浊点系统中青霉素水解物系的分配及固定化青霉素酰化酶的稳定性

引言 青霉素是生产β-内酰胺抗生素关键中间体6-氨基青霉烷酸(6-APA)的重要原材料.常规酶法水解工艺的底物为结晶青霉素,水解工艺和青霉素发酵工艺不协调;水解反应在控制pH 7～8的环境中进行,不但消耗中和剂氨而且产生工业废物[1].     

Modelling of the Hydrolysis of Benzylpenicillin to 6-Aminopenicillanic Acid and Phenyl Acetic Acid by an Immobilised Penicillin Amidase in a Small Pilot Plant Batch Recirculated Reactor

This paper deals with the modelling of the hydrolysis of benzylpenicillin to 6-aminopenicillanic acid (6-APA) and phenyl acetic acid (PAA) in a small pilot plant batch recirculated reactor by an immobilised penicillin amidase preparation. By using the following linearised form for an integrated Michaelis-Menten equation Et/V₀X=α+β| In (1-X)/X| where α and β are reaction kinetic parameters, good correlations are obtained of α and β with linear velocity across the reactor, substrate concentration and temperature of operation. A process to determine α and β from initial velocity measurements is outlined. The applicability of the above equation to published data is also analysed.     

乳状液膜包酶制备6-APA的研究

Production of 6-aminopenicillanic acid (6-APA) by hydrolysis using penicillin acylase (PA) was studied as a model of an enzymatic emulsion liquid membrane (ELM) process. The loss of PA activity was examined for various membrane compositions (organic solvent, surfactant, carrier). The effects of some experimental variables on the stability of emulsion were investigated. It was found that the choice of organic solvent greatly affected tilestability of the emulsion. Increasing the concentration of the carrier in the membrane phase increases the transfer rate of substrate and products but also has a destabilizing effect on the emulsion. The recovery of 6-APA obtained by a di-carrier system (N263-N1923) was much higher than those when either of the di-carriers was used separately.The whole process was controlled both by the enzymatic reaction rate and by the transfer rate of the substrate and the products, however, the ratio of them could be changed by varying the composition of the system. For an optimum condition, it was obtained that the recovery ratio of 6-APA was over 80% and the conversion of benzyl penicillin (PG) was up to 90% in the external phase after 30 minutes. Meanwhile, the breakage percentage of the emulsion was less than 2%.     

固定化青霉素酰化酶生产6-APA的产业化技术

该项目研究成功新型颗粒状固定化青霉素酰化酶,并建立了5t/a的生产能力。用无机氮源替代了有机氮,显著降低了固定化酶的成本;采用比重较大的吸附剂吸附酶蛋白,简化了酶的提取步骤;应用吸附、洗脱、超滤3步工艺,实现了酶的高效分离纯化。产品达到国外同类产品水平,用于6-APA的生产,酶成本低于国外产品。预计国内半合成抗生素生产对固定化青霉素酰化酶的需求量在10t/a左右。     

6-APA制备方法的研究进展

6-APA是半合成青霉素的前体。对6-APA的不同制备方法进行了对比分析,得出直通工艺法更具有经济效益和社会效益。     

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.     

Dynamics of penicillin G hydrolysis in an electro‐membrane reactor

Analysis of penicillin G hydrolysis in a membrane reactor with membrane‐entrapped penicillin G acylase is performed using a mathematical model of the reactor system. An electric field imposed to the reactor is considered to enhance transport rates of reaction components and reaction rate. Diffusion, electrophoretic migration and electro‐osmotic flux across the membrane are considered. The analysis focuses on possible effects of the principal operational parameters (electric field intensity, inlet substrate concentration, membrane thickness) on reactor performance. Multiplicities of steady states are frequently encountered. The membrane reactor performance can be easily targeted towards the required reaction regime by applying a constant or periodically varying electric field to the system. The periodic alternation of the polarity of the electric field substantially increases the effectiveness factor of penicillin hydrolysis compared with the steady state operation. Proper adjustments of electric field intensity may also compensate for the decay in enzyme activity. © 2001 Society of Chemical Industry     

Direct extraction of phenylacetic acid from immobilised enzymatic hydrolysis of penicillin G with cloud point extraction

The enzymatic hydrolysis of potassium salt of penicillin G (Pen G) into phenylacetic acid (PAA) and potassium salt of 6‐aminopenicillanic acid (APA) is inhibited not only by the substrate and the product APA but also by the by‐product PAA. The partitioning behaviour of PAA in a cloud point system, a novel two‐phase partitioning system, was determined. Direct extraction of PAA in the process of immobilised penicillin acylase hydrolysis of Pen G without pH control was achieved. Pen G was hydrolysed almost completely and the product APA concentration in the cloud point system was much higher than in the control, suggesting that the cloud point system may be applied as a novel extractive bioreactor for the enzymatic hydrolysis of Pen G. Copyright © 2006 Society of Chemical Industry     

