聚丙烯酸载体用于青霉素酰化酶的固定

以反应性单体丙烯酸和交联剂二乙烯基苯,以石油醚为致孔剂,通过悬浮聚合制备固定化酶的载体,并用于对青霉素酰化酶的固定。研究了丙烯酸与二乙烯基苯以不同摩尔比对青霉素酰化酶固定活性的影响,以及悬浮聚合时水油相比例的不同所合成的载体对固定化酶性能的影响。当丙烯酸和二乙烯基苯摩尔比为84.2:4时合成的载体固定青霉素酰化酶的酶活为2784U/g,而水油相比为2.75:1（丙烯酸和二乙烯基苯摩洋比为84.2:5）时固定青霉素酰化酶活达到2183U/g。固定青霉素酰化酶可使青霉素转化,得到半合成青霉素的中间体6-氨基青霉烷酸,由此可制成高效、广谱、服用方便的新青霉素。     

青霉素酰化酶在介孔分子筛MCM-41上的固定化研究

通过改变酶固定化时的pH值、温度、时间以及酶用量,研究了固定化条件对MCM-41载体上固定化青霉素酰化酶的酶活及其稳定性的影响,明确了酶活及其稳定性随固定化条件的变化规律,初步分析了青霉素酰化酶在介孔分子筛MCM-41上的固定化过程。     

固定化扩展青霉脂肪酶的制备及其在玉米油转酯反应中的应用

采用吸附法对来源于扩展青霉Penicillium expansum的脂肪酶进行了固定化.从20种不同来源的树脂中筛选出固定化效率高且价格低廉的D4020树脂作为载体,系统研究了固定化条件对固定化效率及固定化酶转酯活力的影响.结果表明,最适加酶量、缓冲液pH和吸附时间分别为0.7 g/g、9.4和4 h.冻干时添加0.5%的半乳糖有助于提高固定化酶的转酯活力.在上述优化条件下,固定化酶的转酯活力为404.0 U/g,而所用的游离酶不能催化该转酯反应.利用该固定化酶催化玉米油转酯反应生产生物柴油时,叔戊醇为适宜的反应介质,其最适添加量为0.5 ml/g;适宜的酶量、加水量和反应温度分别为60.6 U/g、油重的1.2%和35℃.按醇/油摩尔比为1的比例分别在反应0、2和6 h时加入甲醇,在优化反应条件下,反应24 h后甲酯产率达85.0%;固定化脂肪酶具有较好的操作稳定性,反应10批次时,相对酶活力为62.8%.     

固定化青霉素酰化酶新型载体PEI/SiO2的制备及其特性

通过γ-氯丙基三甲氧基硅烷的媒介, 将聚乙烯亚胺(PEI)化学偶联在硅胶微粒表面, 制备了固定化青霉素酰化酶的新型复合载体PEI/SiO2, 最终制得了活性高且稳定性好的固定化青霉素酰化酶. 通过测定复合载体表面PEI的偶合量, 考察了各种反应条件对复合载体制备的影响规律; 通过红外光谱与电导滴定法测定, 对复合载体表面的化学结构与组成进行了表征; 为探索复合载体PEI/SiO2固定化酶的作用机理, 测定了复合载体在固定化酶前的ζ电位. 研究结果表明, 通过氯丙基硅烷偶联剂的媒介, 聚胺大分子PEI可以充分地被化学偶联在SiO2表面, 键合量可达到15%. 偶联反应的适宜条件: 反应温度90-94 ℃; 反应时间5h; PEI的质量浓度0.45-0.50 g/mL. 由于PEI分子链中含有大量氨基, 少量的共价键联与大量的物理吸附相结合, 既可使青霉素酰化酶被快速稳定地固定化, 又能很好地保持酶的构象, 使其具有较高的催化活性与活力回收率, 而且具有良好的连续操作稳定性, 重复使用15次, 固定化酶的活性可稳定地保持在初活性的87.5%水平上.     

青霉素酰化酶在含铁MCM-41介孔分子筛上的固定化研究

制备了具有长程有序结构、孔径分布狭窄的含铁MCM-41介孔分子筛，利用直接法和共价结合法将青霉素酰化酶固定在分子筛表面。结果表明，两种方法制备的固定化酶对青霉素G水解反应的表观活性分别为782U/g和256U／g；经6次连续操作使用，二者保持初始活性的49．4％和81．2％，后者的操作稳定性好于前者。共价结合法制备的固定化酶活性较低，是由于Fe—MCM-41表面修饰后比表面积和孔径明显减小所致。     

介孔材料的修饰及固定青霉素酰化酶的稳定性研究

利用扩孔剂的作用合成出较大孔径(12 nm)的介孔材料SBA-15, 并进行表面氨基修饰, 以此为载体, 以戊二醛为交联剂, 对青霉素酰化酶进行组装固定, 并对固定化青霉素酰化酶(PGA)的稳定性进行了深入的研究. 实验结果表明, PGA与载体交联后仍保持活性. 热稳定性研究结果表明, 制备的固定化青霉素酰化酶在低于60 ℃时保持稳定; pH在6~11范围内保持稳定; 固定化酶重复使用10次之后, 仍具有高达90%的残留活力.     

