Prediction of penicillin V acylase stability in water-organic co-solvent monophasic systems as a function of solvent composition

Hydrolytic activity of penicillin V acylase (EC 3.5.1.11) can be improved by using organic cosolvents in monophasic systems. However, the addition of these solvents may result in loss of stability of the enzyme. The thermal stability of penicillin V acylase from Streptomyces lavendulae in water-organic cosolvent monophasic systems depends on the nature of the organic solvent and its concentration in the media. The threshold solvent concentration (at which half enzymatic activity is displayed) is related to the denaturing capacity of the solvent. We found out linear correlations between the free energy of denaturation at 40 degrees C and the concentration of the solvent in the media. On one hand, those solvents with logP values lower than -1.8 have a protective effect that is enhanced when its concentration is increased in the medium. On the other hand, those solvents with logP values higher than -1.8 have a denaturing effect: the higher this value and concentration, the more deleterious. Deactivation constants of PVA at 40 degrees C can be predicted in any monophasic system containing a water-miscible solvent.     

Cloning and characterization of penicillin V acylase from Streptomyces mobaraensis

We report on the molecular cloning and characterization of penicillin V acylase (PVA) from an actinomycete, Streptomyces mobaraensis (Sm-PVA), which was originally isolated as an acylase that efficiently hydrolyzes the amide bond of various N-fatty-acyl-l-amino acids and N-fatty-acyl-peptides as well as capsaicin (8-methyl-N-vanillyl-6-nonenamide). In addition, the purified Sm-PVA hydrolyzed penicillin V with the highest activity (k(cat)) among the PVAs so far reported, penicillin G, and 2-nitro-5-phenoxyacetamide benzoic acid. The BLAST search revealed that the Sm-PVA precursor is composed of a polypeptide that is characteristic of enzymes belonging to the beta-lactam acylase family with four distinct segments; a signal sequence (43 amino acids), an alpha subunit (173 amino acids), a linker peptide (28 amino acids), and a beta subunit (570 amino acids). The mature, active Sm-PVA is a heterodimeric protein with alpha and beta subunits, in contrast to PVAs isolated from Bacillus sphaericus and B. subtilis, which have a homotetrameric structure. The amino acid sequence of Sm-PVA showed identities to PVA from S. lavendulae, N-acylhomoserine lactone-degrading acylase from Streptomyces sp., cyclic lipopeptide acylase from Streptomyces sp., and aculeacin A acylase from Actinoplanes utahensis with 68, 67, 67, and 41% identities, respectively.     

Substrate specificity of penicillin acylase from Streptomyces lavendulae

The kinetic parameters of several substrates of penicillin acylase from Streptomyces lavendulae have been determined. The enzyme hydrolyses phenoxymethyl penicillin (penicillin V) and other penicillins with aliphatic acyl-chains such as penicillin F, dihydroF, and K. The best substrate was penicillin K (octanoyl penicillin) with a k(cat)/K(m) of 165.3 mM(-1) s(-1). The enzyme hydrolyses also chromogenic substrates as NIPOAB (2-nitro-5-phenoxyacetamido benzoic acid), NIHAB (2-nitro-5-hexanoylamido benzoic acid) or NIOAB (2-nitro-5-octanoylamido benzoic acid), however failed to hydrolyse phenylacetil penicillin (penicillin G) or NIPAB (2-nitro-5-phenylacetamido benzoic acid) and penicillins with polar substituents in the acyl moiety. These results suggest that the structure of the acyl moiety of the substrate is more determinant than the amino moiety for enzyme specificity. The enzyme was inhibited by several organic acids and the extent of inhibition changed with the hydrophobicity of the acid. The best inhibitor was octanoic acid with a K(i) of 0.8 mM. All the results, taking together, point to an active site highly hydrophobic for this penicillin acylase from Streptomyces lavendulae.     

胞外青霉素酰化酶产生菌的选育

利用纸片显色方法,从土壤甲诀速筛选出98株产胞外青霉素酰化酶的菌种,经复筛其中10株酶活力较高,经鉴定均属于巨大芽孢杆菌。经单株分离得46号菌,用这株菌进行了产酶条件的研究,在最适产酶条件下,酶话力比开始提高了3．6倍。在此基础上又进行了物理化学因素处理,得突变株UL-81,酶活力达720u／1 Ooml发酵液。对原株和突变株进行比较,发现UL-81菌落、细胞形态、诱导剂苯乙酸用量及添加时间等明显不同于原株。在500L罐发酵酶活达8 20u／1OOml发酵液,为开始酶活的16倍。     

A Kinetic Examination of Penicillin Acylase Stability in Water-organic Solvent Systems at Different Temperatures

We have studied the thermal stability of penicillin acylase from Streptomyces lavendulae in water-organic solvent monophasic systems at the range of temperatures between 40 and 60°C. We found a linear correlation between the log λP value of the solvent and the activation free energy for denaturation ( ΔG d ) at all temperatures tested. Thermodynamic analysis of the results indicates that solvents with log λP ≤-2.3 have protective effects, whereas solvents with log λP ≥-1.8 are deleterious for penicillin acylase.     

