Molecular biology of β-lactam acylases

-Lactam acylases such as penicillin G acylases, penicillin V acylases and glutaryl 7-aminocephalosporanic acid acylases are used in the manufacture of 6-aminopenicillanic acid, 7-aminodesacetoxycephalosporanic acid and 7-aminocephalosporanic acid (7-ACA). Genetically-engineered strains producing 1050 U/g, 3200 U/g and 7000 to 10,000 U/I of penicillin G acylase, penicillin V acylase and glutaryl-7-ACA acylase, respectively, have been developed. The penicillin G acylase studied to date and the glutaryl-7-ACA acylase from Pseudomonas sp. share some common features: the active enzyme molecules are composed of two dissimilar subunits that are generated from respective precursor polypeptide; the proteolytic processing is a post-translational modification which is regulated by temperature; and the Ser residue at the N-terminus of the -sub-unit (Ser²⁹⁰; penicillin G acylase numbering) is implicated as the active site residue. Protein engineering, to generate penicillin G acylase molecules and their precursors with altered sequences, and the structure-function correlation of the engineered molecules are discussed.The authors are with Research and Development, Hindustan Antibiotics Ltd, Pimpri, Pune 411 018, India;     

Search for Microorganisms Producing Cephalosporin Acylase and Enzymatic Synthesis of Cephalosporins

Microorganisms were tested for production of cephalosporin acylase. Some bacteria showed strong acylase activity for all of cephalexin, cephaloridine, cephalotin, penicillin G and ampicillin. Some showed a rather specific activity for cephalexin. Pseudomonas melanogenum KY 3987 showed specific activity only for cephalexin and ampicillin which contain a side chain of d-phenylglycine. Most of these acylase-producing bacteria had the ability to synthesize cephalexin and other cephalosporins from 7-aminocephem compounds and organic acid esters. Among them, Ktuyvera citrophila KY 7844 was one of the most promising organisms for enzymatic synthesis of cephalosporins. This organism had the ability to catalyze N-acylation of 7-aminocephem compound not only with α-amino acid ester, but also with such acid esters as 1-(1 H)-tetrazolylacetate methylester which has no α-amino group.     

Improvement of the catalytic properties of penicillin G acylase from Escherichia coli ATCC 11105 by selection of a new substrate specificity

Cloned penicillin G acylase (PGA) from Escherichia coli ATCC 11105 was mutagenized in vivo using N-methyl-N-nitrosoguanidine. Mutants of PGA were selected by their ability to allow growth of the host strain E. coli M8820 with the new substrates phenylacetyl--alanyl-l-proline (PhAc-Ala-Pro) phthalyl-l-leucine (Pht-Leu) or phthalylglycyl-l-proline (Pht-Gly-Pro) as sole source of proline and leucine respectively. PGA mutants were purified and immobilized onto spherical methacrylate (G-gel). The immobilized form of mutant PGA selected with (PhAc-gbAla-Pro) hydrolyzed 95% of 9 mmol penicillin G 30% faster than wild-type PGA using the same specific activities. The specific activity of the soluble enzyme was 2.7-fold, and inhibition by phenylacetic acid was halved. Immobilized PGA mutant selected with Pht-Gly-Pro hydrolyzed penicillin G 20% faster than wild-type PGA. The K _m of the soluble enzyme was increased 1.7-fold. Furthermore, the latter two mutants were also 3.6-fold more stable at 45° C than wild-type PGA. The specific activity of the mutant selected with Pht-Leu was 6.3-fold lower, and inhibition by phenylacetic acid was increased 13-fold.     

