【作者】 王涛；

【导师】 付玉杰；

【作者基本信息】 东北林业大学 ， 生物学， 2025， 博士

【摘要】 植物天然产物因其具有丰富的生物活性,广泛应用于医药、农业、食品等多个领域。植物天然产物通过高效生物转化可获得活性好、附加值高的活性成分,是目前国际上植物高值化利用的研究热点和产业需求。本研究以来源广泛、具备生物可降解性和良好的机械性能的纤维素材料作为基材,结合纳米技术、磁性材料及仿生学设计理念,率先构建了多种纤维素基固定化催化剂体系,系统研究了其在催化木本油料酯交换反应以及糖苷类天然产物生物转化的性能,并探讨了生物基催化体系的催化机制,用于白杜和黄连木种子油酯交换转化以及金银花和木姜叶柯中的黄酮糖苷水解转化。研究结果如下:（1）构建了磁性纳米纤维素基磷钨酸催化剂（ILs-HPW@MNC）,用于木本油料白杜种子油的酯交换转化。以天然可再生的微晶纤维素为原料,通过硫酸水解与磁性Fe₃O₄复合制备磁性纳米纤维素（MNC）,利用环氧氯丙烷改性,通过化学接枝的手段分别进行咪唑基、阴离子、磷钨酸（HPW）等多步接枝修饰,成功构建了具有两亲性催化界面的ILs-HPW@MNC,通过扫描电镜、粒径分布、氮气吸-脱附曲线对材料微观表面及孔隙结构测试;傅立叶红外光谱、元素分析、X射线衍射证明各步成功接枝;热重分析表明ILs-HPW@MNC热稳定性良好。循环利用实验表明材料具有良好的重复利用性。在最佳制备条件和转化条件下,催化白杜种子油酯交换反应脂肪酸甲酯转化率达96.7%,循环使用5次后转化率仍可达到75%以上,具备良好的重复利用性。（2）构建了天然纤维素基固定化酶催化体系（CALB-Si-MPCMs）,用于催化木本油料黄连木种子油高效转化生成脂肪酸甲酯。以天然纤维素原料,制备出磁性三维多孔纤维素微球（MPCMs）通过功能化修饰实现南极假丝酵母脂肪酶（CALB）的固定,系统优化固定化工艺参数,SEM表明材料呈现规则的三维球形结构且表面具有多孔特征,FTIR、EDS、XRD对其结构组成进行表征,结果证明CALB-Si-MPCMs成功制备,热重证实了材料具有良好的热稳定性,通过振动磁强计（VSM）进一步证明材料便于回收的特点。实验计算表明该反应符合一级动力学模型并通过阿伦乌尼斯方程计算反应活化能为80.3 k J/mol。该催化体系结合微波辅助生物催化技术成功催化黄连木油酯交换反应,最佳条件下,FAME转化率达到98.7%,并表现出良好的重复性。（3）构建了纤维素基靶向分子印迹-固定化酶双功能催化剂体系（MIH-LDHE）,用于特异性天然产物识别和生物转化,实现了金银花木犀草苷的高效生物转化。以木犀草苷为模板分子,通过模拟计算选择4-VP为功能单体,制备纤维素基分子印迹水凝胶并优化制备工艺,实现了对木犀草苷的靶向吸附(66.90 mg g^-1)。以层状结构类水滑石（LDH）为载体,固定化β-葡萄糖苷酶构建类水滑石固定化酶体系（LDHE）,优化其制备条件。分子对接揭示木犀草苷与β-葡萄糖苷酶结合机制。进一步优化复合条件制备。通过表征实验,分析其理化性质。MIH-LDHE催化木犀草苷转化效率为80.01%,约为LDHE的1.33倍,且对木犀草苷具有靶向识别能力和良好的可重复使用性。动力学与热力学分析证实吸附过程符合伪二级动力学模型与Langmuir单分子层吸附机制,酶促动力学表示MIH-LDHE具有较好的底物亲和力。MIH-LDHE使金银花中木犀草素含量提升5.66倍。（4）构建了天然多糖仿生催化系统（MIP-Fe/PE）,实现木姜叶柯根皮苷的高效生物转化。通过天然纤维素衍生物构建水凝胶基质,进一步硅烷化改性并聚合制备天然多糖分子印迹水凝胶（SCPMIH-Phz）,SCPMIH-Phz对根皮苷的吸附容量达63.07 mg g^-1。以磁性纳米粒子为载体固定化β-葡萄糖苷酶。分子对接揭示了根皮苷与β-葡萄糖苷酶形成强结合。进一步构建MIP-Fe/PE复合催化体系。通过表征证实了材料成功构建。动力学与热力学分析表明其吸附过程符合伪二级动力学模型与Langmuir单分子层吸附机制。酶促动力学分析表明MIP-Fe/PE亲和力优于NIP-Fe/PE。靶向吸附与转化性能研究中,MIP-Fe/PE对根皮苷的相对转化效率高于单一固定化酶Fe/PE,并且具有良好的靶向识别能力以及重复利用性。在MIP-Fe/PE催化下,木姜叶柯叶片中根皮素含量增加45倍。（5）构建了纤维素基分子印迹-酶“识别-转化”双功能系统（MIP-NSBAE）,通过特异性识别富集与酶促反应的协同效应显著提升了根皮苷向根皮素的生物转化效率。利用生物相容性的纤维素进行功能化改性纤维素水凝胶提供接枝位点,结合分子印迹技术实现根皮苷的靶向吸附。通过复合氨基化SBA固定化β-葡萄糖苷酶（NSBAE）技术,实现对底物根皮苷的特异性吸附与高效转化。该体系提升根皮苷转化效率（85.99%）,较单一固定化酶提升1.5倍。证明MIP-NSBAE对底物的预富集作用及酶活性中心的定向传递效应。动力学与热力学分析证实其吸附过程符合伪二级动力学模型与Langmuir单分子层吸附机制,酶促动力学分析显示,MIP-NSBAE对根皮苷的亲和力大于NIP-NSBAE,在靶向吸附与转化实验中,MIP-NSBAE对根皮苷的相对转化效率较高且表现出良好的靶向识别能力。MIP-NSBAE可使在木姜叶柯提取液的根皮素产量提升至25.7μg·g?1,较原料含量增加79.3倍且循环使用6次后效率仍保持82.1%。综上所述,本研究基于天然纤维素为载体,构建多种纤维素基固定化催化剂体系,有效解决了传统催化体系在天然产物转化过程中选择性低、稳定性差及分离回收困难等关键问题。在植物天然产物活性成分定向转化中展现出优异的底物选择性和高效催化性,为功能性油脂开发和植物天然产物生物转化领域提供了创新科技支撑,在植物资源的高值化利用中具有极大的应用潜力。更多还原

【Abstract】 Plant natural products are widely utilized in medicine,agriculture,food,and cosmetics due to their diverse bioactivities.High-value transformation of these compounds via efficient biocatalysis has become a global research focus.This study innovatively developed cellulose-based immobilized catalytic systems by integrating nanotechnology,magnetic materials,and biomimetic design,aiming to address challenges such as low selectivity,poor stability,and difficult recovery in traditional catalytic systems.The catalytic performance and mechanisms of these systems were systematically investigated for transesterification of woody oils and enzymatic hydrolysis of flavonoid glycosides.The research mainly includes the following aspects:（1）Magnetic nano cellulose-based phosphotungstic acid catalyst（ILs-HPW@MNC）was prepared for transesterification of Euonymus maackii Rupr.seed oil.Magnetic nano-cellulose（MNC）was prepared from natural and renewable microcrystalline cellulose by sulfuric acid hydrolysis and magnetic Fe₃O₄ compounding,modified by epichlorohydrin,and modified by chemical grafting,and ILs-HPW@MNC with amphiphilic