猪骨基多孔碳固定化酶的研究

本文以猪骨基多孔碳为载体,采用明胶包埋与戊二醛交联结合的方法,制备固定化过氧化氢酶,并对固定化酶制备条件的优化,酶作用最适条件及其稳定性进行研究。结果表明:以猪骨基多孔碳为载体吸附2h、1%明胶包埋、1%戊二醛交联过氧化氢酶制备的固定化酶,在最适的缓冲液浓度、溶液pH和体系温度下,虽然催化效率有所下降,但其贮存稳定性和操作稳定性都有所增加,重复使用8次后,活力仍保持在初始活力的90%以上。这对于过氧化氢酶进一步在食品工业和纺织工业的推广可能有潜在的应用价值。     

修饰固定化青霉素酰化酶在非水相中的催化

为提高酶的催化水解活力和稳定性,将青霉素酰化酶组装于介孔泡沫二氧化硅(MCFs)中,并应用于水/有机混合体系催化水解。分别考察了有机介质种类和体积分数、葡聚糖(Dex10k)修饰对固定化酶活力的影响,研究了不同条件下固定化酶的稳定性。实验结果显示:体积分数20%石油醚中,添加Dex10k的介孔泡沫硅固定化酶比活力达209.5U/mg,是缓冲液中MCFs固定化酶活力的196.2%。20%石油醚中,经25次连续操作,固定化酶保持初始活力的71.5%。结果表明:石油醚等烷烃形成的水/有机体系是适合青霉素酰化酶催化的二相体系,且添加Dex10k能提高固定化酶在二相体系中的催化活力及稳定性。     

超氧化物歧化酶的固定化及其酶学性质

目的制备固定化超氧化物歧化酶(SOD),并分析其酶学性质。方法以壳聚糖为载体,戊二醛为交联剂,制备固定化超氧化物歧化酶。以邻苯三酚自氧化法分别测定溶液酶与固定化酶的活力,并计算回收率。对固定化酶的温度和pH稳定性、半衰期、重复使用的回收率及米氏常数(Km)进行测定。结果固定化超氧化物歧化酶活力为333U/g,酶活回收率为86.32%,半衰期为43.8d;固定化酶室温保存5d后,相对酶活力仍保持在80%以上,最适反应温度为45℃,使用一次后回收率为70.12%,重复使用两次后回收率为51.72%;固定化酶与溶液酶在pH6时活性最强,Km分别为0.18mmol/L和0.16mmol/L。结论该固定化酶较溶液酶的稳定性得到提高,便于贮存,在食品、药品、日用品等领域有良好的应用前景。     

氨基树脂固定化S-腺苷甲硫氨酸合成酶的研究

以氨基树脂为载体对S-腺苷甲硫氨酸(SAM)合成酶进行固定化,优化了酶的固定化条件并对固定化酶的性质进行了研究。优化的固定化条件为:戊二醛体积分数5%、SAM合成酶添加量20mg·g-1、固定化时间5h。所制备的固定化SAM合成酶的酶活力为476.8U·g-1,酶活力回收率为74.5%。与游离SAM合成酶相比,固定化SAM合成酶的稳定性大幅提高,在50℃孵育5h酶活力仍保留61.2%,而游离SAM合成酶则完全失活;在pH值为6.0~6.5、8.0~9.5的缓冲溶液中,固定化SAM合成酶也更加稳定;固定化SAM合成酶连续催化反应10批次,酶活力保留86.3%;固定化SAM合成酶在4℃储存30d,酶活力保留81.4%。固定化SAM合成酶米氏常数KATPm=0.14mmol·L-1,KLm-Met=0.28mmol·L-1。     

磁性SiO2纳米粒子的制备及其用于漆酶固定化

以氨基修饰的磁性SiO2纳米粒子为载体,通过交联剂戊二醛固定漆酶,对固定化条件进行了优化,比较了固定化酶与游离酶的酶学性质. 结果表明,漆酶固定化的最佳条件为戊二醛浓度8%(w),固定化时间6 h,缓冲液pH值7.0,初始酶液浓度0.15 g/L. 固定化的漆酶的最适pH为4.0,最适温度为20℃. 在60℃条件下保温4 h,固定化漆酶仍能保持酶活力60.9%,在连续10次操作后,酶活力仍能保持55%以上,其热稳定性和操作稳定性均比游离酶高.     

