亲水性环氧聚合物磁性微球的制备及其固定化青霉素酰化酶

通过设计反相悬浮聚合体系,制备了聚甲基丙烯酸缩水甘油酯（GMA）-甲基丙烯酸羟乙酯（HEMA）- N,N′-亚甲基双丙烯酰胺（MBAA）亲水性磁性聚合物GHM微球。球中的Fe₃O₄微晶呈倒立尖晶石结构,在微球表面存在着大量环氧基和亲水性的羟基及酰胺等基团,这些功能性基团为青霉素酰化酶（PGA）的固定化提供了适宜的微环境;同时,GHM微球具有的大孔结构和较高的比表面积,使其制备的固定化酶的载酶量高,这些有利因素使得固定化酶PGA/GHM在37℃下水解青霉素G钾合成6-氨基青霉烷酸的表观活性达748 IU·g^-1。 PGA/GHM经15次重复使用,其催化活性未出现明显的衰减,在使用中,固定化酶在磁场的作用下能够快速沉降与产物分离。     

Immobilization of penicillin G acylase onto amino-modified silica hydrogel

Amino-modified silica hydrogel (N-MSHG) was prepared by a simple sol-gel processing via the co-condensation of commercial silica sol with 3-aminopropyltrie-oxysilane. Penicillin G acylase (PGA), a model enzyme, was covalently immobilized onto the N-MSHG and then was used for the enzymatic synthesis of amoxicillin. The samples were characterized by Nitrogen sorption analysis, FT-IR and thermal gravimetric analysis (TGA). The results showed that the amino-modified gel was a mesoporous material with an average pore size of 12.64±0.17nm. The immobilization process was efficient and the immobilized enzyme showed high catalytic efficiency. The yield of the synthesis of amoxicillin in aqueous media was 38% for 2.5h. This sol-gel preparation is simple and shows prominent potential value in industrial processing.     

Incorporating Lanthanum into Mesoporous Silica Foam Enhances Enzyme Immobilization and the Activity of Penicillin G Acylase Due to Lewis Acid-Base Interactions

Penicillin G acylase (PGA) has been immobilized on a lanthanum-incorporated mesostructured cellular foam (La-MCF) support by using the interaction between the strong Lewis acid sites on the surface of La-MCF and the free amino groups of lysine residues of PGA. The La-MCF support was successfully synthesized in situ through the addition of a citric acid (CA) complexant. The results of pyridine-IR spectroscopy show the presence of strong Lewis acid sites on the surface of the prepared La-MCF (with CA), attributed to the incorporation of lanthanum species into the framework of MCF. Through interaction with the strong Lewis acid sites, the enzymes can be firmly immobilized on the surface of the support. The results indicate that PGA/La-MCF (with CA) exhibits a high specific activity and greatly enhanced operational stability. For the hydrolysis of penicillin G potassium salt, the initial specific activity of PGA/La-MCF (with CA) reaches 10023 U/g. Even after being recycled 10 times, PGA/La-MCF (with CA) retains 89 % of its initial specific activity, much higher than the 77 % of PGA/Si-MCF.     

Immobilization of penicillin G acylase onto amino-modified silica hydrogel

Amino-modified silica hydrogel (N-MSHG) was prepared by a simple sol-gel processing via the co-condensation of commercial silica sol with 3-aminopropyltrieoxysilane. Penicillin G acylase (PGA), a model enzyme, was covalently immobilized onto the N-MSHG and then was used for the enzymatic synthesis of amoxicillin. The samples were characterized by Nitrogen sorption analysis, FT-IR and thermal gravimetric analysis (TGA). The results showed that the amino-modified gel was a mesoporous material with an average pore size of 12.64±0.17 nm. The immobilization process was efficient and the immobilized enzyme showed high catalytic efficiency. The yield of the synthesis of amoxicillin in aqueous media was 38% for 2.5 h. This sol-gel preparation is simple and shows prominent potential value in industrial processing.     