Ability to use ampicillin as a nitrogen source by the marine cyanobacteriumPhormidium valderianum BDU 30501

The marine non-heterocystous, filamentous cyanobacteriumPhormidium valderianum BDU 30501, which failed to grow in the absence of combined nitrogen in the medium, grew when supplemented with ampicillin. Growth in terms of both protein as well as chlorophyll was proportional to the concentration of ampicillin up to 2000 g ml^–1. With the hydroxylamine assay, the organism was found to produce -lactamase extracellularly with activity reaching a maximum within 6 h of incubation. The maximum production and activity of the enzyme in the culture filtrate was at pH 7 at a concentration of 750 g ml^–1 ampicillin and at 25°C. The crude enzyme was thermolabile, because temperatures above 0°C on storage resulted in the loss of activity within hours. The enzyme could be induced both by ampicillin as well as benzyl penicillin, but ampicillin proved to be a more suitable substrate both in terms of induction as well as activity. This is the first report of an antibiotic serving as a nitrogen source for an organism.     

Process of penicillin G hydrolysis catalyzed by penicillin acylase immobilized on acylic carrier

The usefulness of penicillin acylase immobilized onto butyl acrylate — ethyl glycol dimethacrylate (called in this paper acrylic carrier) in penicillin G hydrolysis performed in a stirred tank reactor is shown. The enzyme-acrylic carrier preparation does not deteriorate its own properties in the mixing condition of slurry reactor. The experiments were carried out in a batch and a continuous stirred tank reactor as well as continuous stirred tank reactors in series. It was found to be a satisfactory agreement between experimental and predicted results. It also indicated the optimal substrate concentration range which provides the most effective enzyme operation. A superiority of the three reactors in series over the batch reactor is shown.List of Symbols C_E g/m³ equivalent enzyme concentration - C_SO mol/m³ initial penicillin G concentration - K_A mol/m³ substrate affinity constant - K_iS mol/m³ substrate inhibitory constant - K_iP mol/m³ PhAA inhibitory constant - K_iQ mol/m³ 6-APA inhibitory constant - k₃ mol/g min constant rate of dissotiation of the active complex - r mol/m³ rate of reaction - t min. reaction time - t_j min. maintenance time - degree of conversion - _B, _F dimensionless time - min. residence time - PA penicillin acylase - PG penicillin G - PhAA phenylacetic acid - 6-APA 6-aminopenicillanic acid     

Isolation and mapping of a mutant penicillin G acylase with altered substrate specificity from Escherichia coli

Summary Penicillin G acylase of Escherichia coli ATCC 11105 catalyzes hydrolysis as wellas synthesis of penicillin G. In this work a recombinant penicillin G acylase genewas mutagenized in vivo. A mutant with altered penicillin G acylase was selectedby its ability to grow with phthalyl-L-leucine as sole source of leucine. Themutant enzyme obtained was deficient in hydrolyzing penicillin G. A mutation ofGly359 to aspartic acid was mapped first by construction of chimeric pac genescomposed of wild type and mutant DNA, followed by nucleotide sequencing.     

Polymyxin Acylase: An Enzyme Causing Intramolecular N2⇄N6 Acyl Transfer in N-Monooctanoyl-l-Lysine

Polymyxin acylase from Pseudomonas sp. M-6-3 can deacylate not only polymyxin antibiotics, but also A-fatty acyl-peptides and N-fatty acyl-amino acids. We found that this enzyme causes intramolecular N²?N⁶ acyl transfer in monooctanoyl-l-lysine; when N²-octanoyl-l-lysine is the substrate, N⁶-octanoyl- l-lysine is produced at pH 10.5, but when N⁶-octanoyl- l-lysine is the substrate, N²-octanoyl- l-lysine is produced at pH 8.0. In these reactions, the deacylation proceeded gradually at the final stage and eventually, both N²-octanoyl- l-lysine and N⁶-octanoyl- l-lysine were hydrolyzed to l-lysine and octanoic acid. Furthermore, this enzyme showed intermolecular acyltrans- ferase activity, transferring several N-octanoyl- dl-amino acids to N-octanoyl-hydroxylamine. This acyltransfer ability of polymyxin acylase offers a new method of enzymic N-acylation of compounds containing amino components.     

