Expression, purification and crystallization of penicillin G acylase from Escherichia coli ATCC 11105

The penicillin acylase gene (pac) from Escherichia coli ATCC11105 was cloned into pUC 9 and the resulting vector (pUPA-9),when transformed into E.coli strain 5K, allowed the constitutiveoverproduction of mature penicillin acylase when grown at 28°C.The enzyme ws purified from the periplasmic fraction of E.colipUPA-9 by hydrophobic interaction chromatography and anion exchange.Crystals of penicillin acylase were grown in batch using polyethyleneglycol 8000 as a precipitant. The crystals (space group P1)diffracted to beyond 2.3 Å.     

青霉素酰化酶纳米凝胶的制备与性质

提高青霉素酰化酶的耐热性和耐有机溶剂性对于其工业应用具有重要的意义.采用E.coli Top10F'/pGEMKT-TacPGA-Tag为表达菌株发酵生产青霉素酰化酶,经金属鳌合层析得到比酶活为23200 U · L-1的青霉素酸化酶样品,将青霉素酰化酶样品与丙烯酰琥珀酰亚胺反应在酶分子表面修饰上丙烯酰基,然后加入丙烯...     

An integrated downstream processing strategy for the recovery and partial purification of penicillin acylase from crude media

An integrated process strategy for the recovery of penicillin acylase was developed, based on precipitation of non‐enzymatic proteins directly from Escherichia coli homogenates or crude extracts using Rolquat (quaternary ammonium salt) and adsorption of the enzyme on Amp‐Seph (3.8 µmole ampicillin cm^?3) under pseudo‐affinity conditions. The effect of pH, concentrations of ammonium sulfate and Rolquat, and also concentrations of protein and cell debris on the precipitation of non‐enzymatic proteins from homogenates and crude extracts of penicillin acylase were analysed. The method of addition of Rolquat to homogenates and crude extracts significantly influenced the size of the precipitated particles. Improved results on the specific activity of penicillin acylase were obtained for 22% and 1% (w/v) of ammonium sulfate and Rolquat, respectively, added sequentially to enzyme solutions and at room temperature. Under these experimental conditions, the specific activity of penicillin acylase in homogenates and crude extracts was enhanced 2.5–3.0‐fold. Finally, the integrated process strategy was implemented first by precipitation of non‐enzymatic proteins and recovery of penicillin acylase directly from the enzyme solution treated with Rolquat using an adsorption/filtration system with an overall yield of 86%. This system allows simultaneously the filtration of cell debris and fine precipitated particles, in situ recovery of penicillin acylase by its adsorption on Amp‐Seph, and selective desorption of the enzyme with a specific activity of 11 IU (mg prot)^?1 and a desorption yield of 95%. © 2002 Society of Chemical Industry     

Stability and stabilisation of penicillin acylase

The stability of an oligomeric enzyme, penicillin acylase, was studied in aqueous media. The enzyme was produced by mutant cells of Escherichia coli ATCC 9637, extracted from the periplasmic space by osmotic shock and further purified using a pseudo‐affinity adsorption process. Enzyme stabilisation attempts were performed with salts, alcohols and sugars. The highest levels of retained activity were obtained in the presence of 15% (w/v) ammonium or sodium sulfate. A kinetic model was proposed to describe the inactivation of penicillin acylase, taking into account results obtained in stability assays performed at different temperatures and with different enzyme concentrations. According to this model, the inactivation of penicillin acylase involves an intermediary active precursor of the enzyme, formed prior to dissociation into sub‐units. © 1999 Society of Chemical Industry     

Penicillin acylase release from Escherichia coli cells by mechanical cell disruption and permeabilization

