Production of glycosylated thermostable Providencia rettgeri penicillin G amidase in Pichia pastoris

Penicillin G amidase from Providencia rettgeri is a heterodimer of 92 kDa. We have previously expressed the Pr. rettgeri pac gene coding for this enzyme in Saccharomyces cerevisiae, and now we report the expression and characterization in the methylotrophic yeast Pichia pastoris. The recombinant catalytically active enzyme (rPAC(Pr)) was secreted from shake flask-grown P. pastoris cells into the medium at a level of approximately 0.18 U ml(-1). This yield of rPAC(Pr) was higher, by two orders of magnitude, than that obtained using a single-copy expression plasmid in S. cerevisiae. In addition, the secreted recombinant enzyme was entirely N-glycosylated. The recombinant PAC(Pr) was further characterized in terms of specific activity, kinetic parameters and thermostability. Except the significantly higher thermostability of the glycosylated rPAC(Pr) produced in P. pastoris, the other parameters were very similar to those of the corresponding non-glycosylated enzymes produced in bacteria or in S. cerevisiae. The higher thermostability of this recombinant enzyme has a clear industrial advantage.     

High-level production and covalent immobilization of Providencia rettgeri penicillin G acylase (PAC) from recombinant Pichia pastoris for the development of a novel and stable biocatalyst of industrial applicability

A complete, integrated process for the production of an innovative formulation of penicillin G acylase from Providencia rettgeri(rPAC(P.rett))of industrial applicability is reported. In order to improve the yield of rPAC, the clone LN5.5, carrying four copies of pac gene integrated into the genome of Pichia pastoris, was constructed. The proteinase activity of the recombinant strain was reduced by knockout of the PEP4 gene encoding for proteinase A, resulting in an increased rPAC(P.rett) activity of approximately 40% (3.8 U/mL vs. 2.7 U/mL produced by LN5.5 in flask). A high cell density fermentation process was established with a 5-day methanol induction phase and a final PAC activity of up to 27 U/mL. A single step rPAC(P.rett) purification was also developed with an enzyme activity yield of approximately 95%. The novel features of the rPAC(P.rett) expressed in P.pastoris were fully exploited and emphasized through the covalent immobilization of rPAC(P.rett). The enzyme was immobilized on a series of structurally correlated methacrylic polymers, specifically designed and produced for optimizing rPAC(P.rett) performances in both hydrolytic and synthetic processes. Polymers presenting aminic functionalities were the most efficient, leading to formulations with higher activity and stability (half time stability >3 years and specific activity ranging from 237 to 477 U/g (dry) based on benzylpenicillin hydrolysis). The efficiency of the immobilized rPAC(P.rett) was finally evaluated by studying the kinetically controlled synthesis of beta-lactam antibiotics (cephalexin) and estimating the synthesis/hydrolysis ratio (S/H), which is a crucial parameter for the feasibility of the process.     

Synthesis and secretion of Providencia rettgeri and Escherichia coli heterodimeric penicillin amidases in Saccharomyces cerevisiae

The Providencia rettgeri and Escherichia coli pac genes encoding heterodimeric penicillin G amidases (PAC) were successfully expressed in Saccharomyces cerevisiae. Furthermore, these recombinant enzymes are secreted from the yeast cell into the medium which is in contrast to bacterial hosts, where the enzymes are retained in the periplasm. Contrary to the P. rettgeri PAC-encoding gene, the E. coli pac is poorly expressed in yeast. The highest yield of P. rettgeri PAC was obtained with a multi-copy plasmid, resulting in of 1500units per liter. This yield is higher by an order of magnitude than that obtained in the best recombinant bacterial expression system. The recombinant P. rettgeri enzyme is only partially and selectively O-glycosylated. Only every sixth or seventh alpha-subunit is glycosylated, while the beta-subunit is not glycosylated at all. N-Glycosylation has not been detected.     

The role of the C-terminal region of pulchellin A-chain in the interaction with membrane model systems

Pulchellin is a Ribosome Inactivating Protein containing an A-chain (PAC), whose toxic activity requires crossing the endoplasmic reticulum (ER) membrane. In this paper, we investigate the interaction between recombinant PAC (rPAC) and Langmuir monolayers of dipalmitoyl phosphatidyl glycerol (DPPG), which served as membrane model. Three catalytically active, truncated PACs with increasing deletion of the C-terminal region, possessing 244, 239 and 236 residues (rPAC(244), rPAC(239) and rPAC(236)), were studied. rPAC had the strongest interaction with the DPPG monolayer, inducing a large expansion in its surface pressure-area isotherm. The affinity to DPPG decreased with increased deletion of the C-terminal region. When the C-terminal region was deleted completely (rPAC(236)), the interaction was recovered, probably because other hydrophobic regions were exposed to the membrane. Using Polarization Modulated-Infrared Reflection Absorption Spectroscopy (PM-IRRAS) we observed that at a bare air/water interface rPAC comprised mainly α-helix structures, the C-terminal region had unordered structures when interacting with DPPG. For rPAC(236) the α-helices were preserved even in the presence of DPPG. These results confirm the importance of the C-terminal region for PAC-ER membrane interaction. The partial unfolding only with preserved C-terminal appears a key step for the protein to reach the cytosol and develop its toxic activity.     