Stability and catalytic properties of penicillin acylase in systems with low water content

Stability and catalytic properties of native and immobilized penicillin acylase were studied in systems with low water content. Preparations of both native and immobilized penicillin acylase demonstrated the catalytic activity even in solid-phase systems which contained 3-5 wt. % of water. The stability and catalytic activity of penicillin acylase at low water content depended on the thermodynamic water activity (aw) in the system.     

Beijerinckia indica var.penicillanicum penicillin V acylase: enhanced enzyme production by catabolite repression-resistant mutant and effect of solvents on enzyme activity

Summary Beijerinckia indica var.penicillanicum mutant UREMS-5, producing 168% more penicillin V acylase, was obtained by successive treatment with UV, -irradiation and ethylmethane sulfonate. Penicillin V acylase production by the mutant strain was resistant to catabolite repression by glucose. Incorporation of glucose, sodium glutamate and vegetable oils in the medium enhanced enzyme production. The maximum specific production of penicillin V acylase was 244 IU/g dry weight of cells. Effect of solvents on hydrolysis of penicillin V by soluble penicillin V acylase and whole cells was studied. Methylene chloride, chloroform and carbon tetrachloride significantly stimulated the rate of penicillin V hydrolysis by whole cells.     

Cephalosporin C acylase and penicillin V acylase formation by Aeromonas sp. ACY 95

Aeromonas sp. ACY 95 produces constitutively and intracellularly a penicillin V acylase at an early stage of fermentation (12 h) and a cephalosporin C acylase at a later stage (36 h). Some penicillins, cephalosporin C and their side chain moieties/analogues, phenoxyacetic acid, penicillin V and penicillin G, enhanced penicillin V acylase production while none of the test compounds affected cephalosporin C acylase production. Supplementation of the medium with some sugars and sugar derivatives repressed enzyme production to varying degrees. The studies on enzyme formation, induction and repression, and substrate profile suggest that the cephalosporin C acylase and penicillin V acylase are two distinct enzymes. Substrate specificity studies indicate that the Aeromonas sp. ACY 95 produces a true cephalosporin C acylase which unlike the enzymes reported hitherto hydrolyses cephalosporin C specifically.The authors are with Research and Development, Hindustan Antibiotics Limited, Pimpri. Pune 411 018, India     

A Kinetic Examination of Penicillin Acylase Stability in Water-organic Solvent Systems at Different Temperatures

We have studied the thermal stability of penicillin acylase from Streptomyces lavendulae in water-organic solvent monophasic systems at the range of temperatures between 40 and 60°C. We found a linear correlation between the log &#117 P value of the solvent and the activation free energy for denaturation ( &#106 G d ) at all temperatures tested. Thermodynamic analysis of the results indicates that solvents with log &#117 P &#104 &#109 2.3 have protective effects, whereas solvents with log &#117 P &#83 &#109 1.8 are deleterious for penicillin acylase.     

Enzymatic splitting of penicillin V for the production of 6-APA using immobilized penicillin V acylase

Penicillin V acylase from Fusarium sp. SKF 235 was immobilized on several cation-exchange resins, of which Amberlite CG-50 was preferred. Maximum activity of the immobilized penicillin V acylase was 250 to 280 IU/g dry beads. The pH and temperature optima of the enzyme shifted from 6.5 to 6.8 and 55°C to 60°C, respectively, as a result of immobilization. However, the K _m for penicillin V remained at 10mm. Parameters for producing 6-aminopenicillanic acid were investigated and the immobilized penicillin V acylase was used for 68 cycles in a stirred tank reactor.     

Biosynthesis of penicillin V acylase by Fusarium sp.: effect of culture conditions

Penicillin V acylase was produced, both intracellularly and extracellularly, by Fusarium sp. SKF 235 grown in submerged fermentation. When neopeptone was added to the medium, >95% of the penicillin V acylase was extracellular. In the absence of a complex organic nitrogen source, the fungus produced low levels of totally intracellular penicillin V acylase. MgSO₄ was essential for synthesis of the enzyme, which was induced by phenoxyacetic acid and penicillin V. The maximum yield of penicillin V acylase was 430 IU/g dry cell wt. The optimum pH value and temperature for the penicillin V acylase were 6.5 and 55°C, respectively.     

