Covalent Assembly of Penicillin Acylase in Mesoporous Silica Based on Macromolecular Crowding Theory

To improve the covalent immobilization of penicillin acylase （PA）, macromolecular crowding theory was applied to its immobilization. Influence of mass ratio of enzyme to the silica, as well as, activation time with glutaraldehyde on the activity of assembled PA, was studied. In the mesopores, the effect of fl-cyclodextrin （β-CD） on the immobilization of the enzyme was also investigated. It was remarkable that the coupled yield and relative activity reached 99.5% and 92.3%, respectively, when penicillin acylase assembled covalently in the mesopores. The results here indicate that mimicked macromolecule crowding could significantly ameliorate the performance of covalently immobilized PA.

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

Paramagnetic polymer microspheres were synthesized by the inverse suspension polymerizationmethod through polymerization of glycidyl methacrylate,ally glycidyl ether and methacrylamide onthe surface of silica‐coated Fe3O4nanoparticles using N,N’‐methylene‐bis(acrylamide)as across‐linking agent.Penicillin G acylase(PGA)was covalently immobilized on the surface of theparamagnetic microspheres by reacting the amino groups of the PGA molecules with the epoxygroups of the paramagnetic polymer microspheres.The effect of the SiO2coating and the amount ofparamagnetic Fe3O4nanoparticles on the initial activity and the operational stability of the immobilizedPGA was investigated.The results indicated that SiO2played an important role in the polymerization process and paramagnetic polymer microspheres with a SiO2‐coated Fe3O4nanoparticles mass content of7.5%are an optimal support material for PGA immobilization.Immobilized PGA on the paramagnetic polymer microspheres shows a high initial activity of430U/g(wet)and retains99%of its initial activity after recycling10times.Furthermore,immobilized PGA exhibits high thermal stability,pH stability and excellent reusability,which can be rapidly recycled by the aid of magnet.?2018,Dalian Institute of Chemical Physics,Chinese Academy of Sciences.Published by Elsevier B.V.All rights reserved.

An efficient synthesis of ampicillin on magnetically separable immobilized penicillin G acylase

Penicillin G acylase(PGA) was immobilized on the magnetic hydrophilic polymer microspheres with average pore size of 17.1 nm,specific surface area of 128.2 m2/g and saturate magnetization of 6.4 emu/g.The 96.7%ampicillin yield with 1.60 of the synthesis/hydrolysis(S/H) ratio from 6-aminopenicillanic acid(6-APA) and D-(-)-alpha-phenylglycine methyl ester(D-PGME) can be achieved using the resultant magnetic biocatalyst in ethylene glycol,where only 82.1%yield with 1.40 of the S/H ratio was obtained using the free PGA under the identical reaction conditions.The immobilized PGA can be separated magnetically and recycled for five times without obvious loss of its catalytic activity.

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., Fe304 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 themeans 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 Fe304 nanoparticles and MSNPs were -10 and -90 nm, respectively. The saturation magnetizations of magnetic Fe304 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 carder）, 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 leyel of or higher than that in previous reports. After 10 consecutive operat!ons, ImPGA still mainrained 62% of its initial activity, indicating the＇good recovery property of ImPGA activity. The ionic liquid modified magnetic particles integrate the magnetic properties of Fe304 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.

Poly(GMA/MA/MBAA)Copolymer Beads:a Highly Efficient Support Immobilizing Penicillin G Acylase

The hydrophilic, macroporous and beaded ternary copolymer of glycidyl methacrylate （GMA）/methacrylamide（MA）/N,N＇-methylene-bis（acrylamide）（MBAA）was synthesized using the industrial agents by inverse suspension polymerization. The apparent activity of the immobilized penicillin G acylase reached 1096 IU/g for hydrolysis penicillin G on the beads with diameter of 0.11-0.13 ram, and it changed hardly after 50 cycles. It can be expected to be a good potential in industrial application,

Immobilization of Penicillin G Acylase on Calcined Layered Double Hydroxides

A hydrotalcite-like Mg 2+ /Al 3+ layered double hydroxide(LDH) material was prepared by means of a modified 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 a support 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 enzyme in 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 the immobilized 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. Its original activity(36%) after 15 cycles of reuse of the immobilized enzyme was retained, but no further loss in the activity was observed.