Immobilization of penicillin G acylase on a composite carrier with a biocompatible microenvironment of chitosan

BACKGROUND: Penicillin G acylase (PGA) has been used extensively in the β‐lactam antibiotics industry. As a biocatalyst, it is better to use immobilized enzymes than free enzymes, therefore, the immobilization of PGA on a composite carrier consisting of an adsorbent resin and biocompatible chitosan were investigated. RESULTS: First, FT‐IR, BET and SEM analysis confirmed the structure of the composite carrier. Then, the immobilization process was optimized. The activity of the immobilized PGA on the chitosan–resin (IP‐CsR) was about 1300 U (g dry carrier)^?1 with a protein loading of about 27 mg (g dry carrier)^?1. Compared with the immobilized PGA on unmodified resin (IP‐R), the specific activity of IP‐CsR was enhanced about 2‐fold. The operational, thermal and pH stability were investigated. IP‐CsR maintained more than 75% initial activity after 35 cycles, while IP‐R was active for only 10 cycles. The half‐life at 50 °C increased from 75 to 300 min and the most stable pH was changed from 8.0 to 5.5. CONCLUSION: A novel composite carrier containing a biocompatible chitosan was very effective for PGA immobilization. Copyright © 2008 Society of Chemical Industry     

A thermal study on the use of immobilized penicillin G acylase in the formation of 7‐amino‐3‐deacetoxy cephalosporanic acid from cephalosporin G

Penicillin G acylase (PGA) is an important enzyme for the industrial production of 7‐amino‐3‐deacetoxy cephalosporanic acid (7‐ADCA) from cephalosporin G (Ceph‐G), and 6‐aminopenicillanic acid (6‐APA) from penicillin G (Pen‐G). These products are used for the manufacture of semi‐synthetic cephalosporins and penicillins. In this study, immobilized PGA was utilized to catalyze the conversion of Ceph‐G to 7‐ADCA. The optimal conditions were found to be an operating temperature of 45 °C, 0.2 M phosphate buffer, a substrate concentration of 30 mg cm^?3 and a catalyst particle concentration of 0.01 g cm^?3 (specific activity of 623.2 U g^?1). Up to 45 °C the reaction was characterized by an activation energy of 38.66 kJ mol^?1. Beyond 57.5 °C there was a sharp decline of activity, characterized by a deactivation energy of 235.88 kJ mol^?1. Copyright © 2004 Society of Chemical Industry     

离子液体修饰超顺磁性纳米颗粒用于青霉素G酰化酶固定化(英文)

Functionalized ionic liquids containing ethyoxyl groups were synthesized and immobilized on magnetic silica nanoparticles(MSNP) prepared by two steps,i.e.,Fe3O4 synthesis and silica shell growth on the surface.This magnetic nanoparticle supported ionic liquid(MNP-IL) were applied in the immobilization of penicillin G acylase(PGA).The MSNPs and MNP-ILs were characterized by the means of Fourier transform infrared spectroscopy(FTIR),scanning electron microscopy(SEM),transmission electron microscopy(TEM),and vibrating sample magnetometer(VSM).The results showed that the average size of magnetic Fe3O4 nanoparticles and MSNPs were ~10 and ~90 nm,respectively.The saturation magnetizations of magnetic Fe3O4 nanoparticles and MNP-ILs were 63.7 and 26.9 A?m2?kg?1,respectively.The MNP-IL was successfully applied in the immobilization of PGA.The maximum amount of loaded enzyme was about 209 mg?g?1(based on carrier),and the highest enzyme activity of immobilized PGA(based on ImPGA) was 261 U?g?1.Both the amount of loaded enzyme and the activity of ImPGA are at the same level of or higher than that in previous reports.After 10 consecutive operations,ImPGA still main-tained 62% of its initial activity,indicating the good recovery property of ImPGA activity.The ionic liquid modified magnetic particles integrate the magnetic properties of Fe3O4 and the structure-tunable properties of ionic liquids,and have extensive potential uses in protein immobilization and magnetic bioseparation.This work may open up a novel strategy to immobilize proteins by ionic liquids.     

Efficient Biocatalytic Cleavage and Recovery of Organic Substrates Supported on Soluble Polymers

The applicability of novel solution‐phase supports in combination with enzymes for biocatalytic transformations is reported. Ex novo designed styrene‐based copolymers, bearing a phenylacetic residue in variable loadings and linked as a pendant group to the macromolecular backbone, through a spacer of variable length, have been synthesized and characterized. These derivatives are compatible and can be used as soluble supports in combination with immobilized penicillin G acylase (PGA – EC 3.5.1.11) for the biocatalytic cleavage of the covalently anchored organic substrate in quantitative yields, in water or water/dimethylformamide solvent mixtures, with recovery of the immobilized enzyme with negligible losses in activity.     

