Formation of 6-Aminopenicillanic Acid, Penicillins, and Penicillin Acylase by Various Fungi

Several penicillin-producing fungi were examined for ability to produce 6-aminopenicillanic acid (6-APA) and penicillin acylase. 6-APA was found in corn steep liquor fermentations of Trichophyton mentagrophytes, Aspergillus ochraceous, and three strains of Penicillium sp. 6-APA was not detected in fermentations of Epidermophyton floccosum although penicillins were produced. 6-APA formed a large part of the total antibiotic production of T. mentagrophytes. The types of penicillins produced by various fungi were identified by paper chromatography, and it was found that all cultures produced benzylpenicillin. T. mentagrophytes and A. ochraceous showed increased yields of benzylpenicillin and the formation of phenoxymethylpenicillin in response to the addition to the fermentation medium of phenylacetic acid and phenoxyacetic acid, respectively. Washed mycelia of the three Penicillium spp. and two high penicillin-yielding strains of P. chrysogenum possessed penicillin acylase activity against phenoxymethylpenicillin. A. ochraceous, T. mentagrophytes, E. floccosum, and Cephalosporium sp. also had penicillin acylase activity against phenoxymethylpenicillin. Only two of the above fungi, T. mentagrophytes and E. floccosum, showed significant penicillin acylase activity against benzylpenicillin; in both cases it was very low. The acylase activity of A. ochraceous was considerably increased by culturing in the presence of phenoxyacetic acid. It is concluded that 6-APA frequently but not invariably accompanies the formation of penicillin, and that penicillin acylase activity against phenoxymethylpenicillin is present in all penicillin-producing fungi.     

Ca2+ and calmodulin antagonists inhibit the proton gradients associated with non-cyclic and cyclic photophosphorylation in spinach chloroplasts

The synthesis of benzylpenicillin (BP) after mixing phenyl-acetyl-glycine(PAG), 6-aminopenicillanic acid (6-APA) and free or immobilized penicillin amidase (E.C.3.5.1.11.) was studied as a function of pH and ionic strength. Before the final equilibrium was reached a kinetically controlled synthesis of BP was observed. Then a transient maximum concentration in BP much larger than the final equilibrium content was synthesized in the acyl-transfer process. The factors influencing this maximum have been analyzed. Increasing ionic strength markedly decreased the maximum in BP and the rate of deacylation of phenyl-acetyl-penicillin amidase by 6-APA. The change was largest when the enzyme was immobilized in a positively charged support, where at low ionic strength the concentration of 6-APA around the enzyme is larger than the bulk concentration due to the partitioning of charged solutes.     

Cell aggregates ofEscherichia coli with benzylpenicillin amidase activity

Intact cellsEscherichia coli CCM 2843, exhibiting substantial benzylpenicillin amidase activity, were bound mutually with supporting waste microbial cells, native or treated, to obtain an inexpensive biocatalyst for the production of 6-aminopenicillanic acid (6-APA). The bond was effected by glutaraldehyde (GA) and Sedipur CL-930 (PEI), without any carrier. The optimal concentration of GA was 2%, that of PEI 1%. The optimal biocatalyst was obtained by immobilization of productive cells with their fragments at a mass ratio of 4∶1. The cell aggregates were used for hydrolysis of potassium benzyl-penicillin at a concentration of 5 % to 6-APA. After 25 repeated batch conversions the degree of conversion did not decrease; its average value was 96.4%.     

