hv). The schematic diagram of photocrosslinking is shown in Figure 2. After the reaction, the immobilized enzyme was collected, washed with buffer for 3 times, and stored at 4°C for later use. Changes in the surface chemical structure of the support and immobilized enzymes were detected using Fourier Transform Infrared Spectroscopy (FTIR). The freeze-dried sample was mixed and ground with potassium bromide powder, and after tableting treatment, it was measured by FTIR with a scanning range of 4000-400 cm^-1 . The results are shown in Figure 3. The characteristic peaks of VG are 3060 cm^-1 , 1670 cm^-1 and 1545 cm^-1 . It can be seen from Fig. 3 that the -C=O absorption peak blue-shifted from 1670 cm^-1 to 1722 cm^-1 after VG was attached to the EP surface. The -C=O absorption peak of the photocrosslinking directional immobilized enzyme EP-VG-SI hv decreased, and the peak at 1670cm^-1 also decreased, indicating that -C=O reacted with CH to decrease after irradiation. The successful photocrosslinking of VG and SI made SI immobilized on the surface of the carrier.

实施例4：固定化酶的热稳定性及重复使用性测试Example 4: Thermal stability and reusability test of immobilized enzyme

将游离酶与固定化酶样品置于45℃水浴锅中，在70rpm条件下分别热处理不同的时间(0h，2h，4h，6h，8h)，按照3，5-二硝基水杨酸法(Dinitrosalicylic acid，DNS)测定反应后样品异麦芽酮糖的含量并计算样品不同热处理时间后的残余酶活力。以0h的催化活性为100％，以计算剩余酶活性的百分比。结果见图4，固定化酶活力的降幅明显小于游离酶，在温育2h后EP-VG-SI hv的酶活力在90％以上，且温育4h后能保持40％的初始活性。表明相较于游离酶，该定向固定化技术可以提高酶的抗热应力破坏性。The free enzyme and immobilized enzyme samples were placed in a 45°C water bath, and heat-treated at 70 rpm for different times (0h, 2h, 4h, 6h, 8h), according to the 3,5-dinitrosalicylic acid method ( Dinitrosalicylic acid, DNS) to determine the content of isomaltulose in the samples after the reaction and calculate the residual enzyme activity of the samples after different heat treatment time. The catalytic activity at 0 h was taken as 100% to calculate the percentage of remaining enzymatic activity. The results are shown in Figure 4. The decrease of the immobilized enzyme activity was significantly smaller than that of the free enzyme. The enzyme activity of EP-VG-SI hv was above 90% after incubation for 2 hours, and 40% of the initial activity was maintained after incubation for 4 hours. It is indicated that the directional immobilization technology can improve the thermal stress resistance of the enzyme compared with the free enzyme.

采用循环测定酶活力的方法研究固定化酶的重复使用性。将固定化酶分散于0.1mL磷酸氢二钠-柠檬酸缓冲溶液(pH 6.0，10mM)制得固定化酶溶液，在恒温水浴振荡器中于35℃和70rpm的条件下催化蔗糖的异构化持续反应10min。反应完成后静置使其沉淀，取出全部上清液并立即检测其酶活性。而静置得到的固定化酶(沉淀物)用缓冲溶液反复清洗，以除去未反应的底物和产物。将洗涤后的固定化酶重新分散于0.1mL缓冲溶液中，加入底物进行下一次的催化反应。将上述操作重复11次，连续检测固定化酶在循环多次后的残余酶活力。结果见图5，重复利用11次后，EP-VG-SI hv的催化活性仍保持在50％以上，远高于EP-VG-SI(40％)和EP-SI(30％)。表明发明中光交联短肽的定向吸附作用较好的保留了酶的活性，且进行紫外光辐照后，酶与载体的光交联共价结合提高了EP-VG-SI hv的重复使用性。The reusability of immobilized enzyme was studied by cyclic determination of enzyme activity. Disperse the immobilized enzyme in 0.1 mL of disodium hydrogen phosphate-citric acid buffer solution (pH 6.0, 10 mM) to prepare the immobilized enzyme solution, and catalyze the isomerization of sucrose in a constant temperature water bath shaker at 35 °C and 70 rpm Continue to react for 10min. After the reaction was completed, it was left to settle, and the entire supernatant was taken out and immediately tested for its enzymatic activity. The immobilized enzyme (precipitate) obtained by standing is repeatedly washed with a buffer solution to remove unreacted substrates and products. The washed immobilized enzyme was redispersed in 0.1 mL buffer solution, and the substrate was added for the next catalytic reaction. The above operation was repeated 11 times, and the residual enzyme activity of the immobilized enzyme after repeated cycles was continuously detected. The results are shown in Figure 5. After 11 times of reuse, the catalytic activity of EP-VG-SI hv remained above 50%, much higher than that of EP-VG-SI (40%) and EP-SI (30%). It shows that the directional adsorption of the photocrosslinked short peptides in the invention can better retain the activity of the enzyme, and after ultraviolet light irradiation, the photocrosslinking and covalent binding of the enzyme and the carrier improves the repeated use of EP-VG-SI hv sex.

