排序方式: 共有81条查询结果,搜索用时 218 毫秒
1.
L
P Fonseca J
M
S Cabral 《Journal of chemical technology and biotechnology (Oxford, Oxfordshire : 1986)》2002,77(10):1176-1185
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 相似文献
2.
κ‐Carrageenan hydrogel crosslinked with protonated polyethyleneimine (PEI+) and glutaraldehyde (GA) was prepared and evaluated as a novel biocatalytic support for covalent immobilization of penicillin G acylase (PGA). The method of modification of the carrageenan biopolymer is clearly illustrated using a schematic diagram and was verified by FTIR, elemental analysis, DSC, and INSTRON using the compression mode. Results showed that the gels' mechanical strength was greatly enhanced from 3.9 kg/cm2 to 16.8 kg/cm2 with an outstanding improvement in the gels thermal stability. It was proven that, the control gels were completely dissolved at 35°C, whereas the modified gels remained intact at 90°C. The DSC thermogram revealed a shift in the endothermic band of water from 62 to 93°C showing more gel‐crosslinking. FTIR revealed the presence of the new functionality, aldehydic carbonyl group, at 1710 cm?1 for covalent PGA immobilization. PGA was successfully immobilized as a model industrial enzyme retaining 71% of its activity. The enzyme loading increased from 2.2 U/g (control gel) to 10 U/g using the covalent technique. The operational stability showed no loss of activity after 20 cycles. The present support could be a good candidate for the immobilization of industrial enzymes rich in amino groups, especially the thermophilic ones. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008 相似文献
3.
4.
Alkema WB Hensgens CM Snijder HJ Keizer E Dijkstra BW Janssen DB 《Protein engineering, design & selection : PEDS》2004,17(5):473-480
Penicillin acylase catalyses the condensation of Calpha-substituted phenylacetic acids with beta-lactam nucleophiles, producing semi-synthetic beta-lactam antibiotics. For efficient synthesis a low affinity for phenylacetic acid and a high affinity for Calpha-substituted phenylacetic acid derivatives is desirable. We made three active site mutants, alphaF146Y, betaF24A and alphaF146Y/betaF24A, which all had a 2- to 10-fold higher affinity for Calpha-substituted compounds than wild-type enzyme. In addition, betaF24A had a 20-fold reduced affinity for phenylacetic acid. The molecular basis of the improved properties was investigated by X-ray crystallography. These studies showed that the higher affinity of alphaF146Y for (R)-alpha-methylphenylacetic acid can be explained by van der Waals interactions between alphaY146:OH and the Calpha-substituent. The betaF24A mutation causes an opening of the phenylacetic acid binding site. Only (R)-alpha-methylphenylacetic acid, but not phenylacetic acid, induces a conformation with the ligand tightly bound, explaining the weak binding of phenylacetic acid. A comparison of the betaF24A structure with other open conformations of penicillin acylase showed that betaF24 has a fixed position, whereas alphaF146 acts as a flexible lid on the binding site and reorients its position to achieve optimal substrate binding. 相似文献
5.
酶法合成头孢菌素类抗生素的研究进展 总被引:2,自引:1,他引:2
半合成抗生素的生产在医药工业中占有十分重要的地位,青霉素酰化酶是半合成β-内酰胺类抗生素的重要用酶,由于酶法合成与化学合成相比具有一定的优越性,随着酶工程技术的发展,酶法合成头包菌素类抗生素已日益受到重视。提高合成产率,降低侧链消耗,从而降低生产成本是酶法合成头隐菌素类抗生素研究的关键问题,平衡控制和动力学控制是酶法合成过程的两大研究策略,通过改变底物,降低水活度,添加高聚物,采用固定化技术及新型应体系等措施可改善酶促反应的微环境,改变酶的动力学特性,从而起到强化合成途径的作用,本文综述了该领域的一些研究进展并进行了展望。 相似文献
6.