Characterization of cellulose acetate micropore membrane immobilized acylase Ⅰ

This paper describes an innovative method for the immobilization of acylase I, which was entrapped into the CA-CTA micropore membrane. The most suitable casting solutions proportion for immobilizing the enzyme was obtained through orthogonal experiment. Properties of the enzyme membrane were investigated and compared with those of free enzyme and blank membrane. The thermal stability and pH stability of the enzyme inside the membrane were changed by immobilization. The optimum pH was found to be 6.0, which changes 1.0 unit compared with that of free acylase I. The optimum temperature was found to be about 90℃, which is higher than that of free acylase I (60℃). Experimental results showed that immobilization had effects on the kinetic parameters of acylase I.

Gold Nanoparticles for Colorimetric detection of hydrolysis of antibiotics by penicillin G acylase

A simple inexpensive method of monitoring hydrolysis of an antibiotic penicillin G (pen G) and subsequent enzyme detection using gold nanoparticles is presented. Gold nanoparticles capped with Cetyl trimethyl ammonium bromide (CTAB) are synthesized using chemical route. The particles could be used for detection of Penicillin G acylase (PGA) enzyme by incorporating hydrolysis reaction with pen G. This hydrolysis reaction leads to a shift in the surface plasmon band of gold nanoparticles from 527 nm to 545 nm accompanied by a visual colorimetric change in the solution from red to blue. The process is attributed to aggregation of nanoparticles caused due to displacement of CTAB bilayer by byproducts of the hydrolysis reaction. It is proposed that the presence of 0.007 mg/ml of PGA can be detected by a color change of gold nanoparticles solution without requiring any complicated instrument or highly trained operator to conduct the test. The method could also identify the presence of different penicillins by showing different spectral shifts. Thus the work presented here would be useful not only for the detection of the pharmaceutically important drug Pen G, but also represents a general methodology for the detection of enzymes, eg PGA.

Crystallographic Studies of Cephalosporin Acylase from Pseudomonas sp. Strain 130

The cephalosporin acylases are a group of enzymes that hydrolyze cephalosporin C and/or glutaryl 7 aminocephalosporanic acid to produce 7 aminocephalosporanic acid. The cephalosporin acylase from Pseudomonas sp. strain 130 was crystallized in two different forms suitable for structural studies. A tetragonal crystal form diffracted to 0.24 nm belonged to the space group P4 12 12. There was one αβ heterodimer per asymmetric unit. A second crystal form diffracted to 0.21 nm belonged to the space group P2 1. There was four αβ heterodimers per asymmetric unit. The tetragonal crystal structure of CA 130 was determined using the multiwavelength anomalous diffraction method and the P2 1 crystal structure was then determined using the molecular replacement method.

Rapid Cloning and Expression of Glutaryl-7-Aminocephalosporanic Acid Acylase Genes from Soil Samples

A polymerase chain reaction （PCR）-based strategy was developed to rapidly obtain the gene encoding for an industrially important enzyme, glutaryl-7-aminocephalosporanic acid （GL-7-ACA） acylase.Different soil samples were cultured with a Pseudomonas selective medium to enrich specific microorganisms, and then the genomic DNA was extracted to serve as PCR templates. PCR primers for GL-7-ACA acylase gene amplification were designed on the basis of bioinformatics searches and analyses.The method was used to successfully amplify three GL-7-ACA acylase genes from different soil samples.The GL-7-ACA acylase genes were then cloned and overexpressed in Escherichia coil with a relatively high level of 266 unit · L^-1.

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 cocondensation 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.