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1.
The usefulness of Lilly's kinetic equation to describe penicillin G hydrolysis performed by immobilized penicillin acylase onto the acrylic carrier has been shown. Based on the experimental results characteristic kinetic constants have been estimated. The effect of noncompetitive inhibition of 6-amino penicillanic acid has not been found. Five components of reaction resistance have been defined. These components were also estimated for the reaction of the native enzyme as well as the Boehringer preparation.List of Symbols
C
E g/m3
enzyme concentration
-
C
P,C
Q mol/m3
product concentrations
-
C
S mol/m3
substrate concentration
-
C
SO mol/m3
initial substrate concentration
-
K
A mol/m3
constant which defines the affinity of a substrate to the enzyme
-
K
iS mol/m3
substrate inhibitory constant
-
K
iP mol/m3
PhAA inhibitory constant
-
K
iQ mol/m3
6-APA inhibitory constant
-
k
3 mol/g/min
constant rate of dissociation of the active complex
-
R(1)
concentrational component of reaction resistance
-
R(2)
resistance component derived from substrate affinity
-
R(3)
resistance component due to the inhibition of the enzyme by substrate
-
R(4)
resistance component due to the inhibition of the enzyme by PhAA
-
R(5)
resistance component due to inhibition of the enzyme by 6-APA
-
r = dCs/dt mol/m3 min
rate of reaction
-
t
min reaction time
- (i)
relative resistance of reaction 相似文献
2.
Susana M. S. A. Bernardino Dr. Pedro Fernandes Luís P. Fonseca 《Biotechnology journal》2009,4(5):695-702
The present work focuses on the development and basic characterization of a new magnetic biocatalyst, namely penicillin G acylase (PGA), immobilized in sol-gel matrices with magnetic properties, ultimately aimed for application in cephalexin (CEX) synthesis. A mechanically stable carrier, based on porous xerogels silica matrixes starting from tetramethoxysilane (TMOS), was prepared leading to micro-carriers with medium sized particles of 30 μm, as determined by scanning electron microscopy. An immobilization yield of 95–100% and a recovered activity of 50–65% at 37°C, as determined by penicillin G (PG) hydrolysis (pH STAT method), were observed. These results clearly exceed those reported in a previous work on PGA immobilization in sol-gel, where only 10% of activity was recovered. The values of activity were kept constant for 6 months. Immobilized PGA (682 U/gdry weight) retained high specific activity throughout ten consecutive runs for PG hydrolysis, suggesting adequate biocatalyst stability. The CEX synthesis was performed at 14°C, using the free and immobilized PGA in aqueous medium. Phenylglycine methyl ester was used as acyl donor at 90 mM and 7-aminodeacetoxycephalosporanic acid was the limiting substrate at 30 mM. The CEX stoichiometric yield after 1-h reaction was close to 68% (23 mM CEX/h) and 65% (19 mM CEX/h), respectively. 相似文献
3.
Daniel Hormigo Isabel De La Mata María Castillón Carmen Acebal 《Biocatalysis and Biotransformation》2013,31(4):271-281
Recombinant penicillin acylase from Streptomyces lavendulae was covalently bound to epoxy-activated Sepabeads EC-EP303®. Optimization of the immobilization process led to a homogeneous distribution of the enzyme on the support surface avoiding the attachment of enzyme aggregates, as shown by confocal electron microscopy. The optimal immobilized biocatalyst had a specific enzymatic activity of 26.2IUgwetcarrier?1 in the hydrolysis of penicillin V at pH 8.0 and 40°C. This biocatalyst showed the highest activity at pH 8.5 and 65°C, 1.5 pH units lower and 5°C higher than its soluble counterpart. Substrate specificity of the derivative also showed its ability to efficiently hydrolyze other natural aliphatic penicillins such as penicillins K, F and dihydroF. The immobilized enzyme was highly stable at 40°C and pH 8.0 (t1/2=625 h vs. t1/2=397 h for the soluble enzyme), and it could be recycled for at least 30 consecutive batch reactions without loss of catalytic activity. 相似文献
4.
5.
6.