Covalent immobilization of penicillin G acylase onto amine-functionalized PVC membranes for 6-APA production from penicillin hydrolysis process. II. Enzyme immobilization and characterization

This article describes the covalent immobilization of penicillin G acylase (PGA) onto glutaraldehyde-activated NH₂-PVC membranes. The immobilized enzyme was used for 6-aminopenicillanic acid production from penicillin hydrolysis. Parameters affecting the immobilization process, which affecting the catalytic activity of the immobilized enzyme, such as enzyme concentration, immobilization's time and temperature were investigated. Enzyme concentration and immobilization's time were found of determine effect. Higher activity was obtained through performing enzyme immobilization at room temperature. Both optimum temperature (35°C) and pH (8.0) of immobilized enzyme have not been altered upon immobilization. However, immobilized enzyme acquires stability against changes in the substrate's pH and temperature values especially in the higher temperature region and lower pH region. The residual relative activities after incubation at 60°C were more than 75% compared to 45% for free enzyme and above 50% compared to 20% for free enzyme after incubation at pH 4.5. The apparent kinetic parameters K_M and V_M were determined. K_M of the immobilized PGA (125.8 mM) was higher than that of the free enzyme (5.4 mM), indicating a lower substrate affinity of the immobilized PGA. Operational stability for immobilized PGA was monitored over 21 repeated cycles. The catalytic membranes were retained up to 40% of its initial activity after 10.5 working h. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Cobalt oxide species supported on SBA-15, KIT-5 and KIT-6 mesoporous silicas for ethyl acetate total oxidation

Cobalt oxide modified SBA-15, KIT-5 and KIT-6 mesoporous silicas with different pore size/pore entrances have been synthesized by a conventional wet impregnation method using cobalt nitrate as the precursor. The modified materials were characterized by N₂-physisorption, XRD, TEM-EDX, XPS, FT-IR, UV–vis and TPR-TG with hydrogen. Their catalytic activities in total oxidation of ethyl acetate were evaluated. A good correlation was observed between the catalytic activity, and the presence of spinel-type Co₃O₄ in the materials. Supports with larger mesopores facilitated the formation of such easily reducible spinel particles. However, the interconnectivity of the mesopores and the uniformity of the channel dimensions also had an influence on the catalytic activity, implying that mass-transfer effects, especially in the case of supports with cage-like mesopores.     

新型复合材料MnFe_2O_4@SiO_2-NH_2的制备及其固定青霉素G酰化酶

通过溶剂热法制备出高磁性的聚合物微球MnFe_2O_4,经正硅酸乙酯(TEOS)和3-氨基丙基三乙氧基硅烷(APTS)对微球表面进行改性修饰,制备出新型复合材料MnFe_2O_4@SiO_2-NH_2,并将其用于固定青霉素G酰化酶。在Si/Fe比为7 mmol/g、n(TEOS)∶n(APTS)=1∶1时,固定化酶PGA/MnFe_2O_4@SiO_2-NH_2在37℃下水解青霉素G钾合成6-氨基青霉烷酸,表观酶活为1 660 IU/g、载酶量为107.1 mg/g、比酶活为15.5 IU/mg、活性回收率为46.9%。经过6次重复使用,保留初始酶活的81.3%,在使用中固定化酶在磁场的作用下能够快速沉降与产物分离。     

Improved Stabilization of Genetically Modified Penicillin G Acylase in the Presence of Organic Cosolvents by Co‐ Immobilization of the Enzyme with Polyethyleneimine

Penicillin G acylase (PGA) is an enzyme that hardly interacts with polycationic polymers (e.g., polyethyleneimine, PEI) and thus the enzyme cannot be stabilized against the action of organic solvents by its co‐immobilization with the polymer in the same support, neither covalently attached to the support nor adsorbed on the already immobilized enzyme. However, a new mutant PGA bearing eight additional Glu residues homogenously distributed throughout the enzyme surface may interact with the polymer. The co‐immobilization of the enzyme and PEI on glyoxyl‐agarose allows one to fully take advantage of the stabilization produced by the multipoint covalent attachment and by the protective hydrophilic micro‐environment generated by the polycationic polymer, enabling a significant stabilization of the immobilized PGA in the presence of organic solvents.     