Kinetic studies on the mechanism of the penicillin amidase-catalysed synthesis of ampicillin and benzylpenicillin

Hydrophobic protein chromatography was used to prepare homogeneous fractions of penicillin amidase (EC 3.5.1.11) from E. coli. The apparent ratios of the rate constants for the deacylation of the acyl-penicillin amidase formed in the hydrolysis of phenylacetylglycine or D-phenylglycine methyl ester, by H2O and 6-aminopenicillanic acid (6-APA), were determined at different concentrations of the latter compound. The ratios were obtained from direct measurements of the initial rates of formation of phenylacetic acid and benzylpenicillin or D-phenylglycine and ampicillin. For the semisynthesis of ampicillin as well as of benzylpenicillin the ratio was found to depend on the concentration of 6-APA. This was observed for heterogeneous and homogeneous enzyme preparations. These results show that 6-APA must be bound to the acyl-enzyme before the deacylation, yielding ampicillin and benzylpenicillin, occurs. The dissociation constant KN for the formation of the complex was estimated to be approximately 10mM. This mechanism in which acyl-enzyme with and without bound nucleophile is involved, is in agreement with the principle of microscopic reversibility. Both acyl-enzymes can be deacylated by H2O. The finding that there is a specific binding site for 6-APA adjacent to the binding site for the phenylacetyl-(D-phenylglycyl-) group in the active site of the enzyme is supported by the observation that 6-APA acts as a mixed inhibitor in the hydrolysis of D-phenylglycine methyl ester. The ionic strength dependence indicates that the binding site for 6-APA of the acyl-enzyme is positively charged.     

In vivo transport of the intermediates of the penicillin biosynthetic pathway in tailored strains of <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis>

Penicillium chrysogenum npe10 (Δpen; lacking the 56.8-kbp amplified region containing the penicillin gene cluster), complemented with one, two, or three penicillin biosynthetic genes, was used for in vivo studies on transport of benzylpenicillin intermediates. 6-Aminopenicillanic acid (6-APA) was taken up efficiently by P. chrysogenum npe10 unlike exogenous δ(l-α-aminoadipyl)-l-cysteinyl-d-valine or isopenicillin N (IPN), which were not taken up or were taken up very poorly. Internalization of exogenous IPN and 6-APA inside peroxisomes was tested by quantifying their peroximal conversion into benzylpenicillin in strains containing only the penDE gene. Exogenous 6-APA was transformed efficiently into benzylpenicillin, whereas IPN was converted very poorly into benzylpenicillin due to its weak uptake. IPN was secreted to the culture medium. IPN secretion decreased when increasing levels of phenylacetic acid were added to the culture medium. The P. chrysogenum membrane permeability to exogenous benzylpenicillin was tested in the npe10 strain. Penicillin is absorbed by the cells by an unknown mechanism, but its intracellular concentration is kept low. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Acyl coenzyme A: 6-aminopenicillanic acid acyltransferase from Penicillium chrysogenum and Aspergillus nidulans

A study of the final stages of the biosynthesis of the penicillins in Penicillium chrysogenum has revealed two types of enzyme. One hydrolyses phenoxymethyl penicillin to 6-aminopenicillanic acid (6-APA). The other, also obtained from Aspergillus nidulans, transfers a phenylacetyl group from phenylacetyl CoA to 6-APA. The acyltransferase, purified to apparent homogeneity, had a molecular mass of 40 kDa. It also catalyses the conversion of isopenicillin N (IPN) to benzylpenicillin (Pen G) and hydrolyses IPN to 6-APA. In the presence of SDS it dissociates, with loss of activity, into fragments of ca 30 and 10.5 kDa, but activity is regained when these fragments recombine in the absence of SDS.     