Dr. A. Gebauer Dr. T. Scheper Prof. Dr. K. Schügerl 《Bioprocess and biosystems engineering》1987,2(2):55-58
Enzyme production with E. coli ATCC 11105, in a complex medium using phenylacetic acid as inducer is carried out in a stirred-tank reactor of 10 dm3 and an airlift tower-loop reactor of 60 dm3 with outer loop at a temperature of 27 °C. The optimum inducer concentration was 0.8 kg/m3, which was kept constant by fed-batch operation. The optimum of the relative dissolved O2-concentration with regard to saturation is below 10% in a stirred-tank reactor and at 35% in a tower-loop reactor. It was kept constant by parameter-adaptive control of the aeration rate. In a stirred-tank enzyme productivity is slightly higher than in a tower-loop reactor, and much higher than in a bubble column reactor.List of Symbols CPR kg/(m3 h)
CO2-production rate
- OTR kg/(m3 h)
O2-transfer rate
- OUR kg/(m3 h)
O2-utilization rate
- PAA
phenylacetic acid (inducer)
- RQ = CPR/OUR
respiratory quotient
-
X kg/m3
cell mass concentration
-
m h–1
maximum specific growth rate 相似文献
7.
8.
Summary Cefotiam (7) and cefotaxime (8) are obtained by penicillin acylase hydrolysis of (5) and (6), prepared, in turn, by chemical condensation of 7-DIMAT (3) and 7-ACA (4), respectively, with N-phenacetyl-2-aminothiazol-4-yl acetic acids (1) and (2). 相似文献
9.
Immobilized penicillin acylase preparations have much higher activities per unit volume than immobilized cell preparations. Many parameters of the deacylation reaction are dependent on pH and both reactant and one of the products, 6-aminopenicillanic acid, are acid and alkali labile. Acid is produced as result of the deacylation reaction and must be neutralised. The influence of these pH effects on the design of the catalyst and the reactor is discussed. 相似文献
10.
Penicillin acylase catalysis in the presence of ionic liquids 总被引:2,自引:0,他引:2
Several ionic liquids were used as reaction media for penicillin G acylase catalysis. In all the assayed ionic liquids, [bmim]PF6 proved good media for PGA-catalyzed hydrolysis. A novel [bmim]PF6/water two-phase system is provided for 6-aminopenicillanic acid (APA) production, which will be more benefical than aquous batch systems used widely in industrial production of APA. 相似文献
11.
Enzymatic syntheses of cefaclor by immobilized penicillin acylase under kinetic control were carried out. According to the initial reaction rate ratio of synthesis to hydrolysis (Vs/Vh), penicillin acylase from Alcaligenes faecalis was chosen as the suitable catalyst for the synthesis of cefaclor. The reaction conditions, such as temperature, pH, and substrate concentration were investigated based on their Vs/Vh values. In the process of preparing cefaclor, in situ product removal (ISPR) and acyl donor feeding were used to achieve high yield. At the optimal conditions, the yield of cefaclor was 90%. In addition, the product were separated and purified, the total yield of cefaclor was 61%. 相似文献
12.
V Fitton N Verdoni J Sanchez X Santarelli 《Journal of biochemical and biophysical methods》2001,49(1-3):553-560
The introduction of affinity chromatography has opened a new dimension in protein purification. This article reviews the current techniques used in the penicillin acylase purification process, especially pseudo-affinity chromatography. A profile for a suitable ligand is established. An aromatic ring and the presence of one or several amino groups seem essential for proper interaction. Immobilized metal affinity chromatography now seems to be a good competitor. 相似文献
13.
G. Alvaro R. Fernandez-Lafuente C. M. Rosell R. M. Blanco J. L. Garcia-Lopez J. M. Guisan 《Biotechnology letters》1992,14(4):285-290
Summary We propose a new and integrated method for the evaluation of industrial enzymes. The application of this method to the enzyme penicillin G acylase fromKlyvera citrophila shows very interesting industrial propects. This acylase presents a much better stability agains heat, pH or organic cosovents as compared with the more popular enzyme fromEscherichia coli. In addition, this enzyme is very easy to immobilize through its amine groups and to stabilize through multipoint covalent attachment on activated pre-existing supports. 相似文献
14.
Penicillin acylase (EC 3.5.1.11) catalyses the condensation of phenylacetic acid (PAA) and 6-aminopenicillanic acid (6-APA) to form benzylpenicillin (BP). Both PAA and 6-APA were found to form host-guest complexes with beta-methylcyclodextrin (beta m-CD) and gamma-cyclodextrin (gamma-CD) respectively. The rate of the reaction catalyzed by the enzyme remained unaffected if one of the substrates used was in the cyclodextrin complexed form. However, in this case, the reaction lasted longer and yielded about 20 per cent more products compared to the condensation reaction involving only uncomplexed substrates. There was distinct increase in the rate of formation of the antibiotic, if both substrates used are in CD-complexed form. 相似文献
15.