The release of Penicillin acylase from Escherichia coli cells through mechanical cell disruption using high‐pressure homogenization and sonication was studied. From these cell disruption processes, the enzyme activity was totally released although with low specific activities, 0.1–0.3 IU(mg prot)^?1. Intracellular total soluble protein release was quantified and modelled by a first order kinetic model. The effect of the driving force for each mechanical method, namely acoustic power input and homogenization pressure, on the respective kinetic disruption constants was also analysed. The release of Penicillin acylase by cell permeabilization using osmotic shock was also evaluated. The effects of cell concentration, penicillin acylase activity in E coli cells, type of buffer, pH, hypertonic solution composition, temperature and time used for osmotic shock were evaluated. Using cold osmotic shock, highly selective penicillin acylase release was attained with specific enzyme activities of about 4 IU(mg prot)^?1 and enzyme activity release yields higher than 90%. The high purity of the penicillin acylase was a consequence of the optimized differential enzyme release method which was validated by SDS gel electrophoresis. © 2002 Society of Chemical Industry     

青霉素酰化酶的分离纯化和固定化研究新进展

青霉素酰化酶又称为青霉素酰胺酶或青霉素氨基水解酶,主要从大肠埃希菌胞内酶和巨大芽孢杆菌胞外酶获得,该酶已大规模应用于工业生产β-内酰胺类抗生素的关键中间体和半合成β-内酰胺类抗生素。主要介绍了青霉素酰化酶固定化技术的进展,讨论了不同固定化技术的特点,并展望了固定化青霉素酰化酶的发展前景。     

Hydrolysis of Penicillin G by Penicillin G Acylase Immobilized on Chitosan Microbeads in Different Reactor Systems

The kinetic parameters for penicillin G hydrolysis in systems with penicillin G acylase from Escherichia coli (free and immobilized on activated chitosan microbeads produced by electrostatic extrusion) were determined. The obtained kinetic results indicated that both systems (free and immobilized) are inhibited by high concentrations of the substrate (penicillin G) as well as by products of the reaction (6‐aminopenicillanic acid and phenylacetic acid). The microbeads appeared convenient for penicillin G acylase immobilization reducing negative inhibitory effects. The hydrolysis was also investigated in a packed bed reactor. The derived kinetic model predicted good hydrolysis rates in the reactor while the system with recirculation of the reaction mixture proved to be a potentially favorable solution providing operation at low shear stresses and possibly higher hydrolysis rates than in the packed bed reactor alone.     

基于大分子拥挤原理的介孔二氧化硅中青霉素酰化酶的共价组装

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.     

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.     

Selective reverse micellar extraction of penicillin acylase from E coli

The recovery of penicillin acylase from E coli by a new reverse micellar treatment is described. The results are compared with the cell disruption by ultrasound followed by reverse micellar extraction. The process gave selective extraction of penicillin acylase directly from the periplasmic space of E coli without disrupting the cells. Unlike ultrasonication which breaks open the cells entirely, making subsequent processing difficult and expensive, reverse micellar treatment of cells gave a moderate recovery of 60% of enzyme activity in a highly pure form. © 2001 Society of Chemical Industry     

Enzyme flow microcalorimetry—a useful tool for screening of immobilized penicillin G acylase

Screening of a representative series of immobilized penicillin G acylase biocatalysts (enzyme, cells) using enzyme flow microcalorimetry is described. Immobilized penicillin G acylase biocatalysts were either prepared in the laboratory by various techniques or obtained from four commercial manufacturers. An industrial strain of Escherichia coli was entrapped in (poly)acrylamide gel or hardened calcium pectate gel. Semi-purified enzyme was immobilized in various ways—either by covalent binding to oxirane-acrylic beads or chlorotriazine bead cellulose or by entrapment in (poly)acrylamide gel. The validity of the enzyme flow microcalorimetry results was corroborated by a pH-stat method, showing enzyme flow microcalorimetry to be a suitable method for rapid screening of immobilized biocatalysts regardless of the immobilization technique, carrier type or the biocatalyst source. © 1998 Society of Chemical Industry     

含青霉素酰化酶的重组大肠杆菌的发酵培养

研究了含青霉素酰化酶的重组菌 Ｅ．ｃｏｌｉ Ａ５６ （ｐ Ｐ Ａ２２）发酵过程中环境因素对酶活的影响,考察了发酵液性质变化与酶活表达之间的关系,获得了酶活较高时的优化环境条件。结果表明,青霉素酰化酶在重组菌生长的平衡期后开始大量表达,苯乙酸作为限制性底物对青霉素酰化酶的合成起诱导作用     