catalytic interface was successfully constructed.The microstructure and pore structure of the material were characterized by scanning electron microscope,particle size distribution and nitrogen adsorption-desorption curve.Fourier transform infrared spectroscopy,elemental analysis and X-ray diffraction proved that each step was successfully grafted.Thermogravimetric analysis showed that ILs-HPW@MNC had good thermal stability.The recycling experiment showed that the material had good reusability.Under the optimum preparation and conversion conditions,the conversion rate of fatty acid methyl ester catalyzed by the transesterification of Euonymus maackii Rupr.seed oil reached 96.7%,and the conversion rate could still reach more than 75%after being recycled for five times,which showed good reusability.（2）Natural cellulose-based immobilized enzyme catalytic system（CALB-Si-MPCMs）was prepared to catalyze the highly efficient conversion of Pistacia chinensis seed oil to FAME.Magnetic three-dimensional porous cellulose microspheres（MPCMs）were prepared from natural cellulose.The immobilization of Candida antarctica lipase（CALB）was realized by functional modification,and the immobilization process parameters were systematically optimized.SEM showed that the material showed a regular three-dimensional spherical structure and the surface had porous characteristics.FTIR,EDS,and XRD were used to characterize the structural composition of the material,which proved that the CALB-Si-MPCMs were successfully prepared,and thermogravimetry confirmed the material’s good thermal stability,and the easy recycling of the material was further proved by VSM.Experimental calculations showed that the reaction conformed to the first-order kinetic model and the activation energy was calculated to be 80.3 k J/mol by the Arrhenius equation.The catalytic system combined with microwave-assisted biocatalytic technology successfully catalyzed the transesterification reaction of yellow horn seed oil,and the conversion of FAME under optimal conditions reached98.7%,which demonstrated a good reproducibility.（3）Cellulose-based targeted molecularly imprinted-immobilized enzyme bifunctional catalyst system（MIH-LDHE）was prepared for specific natural product recognition and biotransformation to achieve efficient biotransformation of Luteolin 7-glucoside（Lut-O）from Lonicera japonica Thunb.The cellulose-based molecularly imprinted hydrogel was prepared by using Lut-O as the template molecule,and 4-VP was selected as a functional monomer by simulation,and the preparation process was optimized to achieve the targeted adsorption of Lut-O(66.90 mg g^-1).Layered hydrotalcite-like（LDH）structure was used as a carrier to immobilizeβ-glucosidase to construct a hydrotalcite-like immobilized enzyme system（LDHE）,and the preparation conditions were optimized.Molecular docking revealed the binding mechanism of Lut-O andβ-glucosidase.Further optimize the compound conditions for preparation.The physical and chemical properties of MIH-LDHE were analyzed by characterization experiments,and the catalytic efficiency of MIH-LDHE for Lut-O conversion was 80.01%,which was about1.33 times higher than that of LDHE,and the system possessed the ability of target recognition of Lut-O and good reusability.The kinetic and thermodynamic analyses confirmed that the adsorption process conformed to the pseudo-secondary kinetic model and Langmuir monolayer adsorption mechanism,and the enzyme kinetics indicated that MIH-LDHE had a better affinity for the substrate.The content of luteolin in Lonicera japonica Thunb.was enhanced by 5.66-fold by MIH-LDHE.