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Covalent Immobilization of Penicillin G Acylase onto Fe₃O₄@Chitosan Magnetic Nanoparticles
( Xiao Min Ling ),( Xiang Yu Wang ),( Ping Ma ),( Yi Yang ),( Jie Mei Qin ),( Xue Jun Zhang ),( Ye Wang Zhang ) 한국미생물 · 생명공학회 2016 Journal of microbiology and biotechnology Vol.26 No.5
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℃. 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 0.113 mmol/min/mg-protein and 0.059 mmol/min/mg-protein, 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℃ 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.
Cheng Zhou,Shemin Zhu,Xiuming Wu,Bo Jiang,Tao Cen,Shubao Shen 한국생물공학회 2010 Biotechnology and Bioprocess Engineering Vol.15 No.3
Penicillin acylase (PA) is known to regulate the microenvironment of nanospores. In this study, nanopores containing chemically-modified macromolecules co-assembled with immobilized PA were constructed. We also investigated the various types of functionalized mesocellular siliceous foams (MCFs) commonly used for the immobilization of PA by measuring the catalytic performance and stability of each PA preparation. Amino-MCF activated by p-benzoquinone was chosen as the optimum support for PA immobilization. Successful modification of macromolecules was verified by FT-IR and ultraviolet (UV)spectroscopy. The specific activity of PA co-assembled with dextran 10 k was 99.1 U/mg, which was 1.5-fold that of pristine immobilized PA, while the optimum pH was shifted to neutral. Compared to pristine immobilized and free PA, the optimum temperatures for the modified PA were 5 and 10oC higher, respectively. The residual activity of the ficoll derivative of PA after treatment at 50oC for 6 h was 70%, and this was later increased to 214.5% compared to that of pristine immobilized PA. The dextran 10 k derivative of PA exhibited 90.2% residual activity after 25times of continuous use. The results show that chemicallymodified macromolecules co-assembled with PA in amino-MCF provided a suitable microenvironment for enzyme stability.
( Zhixiang Lv ),( Qingmei Yu ),( Zhou Wang ),( Ruijiang Liu ) 한국미생물생명공학회(구 한국산업미생물학회) 2019 Journal of microbiology and biotechnology Vol.29 No.6
Magnetic Ni<sub>0.7</sub>Co<sub>0.3</sub>Fe<sub>2</sub>O<sub>4</sub> nanoparticles that were prepared via the rapid combustion process were functionalized and modified to obtain magnetic Ni<sub>0.7</sub>Co<sub>0.3</sub>Fe2O<sub>4</sub>@SiO<sub>2</sub>-CHO nanocomposites, on which penicillin G acylase (PGA) was covalently immobilized. Selections of immobilization concentration and time of fixation were explored. Catalytic performance of immobilized PGA was characterized. The free PGA had greatest activity at pH 8.0 and 45 ℃ while immobilized PGA’s a ctivities p eaked at p H 7.5 and 4 5 ℃. Immobilized PGA had better thermal stability than free PGA at the range of 30-50 ℃ for different time intervals. The activity of free PGA would be 0 and that of immobilized PGA still retained some activities at 60 ℃ after 2 h. V<sub>max</sub> and K<sub>m</sub> of immobilized PGA were 1.55 mol/min and 0.15 mol/l, respectively. Free PGA’s V<sub>max</sub> and K<sub>m</sub> separately were 0.74 mol/min and 0.028 mol/l. Immobilized PGA displayed more than 50% activity after 10 successive cycles. We concluded that immobilized PGA with magnetic Ni<sub>0.7</sub>Co<sub>0.3</sub>Fe<sub>2</sub>O<sub>4</sub>@SiO<sub>2</sub>-CHO nanocomposites could become a novel example for the immobilization of other amidohydrolases.
Carola Bahamondes,Lorena Wilson,Carolina Aguirre,Andrés Illanes 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.4
Synthesis of cephalexin with immobilized penicillin acylase at high substrates concentration at an acyl donor to nucleophile molar ratio of 3 was comparatively evaluated in aqueous and ethylene glycol media using a statistical model. Variables under study were temperature,pH and enzyme to substrate ratio and their effects were evaluated on cephalexin yield, ratio of initial rates of cephalexin synthesis to phenylglycine methyl ester hydrolysis,volumetric and specific productivity of cephalexin synthesis,that were used as response parameters. Results obtained in both reaction media were modeled using surface of response methodology and optimal operation conditions were determined in terms of an objective function based on the above parameters. At very high substrates concentrations the use of organic co-solvents was not required to attain high yields and actually almost stoichiometric yields were obtained in a fully aqueous media with the advantages of higher productivities than in an organic co-solvent media and compliance with the principles of green chemistry.
Cheng Zhou,Anming Wang,Zhiqiang Du,Shemin Zhu,Shubao Shen 한국화학공학회 2009 Korean Journal of Chemical Engineering Vol.26 No.4
Macromolecular reagents were co-assembled with penicillin acylase (PA) and immobilized in mesocellular siliceous foams (MCFs) to resemble living cells. Types and concentrations of macromolecules were studied. The catalytic characteristic and stability of PA preparations were also investigated. PA assembled with dextran 10 k in MCFs showed maximum specific activity, 1.32-fold of that of the solely immobilized PA. The optimum pH of dextran and BSA derivatives shifted to neutrality, and the optimum temperature increased by 10 oC. Also, Km of BSA derivative of PA declined 56.7% compared to solely immobilized PA, while the Kcat/Km of PA assembled with BSA was enhanced to 147%. After incubation at 50℃ for 6 h, residual activity of PA assembled with BSA exhibited 53.0%. The ficoll derivative showed 82.8% of its initial activity at 4 oC after 8-week storage. The results indicated that macromolecular reagents assembled with PA in MCFs could dramatically improve the catalytic performance and stability of im- mobilized enzyme.
Synthesis of Cefprozil Using Penicillin G Acylase in Recyclable Aqueous Two-phase Systems
Chaohui Zhu,Xue-Jun Cao 한국생물공학회 2014 Biotechnology and Bioprocess Engineering Vol.19 No.5
Cefprozil is an important semi-syntheticcephalosporin antibiotic. In this study, immobilized penicillinG acylase (PGA) is used to catalyze the acylation of 7-amino-3-(1-propenyl)-4-cephalosporanic acid (7-APRA)and 4-hydroxyphenylglycine methyl ester (HPGME) and arecyclable thermo-pH responsive PNB/PADB aqueous twophasesystem (ATPS) is used to synthesize cefprozil. In thissystem, the partition coefficient of cefprozil was 2.24 with60 mmol/L (NH4)2SO4. In addition, the optimal enzymaticreaction conditions were found to be pH 6.5, 20°C, 78 u/mLimmobilized PGA, 30 mmol/L 7-APRA and 90 mmol/LHPGME. In the PNB/PADB ATPS, the maximal yield ofcefprozil was 75.81% with 60 mmol/L (NH4)2SO4 and inthe single aqueous system the yield was 56.02%. Theyields are thought to improve because there is a reductionin product inhibition.