Optimization of a pseudo-affinity process for penicillin acylase purification

A pseudo-affinity process for penicillin acylase (EC 3.5.1.11) purification using an affinity ligand (Ampicillin) attached on Sepharose 4B-CNBr was optimized. The enzyme adsorption on this affiant (Amp-Seph) is independent of pH between 5.5 and 8.8, in 100?mM phosphate containing 22% (w/v) ammonium sulphate. The desorption of the penicillin acylase from the affinity gels was carried out, the best desorption results being obtained through a non specific eluent, 100?mM phosphate pH 4.6 with 15% (w/v) ammonium sulphate. The best purification results were obtained with an enzymatic extract, produced through osmotic shock of Escherichia coli cells (3.7?IU/mg prot). With this extract and an affinity gel of Sepharose 4B-CNBr derivatized with ampicillin (3.8?μmol/cm³?gel), a maximum activity capacity adsorbed of 20?IU/cm³?gel was obtained for initial values of activity and protein concentration of 1.7?IU/cm³ and 0.4?mg prot/cm³, respectively. With the optimized eluent it was possible to obtain penicillin acylase in only one purification step with a desorption yield of enzyme activity higher than 90%. The penicillin acylase produced with this process was characterized by a maximum purity of 34?IU/mg prot, corresponding to a purification degree higher than 150 in relation to the lowest pure enzymatic extract. The enzyme purity of the eluted fractions was certified by SDS gel electrophoresis and liquid chromatography through a Mono Q column in a FPLC apparatus. The gel electrophoresis presented 4 main stained bands with 2 corresponding to α and β subunits of the penicillin acylase with equivalent molecular weights of 27 and 63?kDa. No external diffusion resistance on penicillin acylase and total protein adsorption on this affiant (Amp-Seph 3.8?μmol/cm³?gel) were observed for continuous adsorption processes performed at two different agitation speeds (120 and 400?rpm).     

Chainia Penicillin V Acylase: Strain Characteristics, Enzyme Immobilization, and Kinetic Studies

Aerobic cultures of an actinomycete were found to produce penicillin V acylase (PVA) (PA, EC-3.5.1.11) extracellularly. The presence of L-2-3 diamino-propionic acid in cell wall and formation of sclerotia on culture media led to its identification as Chainia, a sclerotial Streptomyces. Partially purified acylase was adsorbed on kieselguhr and entrapped in polyacrylamide gel. The immobilized preparation proved effective with respect to retention of enzyme and enzyme activity even after 15 successful cycles. The pH optimum for crude enzyme was in the range of pH 7.5–8.0, and for the (NH4)₂ SO₄ fraction it was pH 8.5. The immobilized enzyme showed maximal activity at pH 9.5. The optimum temperature for acylase activity was at 55°C. The crude enzyme, ammonium sulfate fraction, and immobilized enzyme showed K _m value for penicillin V of 6.13 mM, 14.3 mM, and 17.1 mM, respectively. Received: 11 December 1997 / Accepted: 9 April 1998     

Enhanced production of penicillin V acylase from Streptomyces lavendulae

At 28 °C, Streptomyces lavendulae produced high levels of penicillin V acylase (178 IU/l of culture) when grown on skim milk as the sole nutrient source for 275 h. The enzyme showed catabolite repression by glucose and was produced in the stationary phase of growth. Penicillin V was a good inducer of penicillin V acylase formation, while phenoxyacetic acid, the side-chain moiety of penicillin V, did not alter enzyme production significantly. The enzyme was stable between pH 6 and 11 and at temperatures from 20 °C to 55 °C. This extracellular enzyme was able to hydrolyse natural penicillins and unable to hydrolyse penicillin G. Received: 22 March 1999 / Received revision: 16 June 1999 / Accepted: 20 June 1999     

Identification and purification of O -acetyl-l-serine sulphhydrylase in Penicillium chrysogenum