Stability of hydrolase enzymes in ionic liquids

In this work we attempted to evaluate the stability of penicillin G acylase (PGA) from Escherichia coli in their native form and free Candida antarctica lipase B (CaLB) in ionic liquids (ILs) at low water content. The hydrolysis of penicillin G to 6‐aminopenicillanic acid (6‐APA), and phenyl acetic acid (PAA) catalysed by PGA and the synthesis of butyl butyrate from vinyl butyrate and 1‐butanol catalysed by CaLB were chosen as activity tests. The influence of these new solvents on enzyme stability was studied by incubating the enzyme (PGA or CaLB) in ILs based on dialkylimidazolium cations associated with perfluorinated and dicyanamide anions at a given temperature. Stability studies indicate that CaLB and PGA exhibited greater stability in water‐immiscible ILs than in water‐miscible ILs. Specifically, native PGA shows greater stability in IL media than in organic solvents. For example, a half‐life time of 23 h was obtained in 1‐ethyl‐3‐methylimidazolium bis{(trifluoromethyl)sulfonyl}imide, , which was about 2000‐fold higher than that in 2‐propanol. The higher half‐life time of CaLB was observed in (t_1/2 = 84 h).     

Immobilization of GL-7-ACA acylase for the production of 7-ACA

Glutaryl-7-aminocephalosporanic acid(GL-7-ACA) acylase is an important enzyme for the production of 7-ACA (7-aminocephalosporanic acid). For an efficient immobilization of GL-7-ACA acylase, various carriers were tested. A high-porous hydrophilic carrier (FPHA) among various carriers tested was found to be the best for the immobilization of GL-7-ACA acylase. In order to develop an effective immobilization method of GL-7-ACA acylase, the parameters that affect the immobilization of GL-7-ACA acylase were also investigated under different conditions of buffer solution and different concentrations of glutaraldehyde. The highest value of GL-7-ACA acylase activity (70 Unit/g-matrix) was obtained when immobilized with 1% glutaraldehyde in a 0.1 M Tris buffer (pH 8.0). Also, in order to enhance the activity of the immobilized GL-7-ACA acylase, unreacted aldehyde groups were quenched by reaction with a low molecular weight agent such as L-lysine after immobilization. The highest activity of immobilized GL-7-ACA acylase was obtained at 0.1% of L-lysine. The immobilized GL-7-ACA acylase was tested for long-term stability and it was found that the activity was retained at about 62% of the initial value after 72 times of reuse at 25 ‡C.     

Role of pore size analysis in development of zeolite reforming catalyst

Effect of preparation variables on the aromatization activity of Pt/KL catalysts was studied. The aromatic content as well as C₅⁺ liquid yields were greatly influenced by the nature of binder material used in catalyst preparation. Detailed characterization of the catalysts indicated that two factors, namely (1) nature of binder and (2) size and volume of pores, influenced the catalytic activity. Among the catalysts studied the one prepared by MgO doped basic binder exhibited better performance. Pt loading was also observed to influence the pore volume and aromatization activity of the catalysts, where the catalyst exhibited decrease in pore volume above 0.4 wt.% Pt loading.     

Application of Plackett–Burman screening design to the modeling of grafted alginate–carrageenan beads for the immobilization of penicillin G acylase

Grafted alginate–carrageenan beads were used to immobilize the industrial enzyme penicillin G acylase (PGA). Sixteen factors were screened with the Plackett–Burman design (PBD) to test their significance on the gel beads formation and enzyme immobilization process. The results of PBD showed a wide variation of 30‐fold in the amount of immobilized penicillin G acylase (iPGA) from 11.9 to 354.16 U/g of beads; this reflected the importance of the optimizing process. Among the 16 tested factors, only 3 were proven to be significant. These factors were the enzyme buffer pH (N), enzyme soaking time (Q) with the gel beads, and enzyme concentration (P). The Pareto chart revealed that both Q and P exerted significant positive effects on the amount of iPGA, whereas N had a negative effect. We recommend further study to optimize only these three significant, distinctive enzyme factors. The PGA covalent attachment to the gel beads were proven by Fourier transform infrared spectroscopy, elemental analysis, and NaCl and reusability tests. The best gel bead formula succeeded in the immobilization of 354.16 U/g of beads and proved to be reusable 14 times, retaining 84% of the initial enzyme activity. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014 , 131, 40295.     