A homology model of penicillin acylase from Alcaligenes faecalis and in silico evaluation of its selectivity 总被引:1,自引:0,他引:1
Braiuca P Ebert C Fischer L Gardossi L Linda P 《Chembiochem : a European journal of chemical biology》2003,4(7):615-622
A three-dimensional model of the relatively unknown penicillin acylase from Alcaligenes faecalis (PA-AF) was built up by means of homology modeling based on three different crystal structures of penicillin acylase from various sources. An in silico selectivity study was performed to compare this homology model to the structure of the Escherichia coli enzyme (PA-EC) in order to find any selectivity differences between the two enzymes. The program GRID was applied in combination with the principal component analysis technique to identify the regions of the active sites where the PAs potentially engage different interactions with ligands. These differences were further analyzed and confirmed by molecular docking simulations. The PA-AF homology model provided the structural basis for the explanation of the different enantioselectivities of the enzymes previously demonstrated experimentally and reported in the literature. Different substrate selectivities were also predicted for PA-AF compared to PA-EC. Since no crystallographic data are available for PA-AF to date, the three-dimensional homology model represents a useful and efficient tool for fully exploiting this attractive and efficient biocatalyst, particularly in enantioselective acylations of amines. 相似文献
7.
Functionalized ionic liquids containing ethyoxyl groups were synthesized and immobilized on magnetic silica nanoparticles(MSNP) prepared by two steps,i.e.,Fe3O4 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 the means 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 Fe3O4 nanoparticles and MSNPs were ~10 and ~90 nm,respectively.The saturation magnetizations of magnetic Fe3O4 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 carrier),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 level of or higher than that in previous reports.After 10 consecutive operations,ImPGA still main-tained 62% of its initial activity,indicating the good recovery property of ImPGA activity.The ionic liquid modified magnetic particles integrate the magnetic properties of Fe3O4 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. 相似文献
8.
为提高青霉素酰化酶的催化性能和热稳定性,在酶组装过程中添加小分子试剂对介孔泡沫硅载体表面过量的活化位点进行封闭。详细考察了小分子添加质量分数和种类对青霉素酰化酶负载率、催化活力及热稳定性的影响。实验结果得到:经精氨酸封闭的固定化酶活力提高至1.92倍;甘氨酸封闭的固定化酶5 h的50℃热稳定性提高至2.9倍,甘氨酸和谷氨酸封闭的固定化酶50℃热处理25 h仍保持87.9%和82.2%的残余活力;甘氨酸和谷氨酸封闭的固定化酶最适催化pH值向中性偏移且对pH值的耐受性增强。结果表明,在青霉素酰化酶共价组装过程中添加合适的小分子封闭能显著提高酶的催化性能和热稳定性。 相似文献
9.
Zhilong Wang Yajing Guo Da Bao Hanshi Qi 《Journal of chemical technology and biotechnology (Oxford, Oxfordshire : 1986)》2006,81(4):560-565
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 相似文献
10.
Xin Jin Qi Wu Qing Chen Chun‐Xiu Chen Xian‐Fu Lin 《Journal of chemical technology and biotechnology (Oxford, Oxfordshire : 1986)》2008,83(12):1710-1716
BACKGROUND: Penicillin G acylase (PGA) has been used extensively in the β‐lactam antibiotics industry. As a biocatalyst, it is better to use immobilized enzymes than free enzymes, therefore, the immobilization of PGA on a composite carrier consisting of an adsorbent resin and biocompatible chitosan were investigated. RESULTS: First, FT‐IR, BET and SEM analysis confirmed the structure of the composite carrier. Then, the immobilization process was optimized. The activity of the immobilized PGA on the chitosan–resin (IP‐CsR) was about 1300 U (g dry carrier)?1 with a protein loading of about 27 mg (g dry carrier)?1. Compared with the immobilized PGA on unmodified resin (IP‐R), the specific activity of IP‐CsR was enhanced about 2‐fold. The operational, thermal and pH stability were investigated. IP‐CsR maintained more than 75% initial activity after 35 cycles, while IP‐R was active for only 10 cycles. The half‐life at 50 °C increased from 75 to 300 min and the most stable pH was changed from 8.0 to 5.5. CONCLUSION: A novel composite carrier containing a biocompatible chitosan was very effective for PGA immobilization. Copyright © 2008 Society of Chemical Industry 相似文献