假丝酵母产脂肪酶在壳聚糖和硅胶上的固定化研究

本实验分别壳聚糖和氨基化后的硅胶作为固定化载体,用戊二醛作为交联剂,将脂舫酶共价固定化于载体上,壳聚糖的最佳固定化条件是戊二醛浓度2．5％,给酶量为4125U／g,氨基化硅胶的最佳固定化条件为戊二醛浓度2％,最佳给酶量在3456U／g,固定化酶活为2952U／g,最佳酶活表现率为85％。两种方法所得的固定化酶的耐温性和储藏稳定性都有了显著提高。氨基化硅胶固定化酶在50℃下保温3小时后仍保留有14％的酶活。结果显示假丝酵母产脂肪酶固定于弱亲水性载体上要比固定于强亲水性载体上效果更好。     

胺化聚苯乙烯载体柔性固定化木瓜蛋白酶

提出酶的“柔性固定化”模型,并以Mannich反应得到担载量为0.4～6.0 mmol NH₂•g^-1的胺化聚苯乙烯树脂为载体,以双醛淀粉为柔性链,对木瓜蛋白酶进行柔性固定化,酶活回收率可达40%～50％,相当于手臂固定化酶活力回收率的1.8～2.4倍,且柔性固定化酶稳定性较好.该结果说明,酶的“柔性固定化”模型可以改善传统共价结合固定化酶及手臂固定化酶活力回收率不高的缺陷.     

壳聚糖载体柔性固定化木瓜蛋白酶

用酶柔性固定化模型,以壳聚糖为载体,双醛淀粉为柔性链,对木瓜蛋白酶进行柔性固定化. 通过对固定化条件的优化,得出选用壳聚糖、双醛淀粉制得的柔性载体(Chitosan-DAS50)在酶用量为14.4 mg/g(酶/干球)、pH 8的条件下,固定木瓜蛋白酶18 h,所得的固定化酶活力回收率达72%,相当于采用壳聚糖-戊二醛(Chitosan-GA)手臂载体的3倍. 结果表明,酶的柔性固定化模型可以改善传统共价结合法固定化及手臂固定化酶活力回收率不高的缺陷.     

以MOFs为载体的漆酶固定化研究进展

漆酶是一种环境友好的生物催化剂,在工业污水处理中通常使用固定化的方法来提高游离漆酶的活性、稳定性以及重复利用率。金属有机框架(MOFs)材料是一种性能优异的载体,由于其制备过程的灵活性、材料性能的可控性,MOFs材料能够有效地提高漆酶的负载量,减少使用过程中漆酶活性的损耗。介绍了漆酶的结构及催化机理,并从漆酶的3种固定化方式(吸附法、共价结合法、包埋法)出发,对传统载体材料与MOFs材料用于漆酶固定化的效果进行了比较,着重介绍了MOFs材料作为固定化载体的独特优势。结构可控、性能优良的MOFs材料能够有效推动固定化漆酶的实际应用,促进漆酶在生物类催化剂、传感器领域的发展。     

触角状环氧链结构对固定化脂肪酶(YCJ01)酶活的影响

为了改善酶的刚性固定化对酶蛋白构象的负面影响,以原子转移自由基聚合法（ATRP）合成了亲水性、柔性、触角状环氧化酶固定化载体PS-acyl-P（AM-co-GMA）,通过改变单体总量与引发剂量比例得到不同链长触角状环氧化酶固定化载体,并将其用于耐有机溶剂脂肪酶（YCJ01）的共价柔性固定化,重点考察了链长对固定化酶酶活的影响。结果表明,链长（增重率不超过3200%）越长,酶活越高。     

触角状环氧链结构对固定化脂肪酶(YCJ01)酶活的影响

为了改善酶的刚性固定化对酶蛋白构象的负面影响,以原子转移自由基聚合法(ATRP)合成了亲水性、柔性、触角状环氧化酶固定化载体PS-acyl-P(AM-co-GMA),通过改变单体总量与引发剂量比例得到不同链长触角状环氧化酶固定化载体,并将其用于耐有机溶剂脂肪酶(YCJ01)的共价柔性固定化,重点考察了链长对固定化酶酶活的影响。结果表明,链长(增重率不超过3200%)越长,酶活越高。     