含环氧基亲水性固定化青霉素酰化酶共聚载体的合成与性能研究

环氧基团可以在温和条件下与酶分子的氨基反应使其固定于载体表面.选用含有活性环氧基团的甲基丙烯酸缩水甘油酯(GMA)和亲水性的N-乙烯吡咯烷酮(NVP)两种单体,以N,N′-亚甲基双丙烯酰胺(MBAA)为交联剂,甲醇水溶液作致孔剂,液体石蜡为主介质,通过反相悬浮聚合技术成功地合成了亲水性大孔GMA-NVP-MBAA三元共聚物载体(GNM).通过调节交联剂的用量和单体NVP与GMA的比例,可以调节载体的孔径、比表面积及在水中的溶胀性能.将巨大芽孢杆菌青霉素酰化酶共价偶联于平均孔径为16.5nm、表面环氧基含量为0.906mmol/g的GNM共聚物载体,制成固定化酰化酶,其表观活性高达625U/g,水解青霉素G钾盐的最适宜温度为50℃,pH值为8.0.固定化酶在4℃保存40d,活性保持不变.经3次使用后,活性达到稳定值(601U/g左右),再经12次使用,活性几乎保持不变.     

青霉素酰化酶在甲基丙烯酸缩水甘油酯共聚物上的固定化

 用共价键合法将青霉素酰化酶固定化在珠状多孔的甲基丙烯酸缩水甘油酯（GM）共聚物上，研究了固定化反应时间、温度、pH值和酶液用量对固定化青霉素酰化酶的表观活性、表观偶联效率、活性回收及稳定性的影响．将GM共聚物载体加入到磷酸缓冲液（0．1mol／L，pH10．8）与青霉素酰化酶液（每克干载体用酶液1ml）的混合溶液中，在30℃下反应72h，单位质量（干重）固定化酶的表观活性为348U／g，表观偶联效率为66．7％，活性回收为31．7％．     

大孔阴离子树脂DEAE-E/H固定化氨基酰化酶的研究

以弱碱性大孔阴离子树脂DEAE-E/H为载体固定化氨基酰化酶.通过对影响固定化结果的几个因素,如树脂的离子类型、pH值、温度,以及自由酶液浓度等进行系统研究,得到了适宜的固定化条件:将DEAE-E/H转化为Ac-型;自由酶液浓度120U/ml,pH值6.5;固定化温度为常温.在此条件下制备的固定化氨基酰化酶比酶活可达1200U/g～1500U/g,酶活保留率超过60%.DEAE-E/H作为固定化载体,具有价格低廉,物理性能好,固定化方法简便等特点,具有很好的工业应用前景.     

树枝状大分子聚酰胺-胺用于氨基酰化酶的固定化研究

以树枝状大分子修饰的硅胶为载体,戊二醛为交联剂,对氨基酰化酶进行固定化研究。考察了树枝状大分子的代数、戊二醛浓度、反应温度与时间对氨基酰化酶固定化效果的影响,并且考察了该固定化酶的最佳酶解条件。结果表明,随着树枝状大分子代数的增加,固定化的酶量随之增大,同时,固定后的酶仍然保持较高的活性。     

介孔材料MCFs的合成及组装青霉素酰化酶的性质研究

介孔材料由于具有纳米级规则孔道和巨大的比表面积而在催化、吸附及分离等方面存在较大的应用价值．近年来,由介孔分子筛如MCM-41和SBA-15州等组装功能性材料已成为研究的热点．酶作为高效催化剂有许多优点,但在溶液中易失活,使用后无法回收,有的酶在溶液中还存在自水解问题：将酶组装在介孔材料中制成固定化酶则可解决上述问题．目前已成功地将辣根过氧化物酶     

不同介孔材料固定青霉素酰化酶的稳定性研究

介孔材料由于具有在2～30nm之间可调的纳米级规则孔道、大比表面积和强吸附性能而成为固定化酶的优良载体．将酶固定于介孔材料的孔道中制备成的固定化酶与溶液酶相比,有易于与产物分离,并可回收和反复使用,可降低生产成本,减少酶的自水解和保持酶的活性．青霉素酰化酶（Penicillin acylase,PGA,EC．3．5．1．11）又称为青霉素酰胺酶或青霉素氨基水解酶,该酶属于球蛋白,分子量较大,由2个亚基组成：分子量为19500的含有侧链结合位点的亚基和分子量为60000的含有催化位点的亚基．     

