Immobilization Of Penicillin G Acylase Research Articles

Investigation of Enzyme Immobilization and Clogging Mechanisms in the Enzymatic Synthesis of Amoxicillin.

This study investigated the blocking mechanism of immobilized penicillin G acylase (PGA) during the enzymatic synthesis of amoxicillin. Laboratory observations revealed that the primary cause of clogging was the crystallization of the substrate and product on the enzyme surface. Adjusting key parameters can significantly reduce clogging and improve catalytic efficiency. Methanol can decrease enzyme activity, but isopropyl alcohol cleaners can effectively remove clogs and protect enzyme activity. These findings provide an experimental foundation for optimizing the PGA immobilization process, which is crucial for achieving high efficiency and sustainability in industrial production.

Rational design of biocatalysts based on covalent immobilization of acylase enzymes

Acylases catalyze the hydrolysis of amide bonds. Penicillin G acylase (PGA) is used for the semi-synthesis of penicillins and cephalosporins. Although protein immobilization increases enzyme stability, the design of immobilized systems is difficult and usually it is empirically performed. We describe a novel application of our strategy for the Rational Design of Immobilized Derivatives (RDID) to produce optimized acylase-based immobilized biocatalysts for enzymatic bioconversion. We studied the covalent immobilization of the porcine kidney aminoacylase-1 onto aldehyde-based supports. Predictions of the RDID1.0 software and the experimental results led to the selection of glyoxyl-Sepharose CL 4B support and pH 10.0. One of the predicted clusters of reactive amino groups generates an enzyme-support configuration with highly accessible active sites, contributing with 82% of the biocatalyst’s total activity. For Escherichia coli PGA, the predictions and experimental results show similar maximal amounts of immobilized protein and activity at pH 8.0 and 10.0 on glyoxyl-Sepharose CL 10B. However, thermal stability of the immobilized derivative is higher at pH 10.0 due to an elevated probability of multipoint covalent attachment. In this case, two clusters of amino groups are predicted to be relevant for PGA immobilization in catalytically competent configurations at pH 10.0, showing accessible active sites and contributing with 36% and 44% of the total activity, respectively. Our results support the usefulness of the RDID strategy to model different protein engineering approaches (site-directed mutagenesis or obtainment of fusion proteins) and select the most promising ones, saving time and laboratory work, since the in silico-designed modified proteins could have higher probabilities of success on bioconversion processes.

Immobilization of Penicillin G Acylase on Vinyl Sulfone-Agarose: An Unexpected Effect of the Ionic Strength on the Performance of the Immobilization Process.

Penicillin G acylase (PGA) from Escherichia coli was immobilized on vinyl sulfone (VS) agarose. The immobilization of the enzyme failed at all pH values using 50 mM of buffer, while the progressive increase of ionic strength permitted its rapid immobilization under all studied pH values. This suggests that the moderate hydrophobicity of VS groups is enough to transform the VS-agarose in a heterofunctional support, that is, a support bearing hydrophobic features (able to adsorb the proteins) and chemical reactivity (able to give covalent bonds). Once PGA was immobilized on this support, the PGA immobilization on VS-agarose was optimized with the purpose of obtaining a stable and active biocatalyst, optimizing the immobilization, incubation and blocking steps characteristics of this immobilization protocol. Optimal conditions were immobilization in 1 M of sodium sulfate at pH 7.0, incubation at pH 10.0 for 3 h in the presence of glycerol and phenyl acetic acid, and final blocking with glycine or ethanolamine. This produced biocatalysts with stabilities similar to that of the glyoxyl-PGA (the most stable biocatalyst of this enzyme described in literature), although presenting just over 55% of the initially offered enzyme activity versus the 80% that is recovered using the glyoxyl-PGA. This heterofuncionality of agarose VS beads opens new possibilities for enzyme immobilization on this support.