氨基功能载体固定化酶研究进展

综述了甲壳素、壳聚糖天然氨基功能载体和氨基化硅胶化学合成载体的制备方法,并介绍了利用戊二醛直接固定、载体活化和酶活化固定化酶的方法,最后对氨基功能载体固定化酶的发展趋势加以评述:在利用氨基功能载体固定化酶过程中,有必要有针对性地合成一些新的氨基功能载体,使其反应条件更温和、酶的固载量更大、酶活力回收率更高、稳定性更强。或者针对特定的载体和酶,通过结合配体、添加稳定剂、固定前修饰、固定后修饰和后固定化技术处理等方法,进一步改善固定化酶的性能。     

固定酶法催化合成生物柴油Ⅰ.大孔树脂固定化脂肪酶的制备

研究了用于生物柴油酶催化的大孔树脂固定化脂肪酶的制备过程,考察和优化了脂肪酶固定化方法及条件。结果表明,采用大孔树脂D3520作载体,以载体涂布法固定化脂肪酶的最适固定化条件为:酶用量为酶∶树脂=0.16∶1(质量比),吸附时间1~3 h,pH值范围为9.0~9.4,固定化温度40℃。酶活力可达91.49 U/g,酶活回收率约为54%。     

表面活性剂改性磁性氧化石墨烯共价固定脂肪酶CRL

采用化学共沉淀法制备磁性氧化石墨烯（MGO）,直接在反应体系中加入Span系列表面活性剂,一锅法制备得到表面活性剂改性磁性氧化石墨烯（SMGO）。X射线衍射仪（XRD）、扫描电镜（SEM）和傅里叶变换红外光谱（FTIR）表征结果表明SMGO制备成功,且具有良好的磁分离性能。以1,5-戊二醛（GA）为交联剂,褶皱假丝酵母脂肪酶（CRL）为模型酶,共价固定CRL于SMGO载体上。Span40 MGO固定化酶酶活回收率为116.5%±1.7%,为MGO固定化酶的6倍;比活可达32.5U/mg,为游离酶的1.6倍;k_cat/K_m也有较大的提升,高于游离酶50%。储存稳定性及热稳定性得到提高,用于水解反应6批次后仍然保留73.6%的相对酶活力。初步分析认为MGO经改性后表面从亲水性转为强疏水性,使得共价固定化过程中同时发生疏水性界面活化,这是酶活性提高的原因之一。文章所报道的改性策略可为类似载体改性提供新思路。     

超氧化物歧化酶的固定及其活性检测

目的探讨超氧化物歧化酶(SOD)的固定及固定化SOD活性的检测方法。方法以淀粉为载体,对SOD进行固定,采用改进的邻苯三酚测活法(以Vc为终止剂),并结合过滤或离心的方法去除沉淀,测定固定化SOD的活性。比较过滤法与离心法,及过滤法与经典邻苯三酚法测定酶活性的差异,并检测过滤法的线性、精密性和准确性。结果过滤法检测SOD活性的精密性较离心法高,与经典邻苯三酚法检测结果差异无统计学意义,在SOD浓度为6~60μg/ml范围内,SOD活性值与浓度线性关系良好(r=0.999 5),精密性及准确性良好。固定化SOD的比活性为1 730.07 U/g,酶活回收率为53.3%。结论以淀粉为载体固定化SOD效果好,经改进的邻苯三酚法可用于固定化SOD的活性测定。     