We have demonstrated that Penicillium chrysogenum possesses the l-cysteine biosynthetic enzyme O-acetyl-l-serine sulphhydrylase (EC 4.2.99.8) of the direct sulphhydrylation pathway. The finding of this enzyme, and thus the presence of the direct sulphhydrylation pathway in P. chrysogenum, creates the potential for increasing the overall yield in penicillin production by enhancing the enzymatic activity of this microorganism. Only O-acetyl-l-serine sulphhydrylase and O-acetyl-l-homoserine sulphhydrylase (EC 4.2.99.10) have been demonstrated to use O-acetyl-l-serine as substrate for the formation of l-cysteine. The purified␣enzyme did not catalyse the formation of l-homocysteine from O-acetyl-l-homoserine and sulphide, excluding the possibility that the purified enzyme was O-acetyl-l-homoserine sulphhydrylase with multiple substrate specificity. The purification enhanced the enzymatic specific activity 93-fold in relation to the cell-free extract. Two bands, showing exactly the same intensity, were present on a sodium dodecyl sulphate/polyacrylamide gel, and the molecular masses of these were estimated to be 59 kDa and 68 kDa respectively. The K _m value for O-acetyl-l-serine and V _max of O-acetyl-l-serine sulphhydrylase were estimated to be 1.3 mM and 14.9 μmol/mg protein⁻¹ h⁻¹ respectively. The activity of the purified enzyme had a temperature optimum of approximately 45 °C, which is much higher than the actual temperature for penicillin synthesis. Furthermore, O-acetyl-l-serine sulphhydrylase activity was to have a maximum in the range of pH 7.0–7.4. Received: 20 March 1998 / Received revision: 27 July 1998 / Accepted: 12 August 1998     

Laccase and Manganese Peroxidase Activities of Phellinus Robustus and Ganoderma Adspersum Grown on Food Industry Wastes in Submerged Fermentation

Phellinus robustus produced both laccase (700–4,000 U l⁻¹) and manganese peroxidase (MnP) (1,000–11,300 U l⁻¹) in fermentation of nine food wastes, whereas Ganoderma adspersum produced only laccase (600–34,000 U l⁻¹). Glucose provided high laccase and MnP activity of P. robustus but repressed enzyme production by G. adspersum. Ammonium sulphate and ammonium tartrate increased the P. robustus laccase yield (3-fold), whereas the accumulation of MnP was not enhanced by additional nitrogen.     

Poly(3-hydroxybutyrate) synthesis from glycerol by a recombinant Escherichia coli arcA mutant in fed-batch microaerobic cultures

Poly(3-hydroxybutyrate) (PHB) synthesis was analyzed under microaerobic conditions in a recombinant Escherichia coli arcA mutant using glycerol as the main carbon source. The effect of several additives was assessed in a semi-synthetic medium by the ‘one-factor-at-a-time’ technique. Casein amino acids (CAS) concentration was an important factor influencing both growth and PHB accumulation. Three factors exerting a statistically significant influence on PHB synthesis were selected by using a Plackett–Burman screening design [glycerol, CAS, and initial cell dry weight (CDW) concentrations] and then optimized through a Box–Wilson design. Under such optimized conditions (22.02 g l⁻¹ glycerol, 1.78 g l⁻¹ CAS, and 1.83 g l⁻¹ inoculum) microaerobic batch cultures gave rise to 8.37 g l⁻¹ CDW and 3.52 g l⁻¹ PHB in 48 h (PHB content of 42%) in a benchtop bioreactor. Further improvements in microaerobic PHB accumulation were obtained in fed-batch cultures, in which glycerol was added to maintain its concentration above 5 g l⁻¹. After 60 h, CDW and PHB concentration reached 21.17 and 10.81 g l⁻¹, respectively, which results in a PHB content of 51%. Microaerobic fed-batch cultures allowed a 2.57-fold increase in volumetric productivity when compared with batch cultures. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

Bacterial growth and substrate degradation by BTX-oxidizing culture in response to salt stress