基于膜分离过程的6-APA生产技术

该项目设计了适当的纳滤膜浓缩工艺,以提高6-APA生成率,6-APA成品的平均克分子收率达90.18%;设计了醋酸丁酯-乙醇结晶工艺;选择了合适的溶媒体系及控制pH值以确保6-APA的质量;对溶媒及副产品苯乙酸进行了回收利用,降低了成本,减少了环境污染。纳滤膜分离技术是使低浓度裂解高浓度结晶得以实现的关键技术。用于100t 6-APA生产线上取得良好效果。     

青霉素直通法生产6-APA工业化研究

为了降低6-氨基青霉烷酸（6-APA）的生产成本,采用了直通、脱酯、萃取工艺对6-APA的生产工艺进行了改进,使得总收率和质量得到提高,大幅度降低了6-APA的生产成本,提高了6-APA及后续产品的市场竞争力.     

青霉素制备青霉素亚砜的研究

以青霉素G钾盐与低质量分数过氧乙酸为原料氧化制备青霉素亚砜 ,其最佳工艺条件为 :n(C16 H18N2 O4 SK)∶n(CH3CO3H) =1 .0∶( 1 1～ 1 2 ) ,反应温度 0～ 5℃ ,反应时间 2 .0～ 2 5h ,w(CH3CO3H) =8 5% ,w(CH3CO2 H) =1 0 %。青霉素亚砜收率达 96 8%。同时建立了青霉素亚砜的半定量分析方法 ,以硅胶G为固定相 ,以V(CH3CO2 C4 H9 n)∶V(CH3CO2 H)∶V(NaH2 PO4 -H2 O)∶V(C4 H9OH n) =6.0∶2 .0∶1 .0∶0 5为流动相 ,用TLC法对产品进行半定量分析检测 ,并经IR、MS谱图验证了产品结构。     

Structure and protein separation efficiency of poly(N-isopropylacrylamide) gels: Effect of synthesis conditions

Crosslinked poly(N-isopropylacrylamide) (PNIPA) gels with different crosslink densities in the form of rods and beads were prepared by free-radical crosslinking copolymerization. Solution and inverse suspension polymerization techniques were used for the gel synthesis. The gels were utilized to concentrate dilute aqueous solutions of penicillin G acylase (PGA), bovine serum albumin (BSA), and 6-aminopenicillanic acid (6-APA). The discontinuous volume transition at 34°C observed in the gel swelling was used as the basis of concentrating dilute aqueous protein solutions. PNIPA gels formed below 18°C were homogeneous, whereas those formed at higher temperatures exhibited heterogeneous structures. The water absorption capacity of PNIPA gels in the form of beads was much higher, and their rate of swelling was much faster than the rod-shaped PNIPA gels. It was also found that the polymerization techniques used significantly affect the properties of PNIPA gels. The separation efficiency decreased when the protein molecules PGA or BSA in the external solution were replaced with small-molecular-weight compounds, such as 6-APA. The protein separation efficiency by the gel beads increased to 100% after coating the bead surfaces with BSA. © 1998 John Wiley & Sons, Inc. J Appl Polym Sci 67: 805–814, 1998     

青霉素G亚砜对-硝基苄酯的合成

青霉素G亚砜对—硝基苄酯是合成GCLE的重要中间体 ,可通过以青霉素G为原料 ,用低浓度过氧乙酸氧化 ,再以对—硝基苄氯进行羧基保护获得。本方法既能确保产品质量 ,又有利于安全生产 ,适合于工业化生产     

青霉素G亚砜的合成

以青霉素G钾盐为原料,采用15%左右低浓度过氧乙酸为氧化剂合成青霉素G亚砜,反应时间2～2.5h,n(过氧乙酸):n(青霉素G钾盐)=(1.1～1.2):1.0,反应温度和结晶温度在0～5℃,总收率可达96%以上。所得青霉素G亚砜可直接从溶液中结晶析出,解决了产物与反应体系的分离。产品纯度较高。     