酶自固定化方法研究进展

固定化酶有诸多优势,传统酶固定化方法通常需要外界辅助,但额外的固定化流程无疑会增加酶使用成本。近年来提出的酶自固定化则可在温和条件下实现自身固定化,简化固定流程,为降低酶的成本、促进酶的广泛应用提供了新思路。本文系统、深入介绍了无载体类型、自催化类型和自结合类型酶自固定化的方法,并指出其各自特点：无载体类型无需载体成本;自催化类型能有效降低载体成本,获得机械强度较高的固定化酶;自结合类型可在温和条件下易实现多酶固定。结合本文作者课题组的相关研究,阐述了酶自身催化的自固定化和蛋白或多肽介导的融合酶自固定化最新研究进展。最后指出当前研究的重点应致力于自固定化新方法的研发,为后期理性选择、合理组合自固定化方法奠定基础,以加快自固定化酶实际应用。     

Bioorthogonal chemistry for site-specific labeling and surface immobilization of proteins

Understanding protein structure and function is essential for uncovering the secrets of biology, but it remains extremely challenging because of the high complexity of protein networks and their wiring. The daunting task of elucidating these interconnections requires the concerted application of methods emerging from different disciplines. Chemical biology integrates chemistry, biology, and pharmacology and has provided novel techniques and approaches to the investigation of biological processes. Among these, site-specific protein labeling with functional groups such as fluorophors, spin probes, and affinity tags has greatly facilitated both in vitro and in vivo studies of protein structure and function. Bioorthogonal chemical reactions, which enable chemo- and regioselective attachment of small-molecule probes to proteins, are particularly attractive and relevant for site-specific protein labeling. The introduction of powerful labeling techniques also has inspired the development of novel strategies for surface immobilization of proteins to create protein biochips for in vitro characterization of biochemical activities or interactions between proteins. Because this process requires the efficient immobilization of proteins on surfaces while maintaining structure and activity, tailored methods for protein immobilization based on bioorthogonal chemical reactions are in high demand. In this Account, we summarize recent developments and applications of site-specific protein labeling and surface immobilization of proteins, with a special focus on our contributions to these fields. We begin with the Staudinger ligation, which involves the formation of a stable amide bond after the reaction of a preinstalled azide with a triaryl phosphine reagent. We then examine the Diels-Alder reaction, which requires the protein of interest to be functionalized with a diene, enabling conjugation to a variety of dienophiles under physiological conditions. In the oxime ligation, an oxyamine is condensed with either an aldehyde or a ketone to form an oxime; we successfully pursued the inverse of the standard technique by attaching the oxyamine, rather than the aldehyde, to the protein. The click sulfonamide reaction, which involves the Cu(I)-catalyzed reaction of sulfonylazides with terminal alkynes, is then discussed. Finally, we consider in detail the photochemical thiol-ene reaction, in which a thiol adds to an ene group after free radical initiation. Each of these methods has been successfully developed as a bioorthogonal transformation for oriented protein immobilization on chips and for site-specific protein labeling under physiological conditions. Despite the tremendous progress in developing such transformations over the past decade, however, the demand for new bioorthogonal methods with improved kinetics and selectivities remains high.     

醇脱氢酶在聚乙烯膜表面的固定化研究

以聚丙烯酸（PAA）改性的聚乙烯（PE）膜为载体，研究了醇脱氢酶（ADH）的两种固定化路线，并以甲醛为底物考察了固定化酶的催化性能。路线1用聚乙烯亚胺（PEI）进一步改性，使用戊二醛（GA）固定化ADH。最优固定化pH为6.0，温度为5～15℃，酶浓度为1.0 mg/ml，GA浓度为0.01%(质量)；固定化酶的最适反应pH为6.5，温度为15～30℃，反应速率最高为9.6 μmol/(L·min)；重复利用10次后可保持47.3%的活性。路线2以PAA-PE为载体，用1-(3-二甲氨基丙基)-2-乙基碳二亚胺盐酸盐（EDC）和N-羟基琥珀酰亚胺（NHS）为活化剂，固定化ADH。EDC和NHS最优摩尔比为1∶0.5，固定化时间为24 h；固定化酶的最适反应pH为6.5，温度为20～37℃，反应速率为15.58 μmol/(L·min)；重复利用10次后可保持53.8%的活性。     