Comparative study of poly tannic acid functionalized magnetic particles before and after modification for immobilized penicillin G acylase

In this work, Fe3O4 nanoparticles (NPs) was synthesized by inverting microemulsion method. After that, based on the physical and chemical properties of tannic acid (TA), poly tannic acid (PTA) was coated on Fe3O4 NPs surface. Fe3O4 NPs coated with PTA, on the one hand, was used to immobilize Penicillin G acylase (PGA) by physical adsorption. On the other hand, it was modified by glutaraldehyde (GA). GA grafting rate (Gr-GA ) was optimized, and the Gr-GA was 30.0% under the optimum conditions. Then, through the Schiff base reaction between the glutaraldehyde group and PGA amino group, this covalent immobilization of PGA was further realized under mild conditions. Finally, the structures of every stage of magnetic composites were characterized by Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), vibration magnetometer (VSM) and transmission electron microscopy (TEM), respectively. The results indicated that the enzyme activity (EA), enzyme activity recovery (EAR) and maximum load (ELC) of the immobilized PGA were 26843 U/g, 80.2% and 125 mg/g, respectively. Compared to the physical immobilization of PGA by only coating PTA nanoparticles, further modified nanoparticles by GA showed higher catalytic stability, reusability and storage stability.

Magnetic thermosensitive polymer composite carrier with target spacing for enhancing immobilized enzyme performance

A novel magnetic thermosensitive polymer composite carrier with target spacing was developed. In this strategy, thermosensitive polymer grafted on magnetic Fe3O4 for enhancing immobilized penicillin G acylase (PGA) performance and introduce immobilized target spacing into magnetic carriers for the first time. Fe3O4 nanoparticles were synthesized by a reverse microemulsion method. The modifier used was the silane coupling agent γ-methylacryloxypropyl trimethoxysilane (KH570) and then reacting with a reversible-adaptive fragmentation chain transfer (RAFT) reagent, 2-cyano-2-propyldodecyl trithiocarbonate (CPDTC). The thermo-sensitive nanoparticle-composite carrier of Fe3O4-grafted-poly N, N-diethyl acrylamide-block-poly β-Hydroxyethyl methacrylate-block-random copolymer of glycidyl methacrylate and methyl methacrylate (Fe3O4-g-PDEA-b-PHEMA-b-P(MMA-co-GMA)) were synthesized by RAFT polymerization technique that used N, N-diethyl acrylamide (DEA), β-Hydroxyethyl methacrylate (HEMA), Glycidyl methacrylate (GMA) and Methyl methacrylate (MMA) as monomer, then which were employed as functional carriers for the immobilization of PGA. Within the carrier, the epoxy group of GMA segment was a target immobilization site for PGA and the introduction of MMA reflected the target space of immobilized PGA to improve catalytic activity and catalytic activity recovery rate of the immobilized PGA. Characterizations demonstrated that the triblock copolymers grafted Fe3O4 nanoparticles were successfully fabricated by the structure design. Besides, under these circumstances the enzyme activity (EA), enzyme loading capacity (ELC) and catalytic activity recovery ration (CAR) reached 31235 U/g, 128.39 mg/g and 93.32 %, respectively. The catalytic activity of immobilized PGA maintained 87.4 % of initial value and the recovery ratio (R) of immobilized PGA reached 96.22 % after recycling 12 times. Furthermore, the immobilized PGA exhibited advantages of low temperature homogeneous catalysis and magnetic separation, which indicated broad application prospects in the biocatalysts’ field.

The immobilization of penicillin G acylase on modified TiO2 with various micro-environments

Immobilization of penicillin G acylase (PGA) on carriers is an effective strategy for running away from the drawbacks of free PGA. In this work, modified TiO2 was employed as a carrier for the immobilization of PGA. Firstly, TiO2 was used as a nucleus and modified by two different approaches, one-step modification and two-step modification, to construct and regulate the micro-environment of carrier, particularly by changing the type of functional immobilization groups, and the arm-length of immobilization sites. In the one-step modification approach, TiO2 was altered by glutaraldehyde, 3-glycidoxypropyltrimethoxysilane (3-GCDPTMS) and 3-aminopropyltriethoxysilane (3-APTMS), separately; while in the two-step modification approach, primary TiO2 was firstly altered with a small amount of glutaraldehyde or 3-GCDPTMS, then followed by a secondary modification process using glutaraldehyde and 3-GCDPTMS, respectively. Secondly, the influence of the micro-environments of carrier on the catalytic performance of immobilized PGA, in terms of enzyme loading capacity (ELC), enzyme activity (EA) and activity retention ratio (EAR) was investigated. Results revealed that PGA immobilized on carrier modified by 3-GCDPTMS, with a graft rate of 30 % had the best performance, which had an ELC of 10,800 U, EA of 14,900 U/g. Both of the functional group and arm-length of immobilization site had influence on the catalytic performance of immobilized PGA, and the functional groups of immobilization site was the most essential one responsible for stabilizing PGA and improving its catalytic activity. Then PGA immobilized carrier with longer arm-length had better catalytic performance.