Increasing the synthetic performance of penicillin acylase PAS2 by structure-inspired semi-random mutagenesis

A semi-random mutagenesis approach was followed to increase the performance of penicillin acylase PAS2 in the kinetically controlled synthesis of ampicillin from 6-aminopenicillanic acid (6-APA) and activated D-phenylglycine derivatives. We directed changes in amino acid residues to positions close to the active site that are expected to affect the catalytic performance of penicillin acylase: alpha R160, alpha F161 and beta F24. From the resulting triple mutant gene bank, six improved PAS2 mutants were recovered by screening only 700 active mutants with an HPLC-based screening method. A detailed kinetic analysis of the three most promising mutants, T23, TM33 and TM38, is presented. These mutants allowed the accumulation of ampicillin at 4-5 times higher concentrations than the wild-type enzyme, using D-phenylglycine methyl ester as the acyl donor. At the same time, the loss of activated acyl donor due to the competitive hydrolytic side reactions could be reduced to <20% with the mutant enzymes compared >80% wild-type PAS2. Although catalytic activity dropped by a factor of 5-10, the enhanced synthetic performance of the recovered penicillin acylase variants makes them interesting biocatalysts for the production of beta-lactam antibiotics.     

离子液体反胶团中青霉素酰化酶的水解反应特性

反胶团是表面活性剂在非极性溶剂中自聚形成的微观结构．其亲水头部朝内,疏水尾部朝外,形成可容纳极性溶质的水相空腔,在蛋白质萃取及酶催化方面得到广泛研究．对于用青霉素酰化酶水解青霉素生产6-氨基青霉烷酸（6-APA）的工艺,反胶团兼具高效性与分离便利的优点Ⅲ．目前反胶团的调控主要集中在水含量、pH值与阴离子类型等方面,对表面活性剂头部的作用还缺乏了解．     

Chemistry of some fluorescamine–amine derivatives with relevance to the biosynthesis of benzylpenicillin by fermentation

The reaction of fluorescamine with 6‐aminopenicillanic acid (6‐APA) at pH 4 was investigated for its potential use in the biosynthesis of benzylpenicillin. A number of amino acids and penicillin amine derivatives, that reacted with fluorescamine at pH 7–9, were unlikely to do so to a significant extent at pH 4 and hence were unlikely to interfere in results that led to the biosynthesis of benzylpenicillin. Biosynthesis was followed using Penicillium chrysogenum Wis F3‐64, growing in a corn steep liquor medium in a shake flask fed with phenylacetic acid daily. Analysis of benzylpenicillin formation was effected enzymically and fluorimetrically. A sample of the fermentation broth was treated with buffer at pH 7.8 and an active penicillin acylase solution for 1 h at 37 °C. The pH was then lowered to 4 by swamping with acetate buffer and the solution was treated with fluorescamine. The resulting fluorescence was compared with that of a standard 6‐aminopenicillianic acid solution treated in the same manner. In this case the biosynthesis of benzylpenicillin was found to increase over 6 days. The results were compared with those for a control broth where the penicillin acylase was not added. © 2002 Society of Chemical Industry     

Continuous enzymatic penicillin G hydrolysis in countercurrent water-butyl acetate biphasic systems

Penicillin G (Pen G) was continuously hydrolyzed into phenylacetic acid (PAA) and aminopenicillanic acid (APA) by penicillin acylase in countercurrently contacted water-butyl acetate biphasic systems. A set of three mixer-settlers was used to establish countercurrent contact of the two immiscible liquid phases. The enzymatic reaction was catalyzed by immobilized penicillin acylase. Pen G was fed in the central mixer. APA continuously left the system with the aqueous exit stream and PAA with the organic exit stream. The conversion in the countercurrently contacted biphasic system was significantly higher than the conversion that can be achieved in an equivalent batch system. A mathematical model, which is based on partition and reaction equilibria, was used to predict the concentrations and pH along the countercurrent reactor. Provided that no APA crystallization occurs, the mathematical model predicted the conversion and the pH profile in the mixer-settler cascade accurately.     