Immobilization of alcohol dehydrogenase on the surface of polyethylene membrane

Using polyacrylic acid (PAA) modified polyethylene (PE) membrane as a carrier, two immobilization routes of alcohol dehydrogenase (ADH) were studied, and the catalytic performance of immobilized enzyme was investigated using formaldehyde as a substrate. In the first route, PAA-PE membrane was further modified by polyethyleneimine (PEI) and then ADH was covalently linked by glutaraldehyde (GA) to the surface of PEI/PAA-PE. The results show that the optimal immobilization pH was 6.0, immobilization temperature was 5—15℃, ADH and GA concentrations were 1.0mg/ml and 0.01%(mass). For immobilized enzyme, the optimal reaction pH was 6.5, temperature was 15—30℃, and the highest reaction rate was 9.6 μmol/(L·min), the remaining activity was 47.3% after 10 use cycles. In the second route, ADH was immobilized on PAA-PE membrane with 1-(3-dimethylaminopropyl)-2-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as activators. The results show that the optimal molar ratio of EDC and NHS was 1∶0.5, and the immobilization time was 24 h. For immobilized enzyme, the optimal reaction pH was 6.5, temperature was 20—37℃, and the highest reaction rate was 15.58 μmol/(L·min), 53.8% activity was remained after 10 cycles.     

生物燃料电池酶电极的研究进展

综述了生物燃料电池酶电极的研究进展,尤其是近年来在氧化还原酶的种类、电子介体电极、直接电子传递电极以及固定化酶等方面的研究成果。从提高生物燃料电池的转换效率出发,分析各因素对酶电极性能的影响,包括针对不同底物燃料使用相应的氧化还原酶实现电极之间的电子传递、小分子或聚合物中介体存在下提高电流密度、导电聚合物等修饰电极对直接电子传递效率的贡献,以及物理或化学的酶固定化方法增加酶的稳定性等。因此采用新材料及新工艺构筑酶电极,最大程度上保持酶的活性,提高载酶量及电子传递效率,将成为该领域未来的发展方向。     

纳米TiO2光催化剂固定化技术研究进展

纳米TiO₂光催化剂在废水治理领域有广阔的应用前景,固定化技术是其应用关键。将TiO₂光催化剂在一定基材上进行负载和固定化,解决TiO₂悬浮体系存在的后期分离回收难的问题。介绍了固定化常用的载体和固定化技术方法,指出今后纳米TiO₂光催化剂固定化技术需要解决和研究的问题：寻找合适的载体与固定方法完成对TiO₂的固定化,既能提供较强的牢固性,又能保护甚至提高TiO₂的光催化活性; 研究载体与光催化剂之间的相互作用,探讨固载过程中各个影响因素对光催化效率的影响; 固定化所带来的传质受限问题以及负载所引起比表面积减少的问题还有待进一步研究;由实验室小型反应装置转向大型化高效光催化反应器的设计还缺少工程经验。     

Nickel-Carnosine complex:A new carrier for enzymes immobilization by affinity adsorption

Immobilization is an effective method to promote the application of enzyme industry for improving the stability and realizing recovery of enzyme.To some extent,the performance of immobilized enzyme depends on the choice of carrier material.Therefore,the development of new carrier materials has been one of the key issues concerned by enzyme immobilization researchers.In this work,a novel organic-inorganic hybrid material,nickel-carnosine complex (NiCar),was synthesized for the first time by solvothermal method.The obtained NiCar exhibits spherical morphology,hierarchical porosity and abun-dant unsaturated coordination nickel ions,which provide excellent anchoring sites for the immobiliza-tion of proteins.His-tagged organophosphate-degrading enzyme (OpdA) and ω-transaminase (ω-TA)were used as model enzymes to evaluate the performance of NiCar as a carrier.By a simple adsorption process,the enzyme molecules can be fixed on the particles of NiCar,and the stability and reusability are significantly improved.The analysis of protein adsorption on NiCar verified that the affinity adsorp-tion between the imidazole functional group on the protein and the unsaturated coordination nickel ions on NiCar was the main force in the immobilization process,which provided an idea way for the develop-ment of new enzyme immobilization carriers.