固定化青霉素酰化酶在光-pH敏感可回用两水相中裂解青霉素G为6-APA

两水相体系在发展中存在的关键问题是相体系回收困难.由于生产成本及降低污染的原因, 用过的相体系需要回收和重复使用.用环境敏感型溶解可逆聚合物形成可回用两水相体系是当前是为可行的回收方法。本文在光敏感可回用高聚物PNBC与pH敏感型可回用高聚物PADB形成的两水相体系中进行固定化青霉素酰化酶的相转移催化青霉素G产生6-APA的反应。在这个两水相体系中,通过优化,在1% NaCl 存在下,６－ＡＰＡ的分配系数可达5.78。催化动力学显示,达平衡的时间近7h,反应最高得率约85.3%（pH 7.8, 20℃）。较相近条件下的单水相反应得率提高近20%。在反应过程中,通过底物及产物的分配系数检测,发现底物分配系数变化不大,而产物6-APA及苯乙酸的分配系数发生很大变化,从而引起产物的得率变化。在两水相中,底物及产物主要分配在上相,固定化酶分配在下相,底物青霉素G进入下相经酶催化产生的6-APA及苯乙酸又转入上相,从而解除了青霉素酰化酶催化反应的底物及产物抑制作用,达到提高产物得率的效果。此外,采用固定化酶较固定化细胞效率高,占用下相体积小,较游离酶稳定性高,且完全单侧分配在下相。因此,在两水相中进行固定化酶的催化反应具有明显的优越性。形成两水相的高聚物PNBC通过488 nm 的激光照射或经滤光的450nm 光源照射得到回收;pH敏感型成相聚合物PADB可通等电点 4.1沉淀可实现循环利用,高聚物的回收率在95%-98%之间,按此回收率计算,聚合物可使用60次以上。     

Study of E. coli penicillin amidase. The pH dependence of the equilibrium constant of the enzymatic hydrolysis of benzylpenicillin]

The equilibrium constant for penicillin amidase-catalyzed hydrolysis of benzylpenicillin(Keg =3.00 +/- 0.24 x 10(-3) M at pH 5.0) and the ionization constants for phenylacetic acid (PAA) and the amino groups of 6-aminopenicillanic acid (6-APA) were determined (4.20 and 4.60 under conditions of the kinetic experiments respectively). The experimental data at pH 6.0 satisfactorily correlated with the theoretical pH-dependence for Keg constructed according to the hypothesis that benzylpenicillin synthesis has a thermodynamic optimum at pH 4.4 equal to a half-sum of the pK values for the carboxylic and amino groups of the PAA and 6-APA respectively.     

Penicillin acylase catalyzed synthesis of penicillin-G from substrates anchored in cyclodextrins

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.     

Continuous production of 6-aminopenicillanic acid from penicillin by immobilized microbial cells

Summary For continuous production of 6-aminopenicillanic acid (6-APA) the microbial cells ofEscherichia coli ATCC 9637 having high penicillin amidase (penicillin amidohydrolase, E.C. 3. 5. 1. 11) activity were immobilized by entrapment in a polyacrylamide gel lattice.Enzymatic properties of penicillin amidase of the immobilizedE. coli cells were investigated and compared with those of the intact cells. With regard to optimal pH and temperature, no marked difference was observed. The heat stability was somewhat increased by immobilization of the cells.The enzyme activity of the immobilized cell column was stable, and its half-life was 17 days at 40°C and 42 days at 30°C. From the effluent of the column, 6-APA was easily obtained in a good yield.Abbreviations 6-APA 6-aminopenicillanic acid - BIS N,N-methylenebisacrylamide - DMAPN -dimethylaminopropionitrile - SV space velocity     

Biotransformation of penicillin V to 6-aminopenicillanic acid using immobilized whole cells of E. coli expressing a highly active penicillin V acylase

The production of 6-aminopenicillanic acid (6-APA) is a key step in the manufacture of semisynthetic antibiotics in the pharmaceutical industry. The penicillin G acylase from Escherichia coli has long been utilized for this purpose. However, the use of penicillin V acylases (PVA) presents some advantages including better stability and higher conversion rates. The industrial application of PVAs has so far been limited due to the nonavailability of suitable bacterial strains and cost issues. In this study, whole-cell immobilization of a recombinant PVA enzyme from Pectobacterium atrosepticum expressed in E. coli was performed. Membrane permeabilization with detergent was used to enhance the cell-bound PVA activity, and the cells were encapsulated in calcium alginate beads and cross-linked with glutaraldehyde. Optimization of parameters for the biotransformation by immobilized cells showed that full conversion of pen V to 6-APA could be achieved within 1?hr at pH 5.0 and 35°C, till 4% (w/v) concentration of the substrate. The beads could be stored for 28 days at 4°C with minimal loss in activity and were reusable up to 10?cycles with 1-hr hardening in CaCl₂ between each cycle. The high enzyme productivity of the PVA enzyme system makes a promising case for its application for 6-APA production in the industry.     