Ignacio Prieto Julio Martín Roberto Arche Piedad Fernández Augustín Pérez-Aranda 《Applied microbiology and biotechnology》1990,33(5):553-559
Summary Oligonucleotide-directed mutagenesis has been used to obtain specific changes in the penicillin acylase gene from Kluyvera citrophila. Wild-type and mutant proteins were purified and the kinetic constants for different substrates were determined. Mutations in Met168 highly decreased the specificity constant of the enzyme for penicillin G, penicillin V and phenylacetyl-4-aminobenzoic acid and the catalytic constant k
cat for phenylacetyl-4-aminobenzoic acid. Likewise, the phenylmethylsulphonyl-fluoride sensitivity was significantly decreased. It is concluded that the 168 residue is involved in binding by interaction with the acid moiety of the substrate. A putative penicillin-binding domain was located in penicillin acylase by sequence homology with other penicillin-recognizing enzymes. Lys374 and His481, the conserved amino acid residues that are essential for catalysis in these enzymes, can be changed in penicillin acylase with no changes to the k
cat and phenylmethylsulphonyl fluoride reactivity, but change the K
m.The likelihood of the existence of this proposed penicillin binding site is discussed. The reported results might be used to alter the substrate specificity of penicillin acylase in order to hydrolyse substrates of industrial significance other than penicillins.
Offprint requests to: I. Prieto 相似文献
16.
Penicillin acylase purification with the aid of hydrophobic charge induction chromatography 总被引:2,自引:0,他引:2
Coulon D Cabanne C Fitton V Noubhani AM Saint-Christophe E Santarelli X 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2004,808(1):111-115
The aim of this work was to test a chromatographic support, 4-mercaptoethyl pyridine (4-MEP) Hypercel, for penicillin acylase purification by using pure penicillin acylase and crude extract. Two equilibration buffers with various salt concentrations and different flow rates were tested. The relationships between electrostatic and hydrophobic interactions and proteins are demonstrated. (NH4)2SO4 proved preferable because no salting-in occurred, contrary to NaCl. The recovery and purification fold were similar to those obtained in pseudo-affinity chromatography with a three-fold reduction of the (NH4)2SO4 concentration. 相似文献
17.
18.
W. Kurzątkowski W. Kuryłowicz A. Paszkiewicz 《Applied microbiology and biotechnology》1982,15(4):211-213
Summary The activity of penicillin G production was compared in a well defined medium by native vesicles as well as by calcium alginate gel immobilized vesicles isolated from the protoplasts of Penicillium chrysogenum PQ-96. The activity yield of the immobilized vesicles was 44% in comparison with native vesicles. After 60 h of storage at 4° C the native vesicles showed a rapid decrease in penicillin G production. The storage stability of these vesicles was improved after entrapment inside the calcium alginate gel. After 240 h of storage 1 mg of immobilized vesicular protein catalyzed the production of about 140 nmoles of penicillin G in 1 h. 相似文献
19.
Liu YC ChangChien CC Suen SY 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2003,794(1):67-76
The immobilized metal affinity membrane (IMAM) with modified regeneration cellulose was employed for purification of penicillin G acylase (PGA). For studying PGA adsorption capacity on the IMAM, factors such as chelator surface density, chelating metal, loading temperature, pH, NaCl concentration and elution solutions were investigated. The optimal loading conditions were found at 4 degrees C, 0.5 M NaCl, 32.04 micromol Cu(2+) per disk with 10 mM sodium phosphate buffer, pH 8.5, whereas elution conditions were: 1 M NH(4)Cl with 10 mM sodium phosphate buffer, pH 6.8. By applying these chromatographic conditions to the flow experiments in a cartridge, a 9.11-fold purification in specific activity with 90.25% recovery for PGA purification was obtained. Meanwhile, more than eight-times reusability of the membrane was achieved with the EDTA regeneration solutions. 相似文献
20.
M O Mandel A I K?stner E H Sijmer G I Kleiner L M Elizarovskaya V J Stamer 《Prikladnaia biokhimiia i mikrobiologiia》1975,11(2):219-225
An active insoluble preparation of immobilized benzyl penicillin acylase (IBA) EC 3.5.1.11 has been obtained by its entrapping into polyacrylamide gel lattice. Due to immobilization the preparation maintains up to 87% of its initial activity. The kinetics of IBA at low substrate concentrations obeys the Michaelis-Menten law; however, the apparent KM value decreases and the temperature optimum elevates. The inhibition by the reaction products--6-aminopenicillanic acid and phenylacetic acid--has been found to be 4.3 mM. The resultant IBA preparation proves to be suitable for hydrolysis of 5% benzyl penicillin solutions. 相似文献