综上所述，本发明结合分子对接和分子动力学模拟理性设计了与远离酶活性区域有较高亲和性的光交联短肽，采用紫外光照的方法使酶以共价键的形式牢固结合在载体表面，在保留酶活力的情况下，实现了酶制剂的分离和回收以及热稳定性的提高，延长了酶制剂的使用寿命，其良好的重复使用性和稳定性在应用中可以显著降低酶的成本，在生产操作上具有较高的工业价值。本发明的固定化工艺流程简单高效，反应条件易于操作控制，且不添加化学交联剂，对环境友好。因此该技术适合应用于工业化生产。To sum up, the present invention rationally designs photocrosslinking short peptides with high affinity to the region far from the active region of the enzyme by combining molecular docking and molecular dynamics simulation, and adopts the method of ultraviolet light to make the enzyme firm in the form of covalent bonds. Combined on the surface of the carrier, the separation and recovery of the enzyme preparation and the improvement of thermal stability are achieved while retaining the enzyme activity, which prolongs the service life of the enzyme preparation. Its good reusability and stability can be significantly used in applications. The cost of the enzyme is reduced, and it has high industrial value in the production operation. The immobilization process of the invention is simple and efficient, the reaction conditions are easy to operate and control, and no chemical cross-linking agent is added, which is environmentally friendly. Therefore, the technology is suitable for industrial production.

Claims (9)