Interactions between microbial growth and substrate degradation are important in determining the performance of trickle-bed bioreactors (TBB), especially when salt is added to reduce biomass formation in order to alleviate media clogging. This study was aimed at quantifying salinity effects on bacterial growth and substrate degradation, and at acquiring kinetic information in order to improve the design and operation of TBB. Experiment works began by cultivating a mixed culture in a chemostat reactor receiving artificial influent containing a mixture of benzene, toluene, and xylene (BTX), followed by using the enrichment culture to degrade the individual BTX substrates under a particular salinity, which ranged 0–50 g l⁻¹ in batch mode. Then, the measured concentrations of biomass and residual substrate versus time were analyzed with the microbial kinetics; moreover, the obtained microbial kinetic constants under various salinities were modeled using noncompetitive inhibition kinetics. For the three substrates the observed bacterial yields appeared to be decreased from 0.51–0.74 to 0.20–0.22 mg mg⁻¹ and the maximum specific rate of substrate utilization, declined from 0.25–0.42 to 0.07–0.11 h⁻¹, as the salinity increased from 0 to 50 NaCl g l⁻¹. The NaCl acted as noncompetitive inhibitor, where the modeling inhibitions of the coefficients, K _T(S), were 22.7–29.7 g l⁻¹ for substrate degradation and K _T(μ), 13.0–19.0 g l⁻¹, for biomass formation. The calculated ratios for the bacterial maintenance rate, m _S, to further indicated that the percentage energy spent on maintenance increased from 19–24 to 86–91% as salinity level increased from 0 to 50 g l⁻¹. These results revealed that the bacterial growth was more inhibited than substrate degradation by the BTX oxidizers under the tested salinity levels. The findings from this study demonstrate the potential of applying NaCl salt to control excessive biomass formation in biotrickling filters.     

Storage stabilization of enzyme activity by poly(ethyleneimine)

The effect of mixing penicillin acylase with poly(ethyleneimine) is discussed. The properties of the polymer-enzyme system were evaluated for a wide range of enzyme concentrations (0.3–45.5 mg/cm³) and poly(ethyleneimine) concentrations (0.0001–10% wt). It was shown that addition of poly(ethyleneimine) to crude enzyme preparation caused precipitation of ballast protein and stabilization of the enzyme fraction remaining in the supernatant. The soluble fraction had stable activity for 21 days storage at 37 °C while the native enzyme lost about 80% of its initial activity. Additionally, it was ascertained that the polymer very slightly affected the properties of penicillin acylase in the PEI-enzyme preparations. Finally, possible ways of using the polymer-enzyme preparations in a membrane reactor are suggested.This work was supported by Government Committee of Science: Grant KBN # 3 0321 91 1     

Effect of organic solvents on penicillin acylase-catalyzed reactions: interaction of organic solvents with enzymes

The effects of various organic solvents on penicillin acylase-catalyzed synthesis of β-lactam antibiotics (pivampicillin and ampicillin) have been investigated in water-solvent mixtures. The rates of penicillin acylase-catalyzed reactions were found to be significantly reduced by the presence of a small amount of organic solvent. In particular, the rate of enzyme catalysis was extremely low in the presence of ring-structured solvents and acids while enzyme activities were fully restored after removing the solvents. This indicates that interactions between the solvents and the enzyme are specific and reversible. To correlate the inhibitory effects of organic solvents with solvent properties the influence of solvent hydrophobicities and solvent activity on the rate of pivampicillin synthesis was examined. The reaction rate was found to decrease with increasing solvent hydrophobicities, and a better correlation was observed between the reaction rate and solvent activity. The effects of ionic strength on the synthesis of pivampicillin and ampicillin were also examined. The ionic strength dependence indicates that electrostatic interactions are involved in the binding of ionic compounds to the enzyme. On the basis of the active site structure of penicillin acylase, a possible mechanism for molecular interactions between the enzyme and organic solvents is suggested.     