Developing a Novel Enzyme Immobilization Process by Activation of Epoxy Carriers with Glucosamine for Pharmaceutical and Food Applications

In this paper, we describe the development of an efficient enzyme immobilization procedure based on the activation of epoxy carriers with glucosamine. This approach aims at both creating a hydrophilic microenvironment surrounding the biocatalyst and introducing a spacer bearing an aldehyde group for covalent attachment. First, the immobilization study was carried out using penicillin G acylase (PGA) from Escherichia coli as a model enzyme. PGA immobilized on glucosamine activated supports has been compared with enzyme derivatives obtained by direct immobilization on the same non-modified carriers, in the synthesis of different 3′-functionalized cephalosporins. The derivatives prepared by immobilization of PGA on the glucosamine-carriers performed better than those prepared using the unmodified carriers (i.e., 90% versus 79% cefazolin conversion). The same immobilization method has been then applied to the immobilization of two other hydrolases (neutral protease from Bacillus subtilis, PN, and bromelain from pineapple stem, BR) and one transferase (γ-glutamyl transpeptidase from Bacillus subtilis, GGT). Immobilized PN and BR have been exploited in the synthesis of modified nucleosides and in a bench-scale packed-bed reactor for the protein stabilization of a Sauvignon blanc wine, respectively. In addition, in these cases, the new enzyme derivatives provided improved results compared to those previously described.

Immobilization of penicillin G acylase on paramagnetic polymer microspheres with epoxy groups

Paramagnetic polymer microspheres were synthesized by the inverse suspension polymerization method through polymerization of glycidyl methacrylate, ally glycidyl ether and methacrylamide on the surface of silica-coated Fe3O4 nanoparticles using N,N’-methylene-bis(acrylamide) as a cross-linking agent. Penicillin G acylase (PGA) was covalently immobilized on the surface of the paramagnetic microspheres by reacting the amino groups of the PGA molecules with the epoxy groups of the paramagnetic polymer microspheres. The effect of the SiO2 coating and the amount of paramagnetic Fe3O4 nanoparticles on the initial activity and the operational stability of the immobilized PGA was investigated. The results indicated that SiO2 played an important role in the polymerization process and paramagnetic polymer microspheres with a SiO2-coated Fe3O4 nanoparticles mass content of 7.5% are an optimal support material for PGA immobilization. Immobilized PGA on the paramagnetic polymer microspheres shows a high initial activity of 430 U/g (wet) and retains 99% of its initial activity after recycling 10 times. Furthermore, immobilized PGA exhibits high thermal stability, pH stability and excellent reusability, which can be rapidly recycled by the aid of magnet.

A Facile Preparation Process of Magnetic Aldehyde-Functionalized Ni(0.5)Zn(0.5)Fe₂O4@SiO₂ Nanocomposites for Immobilization of Penicillin G Acylase (PGA).

A facile sol combustion and gel calcination process has been reported for the preparation of core– shell magnetic Zn0.5Fe₂O₄@SiOnanocomposites. The morphology, chemical composition, structure and magnetic property of as-prepared nanocomposites were investigated by XRD, VSM, BET, SEM, and TEM, and the magnetic Ni0.5Zn0.5Fe₂O₄@SiO₂ nanocomposites were characterized with average size of about 25 nm, saturation magnetization of 90.8 Am²/kg and the specific surface area of 67.1 m2/g. The surface of Ni0.5Zn0.5Fe₂O₄@SiO₂ nanocomposites was functionalized with glutaraldehyde to form the aldehyde-functionalized magnetic Ni0.5Zn0.5Fe₂O4@SiO₂ nanocomposites, and penicillin G acylase (PGA) was successfully immobilized onto them. And the immobilized PGA exhibited high effective activity, good stability of enzyme catalyst and good reusability, and could retain 63.5% of initial activity after 12 consecutive operations. The kinetic parameters were determined, and the value of K m for the immobilized PGA (161.7 mmol/L) is higher than that of the free PGA (3.5 mmol/L), while v max (1.626 mmol/min) is also larger than that of the free PGA (0.838 mmol/min), which revealed that the immobilization of PGA onto Ni0.5Zn0.5Fe₂O₄@SiO₂ nanocomposites was an efficient and simple way for preparation of stable PGA.