Translated fromChinese

1.一种通过光交联短肽定向固定化酶的方法，其特征在于包括如下步骤：1. a method for directional immobilization of enzyme by photocrosslinking short peptide is characterized in that comprising the steps:(1)远离酶的活性位点进行光交联短肽的设计；(1) Design of photocrosslinking short peptides away from the active site of the enzyme;(2)将设计好的光交联短肽连接在载体表面；(2) connecting the designed short photocrosslinking peptide to the surface of the carrier;(3)酶溶液与(2)中得到的载体混合进行吸附反应；(3) the enzyme solution is mixed with the carrier obtained in (2) to carry out adsorption reaction;(4)将(3)中得到的混合物进行紫外光照处理，产物经沉淀，洗涤后得到光交联定向固定化酶。(4) The mixture obtained in (3) is subjected to ultraviolet irradiation treatment, and the product is precipitated and washed to obtain a photocrosslinking directional immobilized enzyme.2.根据权利要求1中所述的通过光交联短肽定向固定化酶的方法，其特征在于：所述酶包括却不限于蔗糖异构酶。2 . The method for directional immobilization of enzymes by photocrosslinking short peptides according to claim 1 , wherein the enzymes include but are not limited to sucrose isomerase. 3 .3.根据权利要求1中所述的通过光交联短肽定向固定化酶的方法，其特征在于步骤(1)具体为：使用Gromacs和Auto Dock软件对酶的三维结构进行模拟与光交联短肽设计，设计的短肽优选为能与酶发生稳定吸附结合的光交联短肽，其中光交联短肽含有3～9个天然氨基酸，且C端为光交联性非天然氨基酸(二苯甲酮类)，包括却不限于4-L-苯甲酰基苯丙氨酸。3. The method for directional immobilization of enzyme by photocrosslinking short peptides according to claim 1, characterized in that step (1) is specifically: using Gromacs and Auto Dock software to simulate and photocrosslink the three-dimensional structure of the enzyme Short peptide design, the designed short peptide is preferably a photocrosslinking short peptide that can stably adsorb and combine with the enzyme, wherein the photocrosslinking short peptide contains 3 to 9 natural amino acids, and the C-terminal is a photocrosslinking non-natural amino acid ( benzophenones), including but not limited to 4-L-benzoylphenylalanine.4.根据权利要求1中所述的通过光交联短肽定向固定化酶的方法，其特征在于：步骤(2)中所述载体为表面带有环氧基团的材料，包括环氧树脂、环氧硅球。环氧树脂载体的材料为聚丙烯酸甲酯，粒径为0.2-200μm。4 . The method for directional immobilization of enzymes by photocrosslinking short peptides according to claim 1 , wherein the carrier in step (2) is a material with epoxy groups on the surface, including epoxy resins. 5 . , epoxy silicon ball. The material of the epoxy resin carrier is polymethyl acrylate, and the particle size is 0.2-200 μm.5.根据权利要求1中所述的通过光交联短肽定向固定化酶的方法，其特征在于：步骤(2)中的连接反应的缓冲溶液为磷酸盐缓冲溶液，浓度为10～100mM，pH为7.5～10.0；所述的短肽质量为0.5～5mg，溶解于缓冲溶液中；所述的载体为环氧树脂，树脂表面环氧基团与短肽的摩尔质量比为5～50。5 . The method for directional immobilization of enzymes by photocrosslinking short peptides according to claim 1 , wherein the buffer solution for the ligation reaction in step (2) is a phosphate buffer solution with a concentration of 10-100 mM, 6 . The pH is 7.5-10.0; the mass of the short peptide is 0.5-5 mg, which is dissolved in a buffer solution; the carrier is epoxy resin, and the molar mass ratio of epoxy groups on the resin surface to the short peptide is 5-50.6.根据权利要求1中所述的通过光交联短肽定向固定化酶的方法，其特征在于：步骤(2)中所述的连接反应的反应时间为10-20h，反应温度为20～30℃。6 . The method for directional immobilization of enzymes by photocrosslinking short peptides according to claim 1 , wherein the reaction time of the ligation reaction described in step (2) is 10-20 h, and the reaction temperature is 20-20 h. 7 . 30°C.7.根据权利要求1中所述的通过光交联短肽定向固定化酶的方法，其特征在于：步骤(3)中吸附反应的缓冲溶液为磷酸盐缓冲溶液，浓度为10～100mM，pH为5.0～6.5。所述的酶溶液初始浓度为0.5～10mg/mL，载体的质量为5～20mg。7 . The method for directional immobilization of enzymes by photocrosslinking short peptides according to claim 1 , wherein the buffer solution for the adsorption reaction in step (3) is a phosphate buffer solution with a concentration of 10-100 mM and a pH of 10-100 mM. 8 . 5.0 to 6.5. The initial concentration of the enzyme solution is 0.5-10 mg/mL, and the mass of the carrier is 5-20 mg.8.根据权利要求1中所述的通过光交联短肽定向固定化酶的方法，其特征在于：步骤(3)中，吸附反应过程中，反应温度为0～20℃，反应时间为2～10h。8 . The method for directional immobilization of enzymes by photocrosslinking short peptides according to claim 1 , wherein in step (3), during the adsorption reaction, the reaction temperature is 0-20° C., and the reaction time is 2 ~10h.9.根据权利要求1中所述的通过光交联短肽定向固定化酶的方法，其特征在于：步骤(4)中紫外光照的波长为350～365nm，功率为25w～120w，照射时间为1～30min。9 . The method for directional immobilization of enzymes by photocrosslinking short peptides according to claim 1 , wherein the wavelength of ultraviolet light in step (4) is 350-365 nm, the power is 25w-120w, and the irradiation time is 9. 10 . 1 to 30 minutes.

Images