固定化细胞延胡索酸酶性质的研究

探讨了海灌酸钠固定化普通变形杆菌延胡索酸酶的性质。研究结果表明：酶催化延胡索酸为Ｌ－苹果酸的最适温度为４０℃，最适ｐＨ为７．０。在温度低于４０℃和ｐＨ５。０～８．０的条件下，固定化细胞酶活力稳定性好，显示其热稳定性和ｐＨ稳定性明显优于游离细胞。Ｃｙｓ对固定化细胞酶活力无影响，ＧＳＨ对前激活作用。     

聚乙烯醇包埋固定Burkholderia cepecia JS-02细胞的研究

用聚乙烯醇(PVA)凝胶包埋固定法对BurkholderiacepeciaJS-02细胞进行了固定化,所得凝胶具有良好的机械性和稳定性。固定化凝胶最佳质量分数为9%,最适湿细胞包埋量为0 28g/mL,固定化JS-02细胞酶活回收率为75%,连续反应6批后,固定化细胞活力为初始活力的86%。固定化细胞的最适pH为9 0,最适温度为50℃,固定化细胞的储存稳定性及操作稳定性高于游离细胞。     

转谷氨酰胺酶在聚丙烯微孔膜上的固定化

研究了转谷氨酰胺酶在聚丙烯微孔膜上的化学固定化的影响因素,确定了最佳固定化工艺条件,即为:第一步光照反应6 min,单体质量分数为20%,第二步光照时间25 min,接枝率最高可达35.2%;己二胺质量分数为25%,胺烷基化时间150 min,胺烷基化温度60℃;戊二醛质量分数3%,戊二醛作用时间45 min;酶液浓度10 mg/mL,固定化时间20 h,固定化温度4℃,固定化酶膜的活力最高可达游离酶的45%.并研究了温度、pH、金属离子对固定化酶膜的酶学性质的影响,其贮存性能和操作稳定性也做了初步研究.     

磁性壳聚糖微球的制备及其用于甲酸脱氢酶的固定化

分别采用乳化交联法和共沉淀法制备磁性壳聚糖微球载体,并对形貌结构进行比较,结果表明,采用共沉淀法制备的磁性壳聚糖微球负载Fe3O4的效果好,故将其作为载体固定甲酸脱氢酶。最佳固定化条件:添加酶量9 U.g-1,pH=7.0,固定化时间5 h。游离酶和固定化酶的最适宜反应温度分别为50℃和30℃;游离酶的最适宜pH=7.0,固定化酶的最适宜pH=6.0;将游离酶和固定化酶分别置于60℃恒温水浴放置180 min后,游离酶和固定化酶的相对酶活力分别为0.78%和40.39%;将游离酶和固定化酶置于不同pH的缓冲液中保存1 h后,在强酸(pH=2.0)和强碱(pH=10.0)条件下,固定化酶的相对酶活力分别为11.03%和38.43%,游离酶已全部失活;固定化酶重复使用6次后,相对酶活力为73.53%,表明固定化酶具有较好的热稳定性、酸碱稳定性和操作稳定性。     

以壳聚糖-戊二醛为载体柔性固定假丝酵母脂肪酶candida rugosa lipase

以壳聚糖-戊二醛(Chitosan-GA)为载体固定假丝酵母脂肪酶candida rugosa lipase(CRL)最高酶活可达240μ·g~(-1)。Chitosan-GA固定化酶循环使用4次后保留了20%左右的初始酶活力。固定化酶在水相中保存185d后保留了40%以上初始酶活力。固定化酶在有机溶剂(正庚醇)中浸泡120h后固定化酶酶活保留32.2%,而游离酶只保留原始酶活的8.5%。Chitosan-GA载体固定化脂肪酶在水相和有机相中的保存稳定性都好于游离酶。     