Covalent Immobilization of Penicillin G Acylase onto Fe3O4@Chitosan Magnetic Nanoparticles.

Penicillin G acylase (PGA) was immobilized on magnetic Fe3O4@chitosan nanoparticles through the Schiff base reaction. The immobilization conditions were optimized as follows: enzyme/support 8.8 mg/g, pH 6.0, time 40 min, and temperature 25°C. Under these conditions, a high immobilization efficiency of 75% and a protein loading of 6.2 mg/g-support were obtained. Broader working pH and higher thermostability were achieved by the immobilization. In addition, the immobilized PGA retained 75% initial activity after ten cycles. Kinetic parameters Vmax and Km of the free and immobilized PGAs were determined as 0.91 mmol/min and 0.53 mmol/min, and 0.68 mM and 1.19 mM, respectively. Synthesis of amoxicillin with the immobilized PGA was carried out in 40% ethylene glycol at 25°C and a conversion of 72% was obtained. These results showed that the immobilization of PGA onto magnetic chitosan nanoparticles is an efficient and simple way for preparation of stable PGA.

An approach for the improved immobilization of penicillin G acylase onto macroporous poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) as a potential industrial biocatalyst.

The use of penicillin G acylase (PGA) covalently linked to insoluble carrier is expected to produce major advances in pharmaceutical processing industry and the enzyme stability enhancement is still a significant challenge. The objective of this study was to improve catalytic performance of the covalently immobilized PGA on a potential industrial carrier, macroporous poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) [poly(GMA-co-EGDMA)], by optimizing the copolymerization process and the enzyme attachment procedure. This synthetic copolymer could be a very promising alternative for the development of low-cost, easy-to-prepare, and stable biocatalyst compared to expensive commercially available epoxy carriers such as Eupergit or Sepabeads. The PGA immobilized on poly(GMA-co-EGDMA) in the shape of microbeads obtained by suspension copolymerization appeared to have higher activity yield compared to copolymerization in a cast. Optimal conditions for the immobilization of PGA on poly(GMA-co-EGDMA) microbeads were 1 mg/mL of PGA in 0.75 mol/L phosphate buffer pH 6.0 at 25°C for 24 h, leading to the active biocatalyst with the specific activity of 252.7 U/g dry beads. Chemical amination of the immobilized PGA could contribute to the enhanced stability of the biocatalyst by inducing secondary interactions between the enzyme and the carrier, ensuring multipoint attachment. The best balance between the activity yield (51.5%), enzyme loading (25.6 mg/g), and stability (stabilization factor 22.2) was achieved for the partially modified PGA.

Aldehyde-functionalized mesostructured cellular foams prepared by copolymerization method for immobilization of penicillin G acylase

Aldehyde-functionalized mesostructured cellular foams (A-MCFs) were successfully prepared by copolymerization of vinyl groups on the surface of vinyl-functionalized mesostructured cellular foams (V-MCFs) with methacrolein, and used for covalent immobilization of penicillin G acylase (PGA). Effects of functionalization with the organo-functional groups on the physicochemical properties of V-MCFs and A-MCFs, and the enzymatic activity and the operational stability of the immobilized PGA were investigated. A-MCFs-15% sample retains the characteristic ultra-large and continuous 3D mesoporous structure of MCFs with high surface area and large pore volume, which is beneficial for immobilization of PGA and diffusion of substrates and products, and thus results in high enzymatic activity of the immobilized PGA. PGA is immobilized covalently on A-MCFs sample via the reaction to produce Schiff’s base between the free amino groups of lysine residues of PGA and the aldehyde groups on the surface of A-MCFs, which greatly increases the operational stability of the immobilized PGA. PGA/A-MCFs-15% sample shows the initial enzymatic activity of 9531Ug−1 and retains 87% of its initial activity after recycled for ten times.