Immobilization-stabilization of Penicillin G acylase fromEscherichia coli

We have developed a strategy for immobilization-stabilization of penicillin G acylase from E. coli, PGA, by multipoint covalent attachment to agarose (aldehyde) gels. We hve studied the role of three main variables that control the intensity of these enzyme-support multiinteraction processes: 1. surface density of aldehyde groups in the activated support; 2. temperature; and 3. contact-time between the immobilized enzyme and the activated support prior to borohydride reduction of the derivatives. Different combinations of these three variables have been tested to prepare a number of PGA-agarose derivatives. All these derivatives preserve 100% of catalytic activity corresponding to the soluble enzyme that has been immobilized but they show very different stability. The less stable derivative has exactly the same thermal stability of soluble penicillin G acylase and the most stable one is approximately 1,400 fold more stable. A similar increase in the stability of the enzyme against the deleterious effect of organic solvents was also observed. On the other hand, the agarose aldehyde gels present a very great capacity to immobilize enzymes through multipoint covalent attachment. In this way, we have been able to prepare very active and very stable PGA derivatives containing up to 200 International Units of catalytic activity per mL. of derivative with 100% yields in the overall immobilization procedure.     

Affinity Covalent Immobilization of Glucoamylase onto ρ-Benzoquinone Activated Alginate Beads: I. Beads Preparation and Characterization

ρ-Benzoquinone-activated alginate beads were presented as a new carrier for affinity covalent immobilization of glucoamylase enzyme. Evidences of alginate modification were extracted from FT-IR and thermal gravimetric analysis and supported by morphological changes recognized through SEM examination. Factors affecting the modification process such as ρ-benzoquinone (PBQ) concentration, reaction time, reaction temperature, reaction pH and finally alginate concentration, have been studied. Its influence on the amount of coupled PBQ was consequently correlated to the changes of the catalytic activity and the retained activity of immobilized enzyme, the main parameters judging the success of the immobilization process. The immobilized glucoamylase was found kept almost 80% of its native activity giving proof of non-significant substrate, starch, diffusion limitation. The proposed affinity covalent immobilizing technique would rank among the potential strategies for efficient immobilization of glucoamylase enzyme.     

Immobilization of Penicillin G Acylase on Calcined Layered Double Hydroxides

A hydrotalcite-like Mg2 /Al3 layered double hydroxide (LDH) material was prepared by means of amodified coprecipitation method involving a rapid mixing step followed by a separate aging process. LDH calcined at 500℃ , denoted as CLDH, was characterized by XRD, IR and BET surface area measurements.CLDH has a poor crystalline MgO-like structure with a high surface area and porosity. CLDH was used as asupport for the immobilization of penicillin G acylase(PGA). The effect of varying the immobilization conditions, such as pH, contact time and the ratio of enzyme to support, on the activity of the immobilized enzymein the hydrolysis of penicillin G has been studied. It was found that the activity of the immobilized enzyme decreased slightly with decreasing pH and reached a maximum after a contact time of 24 h. The activity of theimmobilized enzyme increased with increasing the ratio of enzyme to support. It was found that the adsorption of PGA inhibited the expected reaction of CLDH with an aqueous medium to regenerate a LDH phase. Itsoriginal activity(36%) after 15 cycles of reuse of the immobilized enzyme was retained, but no further loss in the activity was observed.     

Di-functional magnetic nanoflowers: A highly efficient support for immobilizing penicillin G acylase

The novel di-functional magnetic nanoflowers (DMNF) which had both epoxy groups and hydrophilic catechol as well as phthaloquinone groups capable of covalently coupling of penicillin G acylase (PGA) were characterized by scanning electron microscopy, transmission electron microscope (TEM), vibrating sample magnetometer, N₂ adsorption, and so on. The studies showed that DMNF possessed “hierarchical petal” structure of nanosheets had specific saturation magnetization of 39.7 emu/g and average pore diameter of 25.4 nm as well as specific surface area of 17.28 m²/g. For hydrolysis of penicillin G potassium catalyzed by the PGA immobilized on DMNF with enzyme loading of 106 mg/g-support, its apparent activity reached 2,667 U/g, which benefited from the “hierarchical petal” and large pore structure of the magnetic DMNF leading to high enzyme loading and fast diffusion of substrate molecules to the immobilized PGA to reaction. The apparent activity of the immobilized PGA could keep 2,408 U/g (above 90% of its initial activity) after repeating use for 10 cycles. The magnetic immobilized PGA exhibited excellent operational stability due to covalently coupling of the enzyme molecules between the support by covalent interaction of the amino groups of PGA and the reactive groups of epoxy, catechol, and phthaloquinone groups on DMNF. Furthermore, the PGA displayed good acid and alkaline resistance as well as thermal stability by immobilization using DMNF.     

高分子载体材料对青霉素酰化酶的固定化作用

介绍了天然高分子材料和合成高分子材料对青霉素酰化酶的固定化作用,着重讨论了高分子材料的制备、性质及其表面修饰对固定化酶活性和使用稳定性的影响。