青霉素酰化酶的生产及分离技术研究

本文讨论了青霉素酰化酶的生产及分离技术方法。简单介绍了青霉素酰化酶的DNA重组技术构建基因工程菌及用基因工程菌结合发酵的方法生产青霉素酰化酶的方法。最后介绍了一些青霉素酰化酶的分离方法。     

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...     

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。     

argE基因的表达优化及Zn2+添加时间和浓度的影响机制分析

确定了目的基因argE在重组菌BL21(DE3)-pET22b-argE中的表达位置,研究了Zn2+对重组菌生长及表达产物活性的影响,并分析了影响机制. 结果表明,argE可在重组菌中高效表达,表达产物N-乙酰鸟氨酸脱酰基酶大多以不可溶的包涵体形式存在,只有少量为有活性的可溶性表达. 1.0 g/L的Mg2+对重组菌的生长及酶活有明显促进作用. Zn2+加入时机及加入量不同,影响结果也不同. 发酵起始加入Zn2+严重抑制菌的生长及酶活,而在1.0%乳糖诱导2.5 h后加入则可解除生长抑制并提高酶活. SDS-PAGE电泳及活力测定证实Zn2+参与形成酶的催化中心,对酶的表达量没有影响.     

谷氨酰胺合成酶腺苷酰化位点的定点突变和产胺的初步研究

为解决谷氨酰胺合成酶腺苷酰化修饰失活的问题,利用基因定点突变的方法将谷氨酸棒杆菌的谷氨酰胺合成酶(Glutamine Synthetase, GS)腺苷酰化位点由Tyr405突变为Phe405,并在大肠杆菌中获得突变后GS的表达. 对比腺苷酰化位点突变前后的重组大肠杆菌pET-3a/GSI和pET-3a/GSIM在高氨环境下的GS活性和谷氨酰胺产量,发现重组菌pET-3a/GSIM在高氨环境下的最大酶活是150 U/L,产谷氨酰胺浓度为17.5 g/L,分别是pET-3a/GSI酶活(30 U/L)的5.0倍和产谷氨酰胺水平(3.4 g/L)的5.1倍,GS定点突变使谷氨酸转化为谷氨酰胺的途径得到强化.     

增强组成型重组大肠杆菌质粒稳定性的发酵策略

质粒稳定性是影响基因工程菌外源蛋白表达的重要因素,同时,外源蛋白的表达又影响着质粒稳定性。与诱导型表达系统相比,在组成型表达系统中,由于外源蛋白的持续表达,导致细胞代谢负担加重,质粒稳定性的控制更加困难。通过对组成型大肠杆菌DH5a／pKKFPGA发酵过程的优化,增强了质粒稳定性,发酵结束时,仍然维持在85％左右,表达产物粪产碱杆菌青霉素酰化酶的酶活单位达到23384U·L^-1。     

青霉素酰化酶基因工程菌 E.coli A_(56)(pPA22)水解青霉素 G 的动力学

用初速度法测定了青霉素酰化酶基因工程菌 E.coli A_(56)(pPA22)游离细胞和固定化细胞水解青霉素 G 的动力学常数,并进行了动力学模型的检验。该菌体酶活高,米氏常数小,固定化后米氏常数和苯乙酸抑制常数增大,底物抑制常数和6-APA 抑制常数没有变化。通过青霉素 G 裂解过程的考察,验证了青霉裂解动力学模型的适用性及动力学常数测定的可靠性。     

产GL-7ACA酰化酶基因工程菌的高活性表达

实现了戊二酰基-7-氨基头孢烷酸(GL-7ACA)酰化酶的组成型表达,并通过替换重组质粒启动子的RBS序列,在摇瓶培养条件下,使新构建的重组质粒pGEMKT-HPRfACY在大肠杆菌JM105中表达GL-7ACA酰化酶酶活达到0.44 U/mL,是替换前的2倍. 使用廉价的玉米浆作为培养基中氮源主要来源,使JM105/pGEMKT-HPRfACY菌株酶活提高到2.98 U/mL. 采用补料批式培养,利用流加营养物控制发酵中后期的pH值,在pH为7.5的条件下,酶活峰值达到6.37 U/mL. 该体系无需诱导,工艺操作过程简单,成本低廉.     

重组枯草芽孢杆菌生产青霉素G酰化酶发酵条件的研究

对产青霉素G酰化酶的温度诱导型重组枯草芽孢杆菌发酵条件进行了研究。结果表明,培养基的最佳氮源和碳源组成为:35gL-1牛肉膏、3gL-1葡萄糖和6gL-1淀粉;最佳诱导时机是细胞对数生长的中后期,诱导前应将pH调节至中性;最佳诱导条件为升高温度至50℃并维持4min;随后将温度降低到34℃进行重组蛋白的表达。在上述条件下,PGA的表达水平可以达到5.8UmL-1。SDS-PAGE电泳分析表明该重组菌能够将绝大部分表达的PGA分泌到细胞外,而且表达的PGA蛋白占有高比例(90%)。     

青霉素酰化酶的生产与应用新进展

介绍了青霉素酰化酶的生产及应用新进展。着重介绍了青霉素酰化酶固定化技术的发展。青霉素酰化酶主要应用于6氨基青霉烷酸的工业生产和半合成的β内酰胺抗生素的合成,是在半合成抗生素的生产上有重要作用的一种酶。此外,青霉素酰化酶也可应用于其它的生物转化,如肽的合成、手性化合物的外消旋混合物的拆分。     

固定化酶用聚偏氟乙烯分离膜的制备及性能表征

为研究膜技术在生物反应器中的应用,选用聚偏氟乙烯为膜材料,聚乙二醇为添加剂,溶于不同的有机溶剂中,以相转化法成膜,以用于酶固定化。文章讨论了挥发时间、凝固浴温度及铸膜液温度等因素的影响,并对聚偏氟乙烯膜的通透性和固定化性能进行了研究。     

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     

矩形错流移动床床内颗粒流速分布的考察

以矩形错流移动床床内颗粒流动为考察对象,在前人工作的基础上,明确了体积膨胀应力参数 ｋ的物理意义,完善了床内颗粒应力分布的理论预测;并提出了颗粒间发生剪切滑移的理论判据,由此可对不同过床气速条件下床内颗粒流速分布区域的分界点作出理论预测。同时选择了与热态实验物料堆比重相近的小米和硅胶珠为冷态实验物料,在矩形错流移动床中实验考察了床内颗粒流速分布随过床气速的变化规律,结果表明,理论预测与实验相符较好,基于此提出了矩形错流移动床床内颗粒流速分布的预测方法     

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     

低高径比喷射环流反应器结构的研究

在 ７０ Ｌ 低高径比釜式喷射环流反应器中,分别研究了单导流筒单喷咀、单导流筒多喷咀以及多导流筒多喷咀三种结构反应器的气液传质性能,得到了相应平均气含率和体积溶氧传质系数的关联式。研究结果表明多导流筒多喷咀是一种具有传质效率高、能耗低和便于放大的高效生化反应器。此外,还研究了 ８００ Ｌ多导流筒多喷咀结构反应器,得到体积溶氧传质系数关联式,其计算值和实验值吻合较好     

Characterization of the {beta}-lactam binding site of penicillin acylase of Escherichia coli by structural and site-directed mutagenesis studies

The binding of penicillin to penicillin acylase was studiedby X-ray crystallography. The structure of the enzyme–substratecomplex was determined after soaking crystals of an inactiveßN241A penicillin acylase mutant with penicillin G.Binding of the substrate induces a conformational change, inwhich the side chains of     

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     

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.     

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.