Aldehydepropyl-functionalized mesostructured cellular foams: Efficient supports for immobilization of penicillin G acylase

Abstract The aldehydepropyl-functionalized mesostructured cellular foams (CHO-MCFs) were prepared by post-synthetical functionalization of MCFs with trimethoxysilylpropanal (TMSP), and used as efficient supports for immobilization of penicillin G acylase (PGA). The physicochemical properties of CHO-MCFs were characterized by SAXS, nitrogen sorption, TEM, elementary analysis, solid state 29 Si MAS NMR, FT-IR spectroscopy and thermogravimetry. The results show that the aldehydepropyl groups have been grafted successfully on the surface of MCFs, and after functionalization, the BET surface area and pore volume of CHO-MCFs decrease, but the ultra-large and continuous 3D mesoporous structure of CHO-MCFs are retained to be beneficial for immobilization of PGA with large size and diffusion of substrates and products. PGA is immobilized covalently on CHO-MCFs via the reaction to produce Schiff's base between the free amino groups of lysine residues of PGA and the aldehyde groups on the surface of CHO-MCFs, which greatly increases the operational stability of the immobilized PGA with little activity loss due to the short-chain groups of aldehydepropyl grafted on the surface of CHO-MCFs. PGA/CHO-MCFs-10 shows the immobilization yield of 57.6%, the specific activity of 22.2 U/mg and the initial enzymatic activity of 8895 U/g, and retains 93.0% of its initial enzymatic activity after recycled for 10 times.

Modulation of the Microenvironment Surrounding the Active Site of Penicillin G Acylase Immobilized on Acrylic Carriers Improves the Enzymatic Synthesis of Cephalosporins

The catalytic properties of penicillin G acylase (PGA) from Escherichia coli in kinetically controlled synthesis of β-lactam antibiotics are negatively affected upon immobilization on hydrophobic acrylic carriers. Two strategies have been here pursued to improve the synthetic performance of PGA immobilized on epoxy-activated acrylic carriers. First, an aldehyde-based spacer was inserted on the carrier surface by glutaraldehyde activation (immobilization yield = 50%). The resulting 3-fold higher synthesis/hydrolysis ratio (vs/vh1 = 9.7 ± 0.7 and 10.9 ± 0.7 for Eupergit® C and Sepabeads® EC-EP, respectively) with respect to the unmodified support (vs/vh1 = 3.3 ± 0.4) was ascribed to a facilitated diffusion of substrates and products as a result of the increased distance between the enzyme and the carrier surface. A second series of catalysts was prepared by direct immobilization of PGA on epoxy-activated acrylic carriers (Eupergit® C), followed by quenching of oxiranes not involved in the binding with the protein with different nucleophiles (amino acids, amines, amino alcohols, thiols and amino thiols). In most cases, this derivatization increased the synthesis/hydrolysis ratio with respect to the non derivatized carrier. Particularly, post-immobilization treatment with cysteine resulted in about 2.5-fold higher vs/vh1 compared to the untreated biocatalyst, although the immobilization yield decreased from 70% (untreated Eupergit® C) to 20%. Glutaraldehyde- and cysteine-treated Eupergit® C catalyzed the synthesis of cefazolin in 88% (±0.9) and 87% (±1.6) conversion, respectively, whereas untreated Eupergit® C afforded this antibiotic in 79% (±1.2) conversion.

Grafted Carrageenan Gel Disks and Beads with Polyethylenimine and Glutaraldehyde for Covalent Immobilization of Penicillin G Acylase

The precursor for production of 65% worldwide antibiotics, 6-aminopenicillanic acid, has been produced using covalently immobilized penicillin G acylase (PGA) onto modified grafted carrageenan gels. Two shapes of gel were prepared, gel disks and gel beads by using the parallel plates and Encapsulator devises, respectively. Different sizes of gel disks and beads were tested to choose the best sizes for both gel types. 2 2 Full-factorial central composite design (CCD) was applied for optimisation of enzyme loading process. The results revealed that maximal enzyme loading of 11.07 and 14.30 U/g of gel disks and beads respectively could be achieved after soaking the gels in an enzyme loading solution of 20 U of PGA for 16 h for both gel types. The generated model was found to be very adequate for PGA immobilization process 91% accuracy was attained for gel disks as the experimental value was 11.07 U/g disk compared to 10.1 U/g disk for the predicted value while 95% accuracy was attained using gel beads as the experimental value was 14.30 U/g beads compared to 14.07 U/g beads for the predicted value. This result is 40% higher than that obtained in our previous work using gel disks. The immobilization of PGA onto gel beads increased the optimum pH from 6 to 6.5–7 while the reaction temperature increased dramatically from 45 � Ct o 75 � C compared to the free enzyme, respectively. Thus, the modified carrageenan-polyethylenimine-glutaraldehyde gel beads could be a good and promising candidate for the industrial use.