介孔二氧化硅KIT-6介观单晶微球的合成

以非离子表面活性剂［聚环氧乙烷（PEO）-聚环氧丙烷（PPO）-聚环氧乙烷三嵌段共聚物,P123］和阳离子聚电解质（聚二甲基二烯丙基氯化铵,PAC）形成的复合物胶束为模板,合成了具有球形形貌的介孔二氧化硅KIT-6介观单晶微球。通过扫描电镜（SEM）、透射电镜（TEM）、X射线衍射（XRD）、氮气物理吸附和热重分析（TGA）等手段对合成材料的形貌及孔结构进行了表征分析。结果表明,以有机复合物胶束为模板合成出的介孔KIT-6二氧化硅材料具有较规整的球形形貌,颗粒直径为2~3 μm,具有较大的比表面积和孔体积（747 m²/g和1.3 cm³/g）,介孔孔径为8.5 nm,且在整个颗粒内部介孔保持高度的有序排列。由于长链聚电解质PAC与硅源有着较强相互作用,样品可以在较高水热温度下（160 ℃）合成,有利于提升介观结构的稳定性。该合成方法对于介孔二氧化硅KIT-6单晶微球的合成及其在催化及吸附分离等领域的应用具有一定的启发意义。     

Ag/P(St-MMA)纳米复合高分子微球固定化青霉素酰化酶的研究

通过溶剂热法和无皂乳液聚合相结合,制备了P(St-MMA)高分子纳米微球.并以吸附沉积的方式在其表面沉积了Ag金属纳米粒子,最后将青霉素酰化酶共价连接在微球表面.初步研究了微球直径、银的质量分数等因素对固定化酶活力的影响.结果显示随着微球直径减小,固定化酶的偶联率和活力逐渐增加;银纳米粒子最多将固定化酶的偶联率和活力分别提高了42%和72%,固定化酶的最大表观活力(以干重记)达到了1 869 u/g,明显高于其它高分子载体固定化青霉素酰化酶的活力;实验证明银纳米粒子在青霉素水解过程中没有催化活力,但能大大提高青霉素酰化酶的催化活力.     

Intensification of enzymatic hydrolysis of penicillin G: Part 2. model for enzymatic reaction with reactive extraction

During the enzymatic hydrolysis of the potassium salt of Penicillin-G (PenGK) into Phenylacetic acid (PAA) and potassium salt of 6-Aminopenicillanic acid (6-APA), the pH of the reaction mixture falls on account of accumulation of PAA. This lowers the stability and activity of the enzyme used, viz., Penicillin-G acylase (PGA). A new approach of extracting the PAA by a long-chain tertiary amine, which is in the dispersed phase, as a liquid ion exchanger (LIX), is presented. A mathematical model has been developed for this slurry phase reactor with PenGK in the continuous aqueous phase, the amine alongwith a diluent in the dispersed organic phase and the immobilized PGA enzyme as the solid catalyst. Effects of various parameters affecting the conversion of PenG have been discussed. The model has been solved for batch and semi-batch modes. It has been shown that the semi-batch mode yields a higher productivity. This approach can also be advantageously used for other intermediates like 7-ADCA for cephalosporins.     

Synthesis of mesoporous silica particles with controlled pore structure

Silica particles, with controllable porosity, were synthesized using two different precursors, tetraethylortosilicate (TEOS) and sodium silicate, but without the addition of template. Characteristics of silica particles (aggregates) prepared by these two methods were compared. The pore structure was tuned only by changing the processing parameters, such as precursor concentration, base concentration, temperature and reaction time. The pore structure of prepared silica particles (aggregates) is strongly influenced by processing conditions and easy controllable in broad range of the specific surface area, pore size, size distribution and pore volume. However, the silica particles synthesized from TEOS have very low total pore volume (ranging from 0.06 to 0.2 cm³/g) and a large portion of pores smaller than 4 nm. On the other side, the silica particles prepared from sodium silicate can be defined as a mesoporous silica with the average pore size up to 20 nm and much higher total pore volume (ranging from 0.8 to 1.5 cm³/g), which are important advantages for their application in encapsulation of enzymes.