颗粒状固定化青霉素酰化酶的研究

将巨大芽孢杆菌 (Bacillusmegaterium)胞外青霉素酰化酶通过共价键结合到聚合物载体EupergitC颗粒环氧基团上 ,制成的颗粒状固定化青霉素酰化酶表现活力达 1 40 0 μ/g左右。固定化酶水解青霉素的最适 pH8 0 ,最适温度为 55℃。在pH6 0～ 8 5、温度低于 40℃时固定化酶活力稳定。在 pH8 0、温度 37℃时 ,固定化酶对青霉素的表现米氏常数Ka为 2×1 0 - 2 mol/L ;苯乙酸为竞争性抑制剂 ,抑制常数Kip为 2 8× 1 0 - 2 mol/L ;6 APA为非竞争性抑制剂 ,抑制常数Kia为 0 1 2 5mol/L。固定化酶水解青霉素 ,投料浓度为 8% ,在使用 2 0 0批后 ,保留活力 80 %左右 ,6 APA收率平均达 89 48%。     

Comparative study of 6-APA production by free and agar immobilized bacteria in nutrient broth culture

In the present study different bacterial samples were isolated from soil of different places of Dibrugarh and screened for biotransformation ability to produce 6-Aminopenicillanic acid. Among ten isolated bacterial samples, three gram positive bacterial samples designated as AKDD-2, AKDD-4 and AKDD-6 showed the production of 6-APA from penicillin G. Assessment of production of 6-APA after incubation in penicillin G (2 mg/ml) by three different samples separately in free and agar immobilization state was done by HPLC analysis. Reusability of immobilized cells was found successful up to 14 days.     

Precipitation of phenyl and phenoxypenicillin from solutions using ammonium sulfate

An easy, rapid, and available method for separating 6-aminopenicillanic acid (6-APA), benzylpenicillin (penicillin G), and other related molecules from aqueous solutions or complex industrial broths is described. A high concentration of ammonium sulphate induces partially or totally the precipitation of the penicillin present in the solutions, while 6-APA, phenylacetic, and phenoxyacetic acid always remain in the supernatant. The filtration through No. 4 Pyrex glass-fiber filter or Whatman 3MM paper permits the separation of the compounds present in the supernatant from the other ones precipitated. The precipitated product was identified, in all cases, as ammonium penicillin. This method is described here for the first time.     

Enzymatic conversion in aqueous two-phase systems: deacylation of benzylpenicillin to 6-aminopenicillanic acid with penicillin acylase

The conversion of benzylpenicillin (BP) to 6-aminopenicillanic acid (6-APA) using penicillin acylase (penicillin amidohydrolase, EC 3.5.1.11) has been studied in aqueous two-phase systems. In a system composed of 8.9% (w/w) PEG 20000/7.6% (w/w) potassium phosphate the enzyme was almost completely partitioned to the bottom phase (K < 0.01), which allowed repeated batch conversions, recirculating the enzyme several times. The initial specific productivities were 0.31–1.47 μmol 6-APA mg protein^?1 min^?1 in repeated conversions over five steps. The yield obtained from the top phase was 0.47–0.71 mol 6-APA mol BP^?1. The results are discussed in relation to recirculating the enzyme by immobilizing it to a solid matrix. Despite the high phosphate concentration in the bottom phase the system needs to be titrated in order for the reaction to proceed. Titration of the top phase alone protected the enzyme from denaturation by strong alkali used for the titration.     

