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.

Penicillin G acylase-responsive near-infrared fluorescent probe:Unravelling biofilm regulation and combating bacterial infections

Antibiotic resistance poses a critical threat to human healthcare,largely driven by bacterial biofilms.These biofilms resist the immune system and antibiotics,rendering enclosed microbial cells 10-1000times more antibiotic-resistant than planktonic cells,leading to severe infections.Therefore,there is an urgent need to develop innovative tools for investigating biofilm regulators and devising novel antibacterial strategies.In this study,we developed Cy-NEO-PA,a near-infrared(NIR)fluorescent probe responsive to penicillin G acylase(PGA),with bacteria-targeting ability.This probe was designed to visualize the influence of environmental factors on biofilm formation in Acinetobacter baumannii(A.baumannii).Our findings demonstrated that glucose suppressed PGA production,leading to enhanced biofilm formation,whereas phenylacetic acid(PAA)stimulated PGA production and inhibited biofilm formation in A.baumannii.These observations highlight the remarkable capability of Cy-NEO-PA to accurately measure PGA dynamics,shedding light on the critical role of PGA in biofilm development.Additionally,Cy-NEO-PA exhibited excellent biocompatibility,potent reactive oxygen species(ROS)generation,efficient photothermal conversion,and bacteria-targeting abilities,making it a promising agent for combating bacterial infections and promoting wound healing through photothermal(PTT)/photodynamic(PDT)therapy.These discoveries emphasize the significant role of PGA in antibacterial therapy and offer valuable insights for the design of effective strategies targeting PGA to combat biofilm-associated infections.

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.

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.