Enzyme action in polymer and salt solutions. I. Stability of penicillin acylase in poly(ethylene glycol) and potassium phosphate solutions in relation to water activity

The stability of penicillin acylase (penicillin aminohydrolase, EC 3.5.1.11) was studied in poly(ethylene glycol) and potassium phosphate solutions. Enzyme stability measured as the half-life of the enzymatic activity and the transition temperature determined by differential scanning calorimetry, correlated well. The enzyme stability could not be related to the water activity as a measure of solute-solvent interaction. It seems to be related more to the concentration of the solutes and much less to the molecular weight of poly(ethylene glycol). The stabilizing effect of poly(ethylene glycol) is also discussed in terms of poly(ethylene glycol)-protein interactions.     

Enzymatic hydrolysis of penicillin V to 6-aminopenicillanic acid byFusarium oxysporum

Summary A strain ofFusarium oxysporum was identified as having an intracellular penicillin V acylase activity (penicillin V amidohydrolase EC 3.5.1.11). Activity was induced by phenoxyacetic acid and had a good tolerance for high substrate and product concentrations. Washed cells could be used repeatedly for the complete hydrolysis of 5% penicillin V solutions. The enzyme was partially purified and concentrated from disrupted cells by fractional precipitation with water miscible solvents.     

Penicillin Acylase Activity of Penicillium chrysogenum

The penicillin acylase activity of Penicillium chrysogenum was studied. Washed mycelial suspensions of a high penicillin-producing and a nonproducing strain were found to be similar in respect to relative acylase activity on benzylpenicillin, 2-pentenylpenicillin, heptylpenicillin, and phenoxymethylpenicillin. The relative rates for both strains, as determined by 6-aminopenicillanic acid formation, were approximately 1.0, 2.5, 3.5, and 6.0 on the penicillins in the order given. The high producing strain formed both 6-aminopenicillanic acid and "natural" penicillins in fermentations to which no side-chain precursor had been added. Therefore, its demonstrated ability to cleave the natural penicillins, 2-pentenylpenicillin and heptylpenicillin, suggests that at least some of the 6-aminopenicillanic acid produced during such fermentations arises from the hydrolysis of the natural penicillins. At pH 8.5, the mycelial acylase activity of the nonproducing strain was about three times that at pH 6.0; at 35 C, it was about 1.5 times as active as it was at 30 C. When tested on penicillin G or V, no differences in either total or specific penicillin acylase activity were observed among mycelia harvested from cultures of the nonproducer to which penicillin G, penicillin V, or no penicillin had been added. Acetone-dried mycelium from both strains displayed acylase activity, but considerably less than that shown by viable mycelium. Culture filtrates were essentially inactive, although a very low order of activity was detected when culture filtrate from the nonproducer was treated with acetone and the acetone-precipitated material was assayed in a minimal amount of buffer.     

ENHANCED PRODUCTION OF 6-AMINOPENICILLANIC ACID IN AQUEOUS METHYL ISOBUTYL KETONE SYSTEM WITH IMMOBILIZED PENICILLIN G ACYLASE

Enzymatic hydrolysis of penicillin G for production of 6-amino-penicillanic-acid (6-APA) was achieved by using penicillin G acylase as catalyst in an aqueous-methylisobutyl ketone (MIBK) system. The optimization was carried out and it was found that the best conversion was improved 10% more than the aqueous system, which was obtained at the conditions: initial pH 8.0, 5.0% (W/V) substrate (penicillin G), and temperature at 35°C, and the ratio of aqueous and organic phase was 3:1. The stability of the biocatalyst was studied at the operational conditions. After 5 cycles of semi-batch reactions, the residual activity of penicillin G acylase was 69.2% of the initial activity. There was no apparent loss of the yield of product. This process has a potential application in the industrial scale production of 6-APA because it simplifies the process effectively.     

Modification of Enzyme Properties by the use of Inhibitors During Their Stabilisation by Multipoint Covalent Attachment

The effect of a number of inhibitors adsorbed at the active site of penicillin acylase during immobilisation/stabilisation is reported. Each inhibitor, when it is adsorbed at the active centre of penicillin acylase promotes a specific enzymatic conformation which remains fixed after the stabilisation process by multipoint covalent attachment to pre-existing supports. A number of inhibitors: penicillin sulfoxide, phenylacetic acid, mandelic acid, and phenylglycine were employed to induce conformational changes. The activity towards different substrates of the enzyme derivative (in hydrolysis and in synthesis) was determined. The stability of the derivatives was also measured. This technique provides a broad spectrum of enzymatic derivatives with a range of activity/stability depending on the inhibitors used in their stabilisation. The resulting choice offers a considerably increased potential for the use of the enzyme since one can select a derivative which will specifically catalyse the reaction of interest.     