Stabilization of entrapped catalase using photo‐crosslinked resin gel for use in wastewater containing hydrogen peroxide

Catalase immobilized using photo‐crosslinkable resin was applied to treatment of wastewater containing a low concentration of hydrogen peroxide. The structure of photo‐crosslinked resin gel was stable even in the acceleration test of concentrated hydrogen peroxide for a long period. Accordingly, the most important subject of this process was maintaining long term catalase activity. The stabilization of immobilized catalase was investigated by modifying gel carriers with some prepolymers and functional monomers, and adding some stabilizing materials without modification of catalase itself. But these gel carriers did not improve the stability beyond 20%. Addition of Tween 80, polyoxyethylene (5) sorbitan monooleate (hydrophilic/lipophilic balance, HLB: 10.0), to catalase was most effective for maintenance of the activity when the enzyme was immobilized in hydrophilic photo‐crosslinked resin, resulting in 80% of its initial activity. It was assumed that this surfactant did not influence the permeation of hydrogen peroxide into gel, but acted as a stabilizer for catalase in the gel carrier. © 2000 Society of Chemical Industry     

一种嗜热嗜碱过氧化氢酶的分离纯化和固定化

引言过氧化氢酶(catalase,简称CAT,EC1.11.1.6)是一种高效催化分解过氧化氢的酶,广泛存在于好氧微生物和动植物体内。在纺织印染工业、造纸工业、废水处理、纸浆、牛奶保鲜以及临床分析等方面有着广泛的应用[1-3]。     

Enhancing the stability of immobilized catalase on activated carbon with gelatin encapsulation

Gelatin (Gel) encapsulation onto activated carbon (AC) with catalase (CAT) was developed as an alternative method for CAT immobilization in this study. The immobilized CAT with AC encapsulated by Gel as the supporter accounted for 65.69% of the native enzyme activity. Furthermore, the properties of the immobilized CATs were characterized by Fourier transform infrared spectroscopy and scanning electron microscopy. Among free CAT and the two immobilized CATs, the immobilized CAT with AC encapsulated by Gel as the supporter showed the highest relative enzymatic activity and a high stability in a broad range of pH values and temperatures, and its residual activity was 80% after 15 uses, whereas the immobilized CAT with AC as the supporter was retained at a level of only 50% under the same conditions. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 130: 1498–1502, 2013     

Immobilization of catalase by polyion complex

Catalase was immobilized in a polyion complex from poly(sodium 2-acrylamide-2-methyl-1-propanesulfonate) and poly[2-(trimethylammonioethyl) methacrylate chloride]. Polyion complex formation between the two polyions was found to proceed stoichiometrically by the turbidimetric titration. Immobilization of catalase was achieved by conducting the complexation in the presence of the enzyme, which was established to be incorporated in the polyion complex networks. The fixed catalase showed higher thermal stability than the original enzyme. Furthermore, the stability and the activity of the fixed catalase were improved by annexing inert components such as albumin, histone, or lecithin at the time of polyion complex formation.     

Improved catalytic activity by catalase immobilization using γ‐cyclodextrin and electrospun PCL nanofibers

Nanofibrous structures are promising for biocatalyst immobilization due to their large surface area which facilitates the enzyme attachment, stability, ease of separation, and fine porous structure. There is limited research available on the change in enzyme activity following interaction with cyclodextrin. In this study, catalase enzyme was immobilized into nanofibrous structures by various techniques, with and without γ‐CD addition, and the enzymatic activity of catalase was evaluated. In addition, catalase‐γ‐CD complex containing PEO polymer solution was electrospun in between PCL nanofibrous layers as a newly developed technique. The enzyme immobilized nanofibrous structures were characterized by SEM, XRD, and FT‐IR analysis methods. Among all the activity tests, best enzyme activity was recorded with catalase‐γ‐CD physical mixture encapsulated PCL nanofibrous layers. Moreover, the test results indicated that the use of cyclodextrin in immobilization process considerably improves the catalytic activity of the enzyme. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134, 44404.     