Preparation,properties and uses of polymer-enzyme conjugates

Enzymes can be immobilized by gel entrapment, by microencapsulation, by physical or ionic adsorption, by covalent binding to inorganic or organic carriers, or by whole cell immobilization. Of particular interest is the large number of chemical reactions developed for the covalent binding of enzymes via their nonessential functional groups to inorganic carriers such as glass, ceramics and iron, to natural polymers such as cellulose and Sepharose, and to synthetic polymers such as nylon, polyacrylamide, and other vinyl polymers and copolymers possessing reactive chemical groups. The stability of certain enzymes is markedly increased on their immobilization. It was thus possible to transform the biologically active polymer derivatives into active enzyme beads, enzyme capsules, enzyme columns and enzyme membranes and these enabled the construction of enzyme reactors such as the batch-stirred tank reactors, the continuous packed bed reactors, and fluidized bed reactors. So far mainly immobilized hydralases and isomerases are being used in industry on a large scale. It seems likely, however, that once adequate techniques become available for cofactor recycling, the use of immobilized enzymes will be extended to other organic reactions, particularly those involving stereospecific synthesis of simple or complex organic molecules. Among the industrial processes in which immobilized enzymes are being used, it is worth mentioning the industrial-scale continuous production of fructose enriched syrup from glucose by immobilized glucose-isomerase, the batch process for the production of 6-aminopenicillanic acid (6-APA) from penicillin G with the aid of immobilized penicillin amidase; the production of aspartame from aspartic acid and phenylalanine by immobilized thermoase; the large scale production of optically active amino acids with immobilized amino acid acylase; and the large scale production and application of immobilized lactase for the hydrolysis of lactose. The recently developed process for acrylamide production using immobilized nitrilase containing microbial cells should also be referred to. The successful use of an NAD-polyethylene glycol conjugate (NAD-PEG) as a nondialyzable water-soluble coenzyme derivative in the enzymic synthesis of leucine from α-ketoisocaproic acid and ammonia, in a membrane-enclosed reactor containing L-leucine dehydrogenase, NAD-PEG, formate and formate dehydrogenase, illustrates the new possibilities opened up by making use of cofactor-polymer conjugates. The use of enzyme-polymer conjugates in analytical and clinical is also illustrated.     

控制技术在化学制药中的应用

以青霉素工业盐裂解生产6-APA为例，介绍了控制技术在制药生产的应用及特点。     

Separation of phenylacetic acid, 6-aminopenicillanic acid and penicillin G with electrodialysis under constant current

The separation behavior of phenylacetic acid (PAA), 6-aminopenicillanic acid (6-APA) and penicillin-G (Pen-G) with electrodialysis under constant current was studied. The effects of ionic concentrations and current density on their separation behavior were investigated. The sorption of PAA, 6-APA and Pen-G and in the anion exchange membrane, and the variations of the applied voltage and current efficiencies with time during electrodialysis were also examined. It was found that the molecular size and the affinity toward the anion exchange membrane played a key role for the electrodialysis of PAA, 6-APA and Pen-G. Phenylacetic acid had the lowest affinity toward the membrane but the fastest transport rate. Penicillin-G had the highest affinity toward the membrane but the slowest transport rate. With the increase of Pen-G concentration, the concentration polarization of Pen-G in the vicinity of anion exchange membrane became very serious, which severely retarded the transport of PAA and 6-APA. Although the increase of current density accelerated the transport of PAA, 6-APA and Pen-G, more serious concentration polarization of Pen-G occurred and the separation ratio of PAA to 6-APA decreased.     

新型复合材料MnFe_2O_4@SiO_2-NH_2的制备及其固定青霉素G酰化酶

通过溶剂热法制备出高磁性的聚合物微球MnFe_2O_4,经正硅酸乙酯(TEOS)和3-氨基丙基三乙氧基硅烷(APTS)对微球表面进行改性修饰,制备出新型复合材料MnFe_2O_4@SiO_2-NH_2,并将其用于固定青霉素G酰化酶。在Si/Fe比为7 mmol/g、n(TEOS)∶n(APTS)=1∶1时,固定化酶PGA/MnFe_2O_4@SiO_2-NH_2在37℃下水解青霉素G钾合成6-氨基青霉烷酸,表观酶活为1 660 IU/g、载酶量为107.1 mg/g、比酶活为15.5 IU/mg、活性回收率为46.9%。经过6次重复使用,保留初始酶活的81.3%,在使用中固定化酶在磁场的作用下能够快速沉降与产物分离。     

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.     

Reactive extraction of 6-aminopenicillanic acid with aliquat-336: Equilibrium and kinetics

The reactive extraction of 6-aminopenicillanic acid (6-APA) from aqueous buffer solution has been studied using a liquid anion exchanger, tricaprylylmethyl ammonium chloride (Aliquat-336) in n-butyl acetate as the solvent. The extraction equilibrium constant and partition coefficient increase with increase in pH up to a limiting value of pH, beyond which extraction decreases due to coextraction of OH^? and buffer anions and hydrolytic decomposition of 6-APA. The experimental data could be predicted from an equilibrium model which takes into account the ideal behaviour of the two liquid phases. The coextraction of the buffer anion under low pH conditions was found to be negligible. The extraction rate measured in a stirred cell of constant interfacial area appears to be dependent on the interfacial mass transfer of the reactive species as well as on the interfacial reaction through its dependence on the concentration of 6-APA in the aqueous and organic phases, respectively.