（4）Natural polysaccharide biomimetic catalytic system（MIP-Fe/PE）was constructed for the efficient biotransformation of phlorizin（Phz）in Lithocarpus litseifolius（Hance）Chun.The hydrogel matrix was constructed by natural cellulose derivatives,and the natural polysaccharide molecularly imprinted hydrogel（SCPMIH-Phz）was further modified by silanization and polymerized,the adsorption capacity of SCPMIH-Phz for Phz reached 63.07 mg g^-1.β-Glucosidase was immobilized with magnetic nanoparticles as the carrier.Molecular docking revealed that Phz formed strong binding withβ-glucosidase.The MIP-Fe/PE composite catalytic system was further prepared.The successful construction of the material was confirmed by characterization.Kinetic and thermodynamic analyses showed that the adsorption process was consistent with the pseudo-secondary kinetic model and Langmuir monolayer adsorption mechanism.Enzyme kinetic analysis showed that the affinity of MIP-Fe/PE was better than that of NIP-Fe/PE.In the target adsorption and conversion performance study,the relative conversion efficiency of MIP-Fe/PE to Phz was higher than that of the single immobilized enzyme Fe/PE,and it possessed a good target recognition ability as well as reusability.The phloretin（Phl）content in Lithocarpus litseifolius leaves catalyzed by MIP-Fe/PE increased 45-fold.（5）Cellulose-based molecularly imprinted-enzyme“recognition-conversion”bifunctional system（MIP-NSBAE）was constructed,and the bioconversion efficiency of Phz to Phl was significantly enhanced by the synergistic effect of specific recognition and enrichment and enzymatic reaction.Functionalized modified cellulose hydrogel using biocompatible cellulose provided grafting sites and molecular imprinting technology was used to achieve specific recognition of Phz.Through the immobilizedβ-glucosidase（NSBAE）technology of compound amination SBA,the specific adsorption and efficient transformation of Phz were realized.The system improved the conversion efficiency of Phz（85.99%）,which was 1.5 times higher than that of single immobilized enzyme.It is proved that MIP-NSBAE can preconcentrate the substrate and the directional transfer effect of enzyme activity center.Kinetic and thermodynamic analysis confirmed that the adsorption process conformed to the pseudo-second-order kinetic model and Langmuir monolayer adsorption mechanism.Enzyme kinetic analyses showed that the affinity of MIP-NSBAE for Phz was greater than that of NIP-NSBAE,and in the targeting adsorption and conversion experiments,the relative conversion efficiency of MIP-NSBAE for Phz was higher and it showed good target recognition ability.MIP-NSBAE increased the yield of Phl in the extract of Lithocarpus litseifolius（Hance）Chun to 25.7μg g^-1,which was79.3 times higher than that of the raw material,and the efficiency remained 82.1%after being recycled for 6 times.In summary,this study constructed a variety of cellulose-based immobilized catalyst systems based on natural cellulose as the carrier,which effectively solved the key problems of traditional catalytic systems,such as low selectivity,poor stability and difficulties in separation and recovery during the conversion of natural products.It shows excellent substrate selectivity and efficient catalytic properties in the directed conversion of plant natural products,which provides innovative scientific and technological support for the development of functional lipids and the bioconversion of plant natural products,and has great potential for application in the high-value utilization of plant resources.更多还原