Penicillin Acylase of Pseudomonas melanogenum KY 3987

Partially purified penicillin acylases (EC 3.5.1.11) were prepared from Pseudomonas melanogenum KY 3987 and Kluyvera citrophila KY 3641 capable of synthesizing d(–)-α-amino-benzylpenicillin (APc) from 6-aminopenicillanic acid (6-APA) and phenylglycine methyl ester. As the cell-free extract of P. melanogenum contained high levels of penicillinase (EC 3.5.2.6), the acylase was separated completely from the penicillinase by use of Sephadex column chromatography or electrofocusing. The most salient property of the P. melanogenum penicillin acylase was its substrate specificity to penicillin substrates: it could form 6-APA only from APc but not from penicillin G, penicillin V and p-aminobenzylpenicillin, whereas the K. citrophila acylase acted on all of these penicillins. The P. melanogenum enzyme is hence considered a novel type of penicillin acylase.     

Purification of His6–organophosphate hydrolase using monolithic supermacroporous polyacrylamide cryogels developed for immobilized metal affinity chromatography

Organophosphate hydrolase containing hexahistidine tag at the N-terminus of recombinant protein (His₆-OPH) and expressed in Escherichia coli cells was purified using supermacroporous polyacrylamide-based monolith columns with immobilized metal affinity matrices [Me²⁺-iminodiacetic acid (IDA)–polyacrylamide cryogel (PAA) and Me²⁺-N,N,N’-tris (carboxymethyl) ethylendiamine (TED)–PAA]. Enzyme preparation with 50% purity was obtained by direct chromatography of nonclarified cell homogenate, whereas the combination of addition of 10 mM imidazole to buffers for cell sonication and sample loading, the use of precolumn with IDA–PAA matrix noncharged with metal ions, and the application of high flow rate provided the 99% purity of enzyme isolated directly from crude cell homogenate. Co²⁺-IDA–PAA provided the highest level of selectivity for His₆-OPH. Comparative analysis of purification using Co²⁺-IDA–PAA and Ni-nitrilotriacetic acid–agarose showed obvious advantages of the former in process time, specific activity of purified enzyme, and simplicity of adsorbent regeneration.     

Enhanced production of penicillin G acylase from a recombinant Escherichia coli

Penicillin G acylase (pac) gene was cloned into a stable asd ⁺ vector (pYA292) and expressed in Escherichia coli. This recombinant strain produced 1000 units penicillin G acylase g^–1 cell dry wt, which is 23-fold more than that produced by parental Escherichia coli ATCC11105. This enzyme was purified to 16 units mg^–1 protein by a novel two-step process.     

Novel reactive perstraction system applied to the hydrolysis of penicillin G

The activity of penicillin acylase has been studied in aqueous and organic solvents, as free enzyme as well as immobilized within the membrane of liquid-core capsules. The activity of the enzyme is inhibited by the accumulation of the products of the hydrolysis reaction, namely phenyl acetic acid (PAA). In order to overcome this inhibition a range of organic solvents were tested for use in in situ product recovery. Of these solvents dibutyl sebacate (DBS) was chosen due to the rapid extraction rate, the high logP and to facilitate capsule production. The extraction efficiency at pH 3.5 for PAA was >80% for phase ratios of >50% free solvent with partition coefficients of 8 and 0.7 for PAA and penicillin G (PenG), respectively, thereby showing that PAA could be selectively extracted at pH 3.5 and 25 degrees C. Liquid-core capsules containing DBS were shown to efficiently remove PAA selectively and the PAA could be effectively back-extracted and the capsules re-used in a three-stage process resulting in high product separation. Immobilization of penicillin acylase onto the capsule membranes resulted in increased operational stability of the enzyme and a very high enzyme activity. Over 53.3% of the PAA formed could be recovered in the capsule core with a concentration over sevenfold higher than in the aqueous phase. Higher extraction efficiencies could be obtained by varying the substrate concentration and number of capsules. The enzyme immobilized on capsules could be stored for over 4 months at pH 8 and 4 degrees C with no loss of activity. Over 80% of the initial activity could be recovered over five repeated batch cycles of the bioconversion process. The importance of capsular perstraction and reactive capsular perstraction has been clearly demonstrated.