Oriented Immobilization of Penicillin G Acylase on the Epoxydized Magnetic Hydroxyl Particles by Phenyl Acetic Acid

Magnetic hydroxyl microspheres were prepared by suspension polymerization and activated by epoxy chloropropane. The magnetic particles were characterized in terms of chemical composition, particle size and electrophoretic mobility. These epoxy-activated magnetic particles were assessed as a new carries for immobilized penicillin G acylase (PGA) by covalent coupling. The oriented PGA immobilization was achieved by employing the interaction electrostatic repulsion between PGA and magnetic supports through adding the phenyl acetic acid (PAA), which resulted in a 1.194-fold increase in the enzyme activity yield as compared to that of untreated PGA. No activity of immobilized PGA was lost after 20 cycles and about 94.28% enzyme activity was retained at the end of the 80th cycle in the batch reaction system.

Improving the Stability of Immobilized Penicillin G Acylase via the Modification of Supports With Ionic Liquids

With the aim of improving the stability of penicillin G acylase (PGA), functional ionic liquids (ILs) were used to modify the surface chemistry of the supports on which they were physically adsorbed. Four kinds of ILs—specifically, 1-methyl-3-(triethoxysilylpropyl)-imidazolium salts, with Cl–, BF4–, PF6–, and Tf2N– as the anions (IL-Cl–, BF4–, PF6–, and Tf2N–, respectively)—were used to tune the hydrophilic or hydrophobic properties of the ILs. The synthesized ILs were first immobilized on magnetic silica nanoparticles (Fe3O4/SiO2), and the composite material (Fe3O4/SiO2–IL) was then applied to immobilize PGA via physical adsorption. The amount of protein loading, the specific activity, the immobilization yield, and the stability of immobilized PGA were investigated to evaluate the effects of the ILs on the PGA immobilization. The results showed that PGA immobilized on Fe3O4/SiO2–ILs was more stable than that immobilized on Fe3O4/SiO2 with no IL modification. Among the four kinds of ILs tested, the hydrop...

Immobilization of Penicillin G Acylase on Magnetic Nanoparticles Modified by Ionic Liquids

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·m−2·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 immobilize 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 maintained 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.

Development of the reversible PGA immobilization by using the immobilized metal ion affinity membrane

In this study, an immobilized metal ion affinity membrane (IMAM) was prepared and used to immobilize penicillin G acylase (PGA). The stability of storage of the immobilized PGA membrane (IPM) was tested. When stored in deionized water (DI) at 4°C, only 19% residual activity of IPM was retained for 10 days. However, when stored in 10mM phosphate buffer (PB, pH 8) with 0.1% NaN3, the IPM can retain 99% of its activity for a 10-day reaction and storage test. The feasibility of the IPM regeneration after a long period of reaction was investigated. When using 100mM EDTA as the stripping solution, the re-immobilized capacity for PGA is only 52% of the original one after 4 regenerations. When the regeneration process was modified by using the stripping solution (300mM NaCl, 25mM EDTA, 20mM PB, pH 8) for 30min, then immersed in 0.5M HCl and 0.5M NaOH respectively for 10min, the resulting IMAM can reserve 99% PGA immobilized activity after 5 regenerations.

Covalent immobilization of penicillin G acylase onto chemically activated surface of poly(vinyl chloride) membranes for 6‐penicillic acid production from penicillin hydrolysis process I. Optimization of surface modification and its characterization

AbstractThe covalent immobilization of penicillin G acylase (PGA) onto the surface of NH2–poly(vinyl chloride) (PVC) membranes was studied. PGA was chosen because it plays a relevant role in the pharmaceutical industry, catalyzing the production of an important intermediate for the industrial production of semisynthetic penicillin and cephalosporine. Because PVC has no functional groups in its structure, in this work, we focused on the functionalization of PVC with primary amine functional groups for the covalent immobilization of PGA. This goal was achieved through an aminoalkylation process of the surface of the PVC membranes with ethylene diamine followed by activation with glutaraldehyde to finally immobilize the enzyme. Different factors affecting the modification and activation processes were studied, and their impacts on the catalytic activity of the immobilized PGA were followed. The functionalized membranes were characterized with Fourier transform infrared spectroscopy, thermogravimetric analysis, and scanning electron microscopy to verify the modification process. In addition, the changes resulting from the modification in physical characteristics, such as surface roughness, water uptake, and mechanical properties, were monitored. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012