Mutant Escherichia coli penicillin acylase with enhanced stability at alkaline pH

Increased stability at alkaline pH should be a valuable attribute for the utilization of penicillin acylase in bioreactors employed to convert penicillins into 6-aminopenicillanic acid, a precursor of semisynthetic penicillins. In these systems, base is added for pH control, which results in local alkaline conditions that promote enzyme inactivation. Hydrolysis and synthesis reactions are also pH dependent. Here, we report work in which the gene coding for Escherichia coli penicillin acylase was subjected to oligonucleotide-directed random mutagenesis at regions coding for amino acids predicted to be at the surface of the enzyme. The resulting mutant library, cloned in E. coli, was screened by a filter paper assay of the colonies for the presence of penicillin acylase activity with enhanced stability at alkaline pH. Characterization of one of the selected clones revealed the presence of a mutation, Trp431-Arg, which would presumably alter the surface charge of the protein. In vitro experiments demonstrated a near twofold increase in the half-life of the mutant enzyme when stored at pH 8.5 as compared with the wild-type enzyme, with a comparable specific activity at several pH values. In general, the mutant displayed increased stability toward the basic side in the pH-stability profile. (c) 1995 John Wiley & Sons, Inc.     

Specificity of Penicillin Acylase of Fusarium and of Penicillium chrysogenum

Extracts containing penicillin acylase were obtained by shaking the mycelium of Fusarium avenaceum and of Penicillium chrysogenum in 0.2 M sodium acetate or sodium chloride solution. The optimum pH for conversion of penicillin V into 6-aminopenicillanic acid (6-APA) by the enzyme of Fusarium was about 7.5, and the reaction velocity was increased by a rise in temperature from 27 to 37 C. Penicillin G and penicillins with an aliphatic side chain were cleaved much less readily than was penicillin V. With the enzyme preparation obtained from a nonpenicillin-producing strain of P. chrysogenum, the reaction rate was higher at pH 8.5 than at pH 7.5 and pH 6.5. The acylase of P. chrysogenum hydrolyzes penicillin V more readily than penicillin G. In a series of aliphatic penicillins, the amount of 6-APA formed through the action of this enzyme increased with the number of carbon atoms of the side chain. Penicillins with a glutaryl or an adipyl group as side chain were unaffected by the enzyme of Fusarium and of Penicillium. No reaction was observed upon incubation of penicillin N (with a D-aminoadipyl side chain) or isopenicillin N (with an L-aminoadipyl side chain) with Fusarium and Penicillium extract. When the carboxy group of the side chain of these penicillins was esterified, formation of 6-APA was observed upon incubation with Penicillium extract, whereas no 6-APA or only very small amounts were obtained by acylase of Fusarium.     

重组青霉素G酰化酶拆分制备（S）-邻氯苯甘氨酸

对产青霉素G酰化酶的重组枯草芽胞杆菌发酵产酶条件进行优化,确定优化后的发酵条件：可溶性淀粉10g/L、蛋白胨12g/L、酵母粉3g/L、NaCl10g,／L;pH7．5、培养温度37℃、装液量80mL（500mL三角瓶）、培养28h,青霉素G酰化酶的表达水平由最初的7．34U／mL提高至18．23U／mL。以表达青霉素G酰化酶的枯草芽胞杆菌发酵液为酶源,在水相中对映选择性催化N-苯乙酰-（R,S）-邻氯苯甘氨酸制备（S）-邻氯苯甘氨酸,当底物浓度为100mol／L时转化4h,转化率达44．2％。对底物浓度为80mmoL／L反应液中的（S）-邻氯苯甘氨酸进行分离,达到理论收率的94．29％（以N-苯乙酰-（R,S）-邻氯苯甘氨酸的0．5倍摩尔量为理论产率）,e．e．值大于99．9％。170℃条件下,N-苯乙酰-（R）-邻氯苯甘氨酸与苯乙酸共熔消旋为N-苯乙酰-（R,S）-邻氯苯甘氨酸可用于循环拆分。     

Preliminary studies on continuous recovery and purification of the penicillin acylase under pseudo-affinity conditions using phenyl-Sepharose gel

A continuous system for the recovery and purification of the penicillin acylase from crude extracts by recycling phenyl-Sepharose gel through three agitated vessels with disc filters of stainless steel is presented. The penicillin acylase present in the crude extract was absorbed into the phenyl-Sepharose gel under pseudo-affinity conditions (16% w/v of ammonium sulphate). After gel washing under the same conditions in the second vessel, enzyme desorption was performed using the same salt but at a lower concentration (6% w/v) in the third vessel. The preliminary studies reported here occurred without experimental difficulties, even at a gel concentration as high as 40% (v/v). The recovery of the penicillin acylase was achieved with high yield (74%), but a low purification factor of 2.4 was obtained owing to the use of a crude extract with low specific activity.