Penicillin Acylase‐Catalyzed Solid‐State Ampicillin Synthesis

The ability of immobilized penicillin acylase from E. coli to retain a remarkable catalytic activity in solid‐state systems has been demonstrated. Stabilization of immobilized penicillin acylase by inorganic salt hydrates allowed us to exploit nearly the whole catalytic activity of the enzyme at a very low water content. Using this technique, enzymatic synthesis of ampicillin in solid‐state systems was performed with high yields (up to 70% starting from equimolar mixture of reagents) and rates comparable to the corresponding values in homogeneous solutions and heterogeneous systems, “aqueous solution‐precipitate”. Peculiarities of the enzymatic solid‐state acyl transfer process, such as absence of the clear‐cut maximum on the ampicillin accumulation curves and dependence of the synthetic efficiency on the enzyme loading, have been observed. The space‐time yield of solid‐state enzymatic ampicillin synthesis was shown to be up to ten times higher compared to the homogeneous solutions and heterogeneous “aqueous solution‐precipitate” systems.     

Immobilization of penicillin acylase on copolymer of butyl acrylate and ethylene glycol dimethacrylate

The effects of glutaraldehyde, enzyme concentrations and reactants volumes, ionic strength, pH value and carrier particle diameter on immobilization of penicillin acylase onto acrylic carriers were studied. The activity of immobilized enzyme preparations was also studied over a range of pH values and temperatures and thermal and pH stabilities were determined. The use of the immobilized preparation for penicillin G hydrolysis in a batch reactor was investigated. The immobilized enzyme gave a significant reduction in hydrolysis time compared to hydrolysis by the native enzyme.     

A polyacrylic acid as a carrier for immobilization of penicillin acylase

A copolymer of acrylic acid with divinylbenzene was synthesized by suspension polymerization. This polymer is an effective carrier. Penicillin acylase was immobilized on this carrier to convert benzylpenicillin to 6‐aminopenicillanic acid, which may be employed in the manufacture of semisynthetic penicillins. Factors that affect the activity of immobilized penicillin acrylase, such as temperature, pH, and amount of native enzyme, were studied. Under suitable conditions, the activity and activity recovery of the immobilized enzyme were 3100 U/g (dry carrier, p‐dimethylaminobenzaldehyde method) and 59.7%, respectively. The immobilized penicillin acylase shows a remarkable increase in stability. At 40°C and pH 8.0 the value of the kinetic Michaelis–Menten constant (K_m) of the immobilized enzyme is 2.8 × 10^?3 mol/L, and the value of activation energy of enzyme catalysis is 71.5 kJ/mol. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 2067–2069, 2002     

Stabilization of native penicillin G acylase by ionic liquids

Five different ionic liquids, based on dialkylimidazolium cations associated with perfluorinated and bis{(trifluoromethyl)sulfonyl}imide anions, were used to investigate the scope and limitations of these new solvents as media for penicillin G acylase‐catalyzed reactions. Deactivation of the native enzyme in ionic liquids (ILs) and in organic solvents (toluene, dichloromethane and 2‐propanol) at low water content and 40 °C was investigated using the hydrolysis of penicillin G as activity test. Native penicillin G acylase 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, [emim⁺][Tf₂N^?], which was about 2000‐fold higher than that in 2‐propanol. An enhancement of the PGA stability was observed by the presence of substrate in ionic liquids based on tetrafluoroborate and hexafluorophosphate anions, achieving the highest increase of the half‐life time in 1‐butyl‐3‐methylimidazolium hexafluorophosphate ([bmim⁺][PF₆^?]), which was about 9‐fold higher than the half‐life time in the absence of substrate. Copyright © 2007 Society of Chemical Industry