Immobilization of catalase in poly(isopropylacrylamide-co-hydroxyethylmethacrylate) thermally reversible hydrogels

Catalase was entrapped in thermally reversible poly(isopropylacrylamide-co-hydroxyethylmethacrylate) (pNIPAM/HEMA) copolymer hydrogels. The thermoresponsive hydrogels, in cylindrical geometry, were prepared in an aqueous buffer by redox polymerization. It was observed that upon entrapment, the activity retention of catalase was decreased between 47 and 14%, and that increasing the catalase loading of hydrogel adversely affected the activity. The kinetic behaviour of the entrapped enzyme was investigated in a batch reactor. The apparent kinetic constant of the entrapped enzyme was determined by the application of Michaelis–Menten model and indicated that the overall reaction rate was controlled by the substrate diffusion rate through the hydrogel matrix. Due to the thermoresponsive character of the hydrogel matrix, the maximum activity was achieved at 25 °C with the immobilized enzyme. The K_m value for immobilized catalase (28.6 mM) was higher than that of free enzyme (16.5 mM). Optimum pH was the same for both free and immobilized enzyme. Operational, thermal and storage stabilities of the enzyme were found to increase with immobilization. © 1999 Society of Chemical Industry     

Attachment of Lipase on Amino Functionalized Titania Submicrospheres via Covalent Binding

A facile and effective method for immobilized lipase was presented. The titania submicrospheres were synthesized via a modified sol-gel method followed by amino functionalization through the chelation between dopamine and titania. Lipase was covalently attached on the functionalized titania surface using glutaraldehyde as the cross linking agent. The loading ratio and relative activity of the immobilized lipase were 230 mg/g titania submicrospheres and 65%, respectively. The kinetic parameters including the Michaelis constant (K_m) and the maximum reaction rate (V_max) changed slightly after immobilization. Compared to free lipase, the immobilized lipase showed favorable pH stability, thermostability, recycling stability and storage stability. The immobilized lipase retained 90% activity after incubation at 50 ℃ for 2 h, while the free lipase retained only 60% activity. The immobilized lipase retained more than 80% activity after 8 batches.     

海藻酸钠-明胶协同固定化S-腺苷甲硫氨酸合成酶的研究

以海藻酸钠和明胶为载体,对S-腺苷甲硫氨酸合成酶进行固定化。再用戊二醛对其进一步交联,增强固定化酶的稳定性。考察了海藻酸钠和明胶质量分数、CaCl2质量分数、酶和载体比例以及交联剂戊二醛体积分数等因素对固定化酶的影响。结果表明,最佳固定化条件为:海藻酸钠质量分数2.0%、明胶质量分数1.0%、CaCl2质量分数4.0%、固定化酶量为2.5 g/L凝胶、戊二醛体积分数0.6%。交联固定化酶热稳定性得到大幅度提高,在50℃下保温5 h仍保留72%的活力,而游离酶则完全失活。交联固定化酶在碱性溶液中的稳定性较高,在pH=8.0～9.0的缓冲液中4℃保温10 h酶活性仍保留87%以上。将交联固定化酶用于S-腺苷甲硫氨酸的合成,连续反应8批次后酶活性仍保留65%。     

Production of Indigo by Immobilization of E. coli BL21 （DE3） Cells in Calcium-Alginate Gel Capsules

The ability of catalyzing indole into indigo of gene engineering strain expressing P450 BM3 immobilized by entrapment in calcium-alginate gel capsules was examined,and various characteristics of immobilized cells were assessed.Optimum conditions for cells activity were not affected after immobilization,and pH and tempera- ture for both free and immobilized cells were found to be pH 7.5 and 35℃,respectively.The immobilized cells exhibited a markedly improved thermal stability than free cells.After five repeated experiments,the yield of indigo with the immobilized cells retained over 94%of their original activity,which indicated that the operational stability for recycling in batch processes was improved.