Performance of a cyclic operated multicolumns recirculated packed bed batch reactor for 6-APA production

The methodology for designing a multicolumns recirculated packed bed batch reactor (MRPBR) system is developed. The performance of the system employing immobilized penicillin-G amidase (IPGA) for the production of 6-APA is studied. The total IPGA activity in the cyclic operated MRPBR system is maintained within a constant range by periodically replacing the oldest column in the system. The reaction time for each cycle of batch reaction is, therefore, maintained within a constant range by employing a sufficient number of columns and independent on the cycle number. The variations of 6-APA production rate is held to a desired level. The production yield of IPGA increases and 6-APA production rate decreases with the number of column installed in the MRPBR system. Both the production yield and production rate increase with the IPGA lifetime in the system. The production yield can be enhanced significantly by extending the batch reaction time. For a system designed to allow the batch reaction to be completed within 240 min, the production yield of IPGA can be 80% higher than that in the stirred tank reactor (STR) system. However, the 6-APA production rate decreases to 55% of that in STR system.     

An integrated process for the production and biotransformation of penicillin.

The extraction of Penicillin G (PG) from the filtered cultivation medium of Penicillium chrysogenum and its conversion into 6-amino penicillanic acid (6-APA) and phenyl acetic acid (PhA) at pH 8 was performed in a 10 l kühni extractor during the production by means of penicillin-G-amidase immobilized in a liquid membrane carrier system (LM). 6-APA was enriched in LM, and the PhA returned to the cultivation medium. After electrocoalescence of LM, the 6-APA was converted into ampicillin with the same enzyme at pH 6, while the liquid membrane phase and enzyme were recycled and reused.     

Agro industrial by-products as flavinogenic stimulators for riboflavin production

Plate and frame filter used as a recirculated batch reactor for the production of 6-APA was studied. Penicillin-G solution was recirculated between the immobilized penicillin-G amidase (IPGA) loaded filter and the pH controlled neutralization tank. As penicillin-G solution flowed convectively through the frames loaded with IPGA particles, penicillin-G was hydrolyzed by IPGA and 6-APA was produced. Because of the large filtration area and short filtration depth of plate and frame filter, the batch hydrolysis reaction was operated at high recirculation flow rate without causing high pressure drop. The effect of recirculation flow rate on 2% penicillin-G hydrolysis was stronger than that on 8% penicillin-G hydrolysis. The amount of IPGA loaded in filter had no significant effect on the production yield of IPGA. The operational stability of IPGA in the filter was very satisfactory. There was no appreciable activity decrease after 25 batches of reaction.     

Immobilization of Escherichia coli cells with penicillin-amidohydrolase activity on solid polymeric carriers

Whole cells of Escherichia coli containing the enzyme penicillinamidohydrolase EC 3.5.1.11 were immobilized on the surface of modified macroporous copolymers of glycidylmethacrylate with ethylenedimethacrylate and of copolymers of methacrylaldehyde (MA) with divinylbenzene (DVB) by means of glutaraldehyde. These polymeric carriers were modified before cell binding by using ammonia or polyamines, especially ethylenediamine and hexamethylenediamine (HMDA). The highest specific activity and the largest yield in cell immobilization were achieved with the macroporous copolymer of MA and DVB modified with HMDA. The material thus obtained was used in repeated conversions of benzylpenicillin to 6-aminopenicillanic acid in a stirred batch reactor.     

Effect of penicillin precursors on antibiotic biosynthesis in various strains]

The regularities of biosynthesis of 6-aminopenicillanic acid (6-APA), benzylpenicillin (BP) and phenoxymethylpenicillin (PMP) by the strains under the investigation did not significantly differ. In the absence of the precursor both the strains mainly synthesized 6-APA. Phenylacetic acid (PAA) and phenoxyacetic acid (POAA) provided directed biosynthesis: the fungus synthesized BP or PMP depending on the precursor nature. When the amount of the precursors was not sufficient, 6-APA was synthesized along with the penicillins. PAA proved to be a more active precursor than POAA. When both precursors were present in the fermentation broth, only BR was synthesized. An important distinction of strain 316A was its increased sensitivity to PAA especially in the initial period. After an increase in the PAA concentration the growth rate of strain 316A lowered to a greater extent than that of strain 284A. This was likely to determine the higher levels of penicillin production by strain 316A in the presence of POAA, a nontoxic precursor. A procedure for supplying the precursors was developed. Under the laboratory conditions it provided high levels of the penicillin production.