Fragments of pro-peptide activate mature penicillin amidase of Alcaligenes faecalis.

Penicillin amidase from Alcaligenes faecalis is a recently identified N-terminal nucleophile hydrolase, which possesses the highest specificity constant (kcat/Km) for the hydrolysis of benzylpenicillin compared with penicillin amidases from other sources. Similar to the Escherichia coli penicillin amidase, the A. faecalis penicillin amidase is maturated in vivo from an inactive precursor into the catalytically active enzyme, containing one tightly bound Ca2+ ion, via a complex post-translational autocatalytic processing with a multi-step excision of a small internal pro-peptide. The function of the pro-region is so far unknown. In vitro addition of chemically synthesized fragments of the pro-peptide to purified mature A. faecalis penicillin amidase increased its specific activity up to 2.3-fold. Mutations were used to block various steps in the proteolytic processing of the pro-peptide to obtain stable mutants with covalently attached fragments of the pro-region to their A-chains. These extensions of the A-chain raised the activity up to 2.3-fold and increased the specificity constants for benzylpenicillin hydrolysis mainly by an increase of the turnover number (kcat).     

Enzymes in polyelectrolyte complexes. The effect of phase transition on thermal stability

Penicillin amidase, alpha-chymotrypsin and urease have been immobilized in water-soluble nonstoichiometric polyelectrolyte complexes (N-PEC). N-PEC are formed by modified poly(N-ethyl-4-vinyl-pyridinium bromide) (polycation) and excess poly(methylacrylic acid) (polyanion). N-PEC are a new class of polymers capable, characteristically, of phase transitions solution in equilibrium precipitate induced by slight change in pH or ionic strength. Neither the chemical structure of the carrier nor the number of cross-linkages between an enzyme and a carrier change on phase transition. That gives an unique opportunity to elucidate the difference between enzymes immobilized on water-soluble and water-insoluble supports. A detailed study of the phase transition effect on thermal stability of the enzymes and protein-protein interactions has been carried out. The following effects were found. Pronounced thermal stabilization of penicillin amidase and urease may be achieved on two conditions: the enzyme is in the precipitate; (b) the enzyme is linked to the N-PEC nucleus. Then the thermal stability of N-PEC-bound penicillin amidase increases 7-fold at pH 5.7, 60 degrees C, and 300-fold at pH 3.1, 25 degrees C, compared to the native enzyme. For urease, the thermal stabilization increases 20-fold at pH 5.0, 70 degrees C. The localization of enzyme on N-PEC has been established by titration of alpha-chymotrypsin bound to a polycation or polyanion with basic pancreatic trypsin inhibitor. Both in solution (pH 6.1) and in N-PEC precipitate (pH 5.7), an alpha-chymotrypsin molecule bound to a polyanion is fully exposed to the solution. If the enzyme is bound to a polycation, only 20% of alpha-chymotrypsin molecules in the precipitate and 40% in solution retain their ability for protein-protein interactions. This means that a polycation-bound enzyme is localized in the hydrophobic nucleus of the complex, whereas the polyanion-bound enzyme sits on the hydrophilic shell of the complex. On pH-induced phase transition (pH decreases from 6.1 to 5.7), there occurs a stepwise decrease in penicillin amidase activity which is due to a 9.8-fold increase in the Km for 2-nitro-4-phenylacetamidobenzoic acid. Change of the catalytic activity and thermal stability of N-PEC-bound penicillin amidase is fully reversible and reproducible. Such soluble-insoluble immobilized enzymes with controllable thermal stability and activity may be used for simulating events in vivo and in biotechnology.     

Hyper production of enzyme penicillin amidase by locally isolated thermo-tolerantBacillus sp. MARC-0103 from rice starch in cheese whey

The present study concern with the extracellular production of penicillin amidase in a cost-effective cheese whey medium under submerged fermentation. ABacillus sp. MARC-0103 producing a high level of extra cellular penicillin G amidase was isolated from rice starch by heat shock method. The penicillin G amidase production in the strain was induced by phenyl acetic acid. The culture medium was optimized by using Plackett-Burman and central composite experimental designs for enhanced production of penicillin amidase. The factorial design indicated that the main factors that positively affect penicillin amidase production were casein hydro-lysate, CaCl2·2H2O, FeCI3·6H2O, Na2SO4 and cheese whey, whereas the presence of calcium carbonate and magnesium chloride in the medium had no effect on enzyme production. Phenyl acetic acid concentration and time of addition was found critical for enzyme pro duction. Enzyme production was enhanced very much by multiple addition of inducer. Other cultural condition such as pH, temperature, inoculum size and age were also optimized. More than two fold increase in enzyme production (40.7 U/ml/min) was observed under optimized cultural conditions. The molecular mass was estimated to be 40.0 kDa by SDS-PAGE.     

Alteration of the catalytic efficiency of penicillin amidase from Escherichia coli

Ampicillin and cephalexin are beta-lactam antibiotics that are synthesized by the condensation of D-(-)-alpha-aminophenylacetic acid with 6-aminopenicillanic acid or 7-aminodeacetoxycephalosporanic acid, respectively. The rates at which the penicillin amidase of Escherichia coli catalyzes these reactions are too low to be of practical use. The objective of this study was to determine whether it is possible to alter the substrate specificity of penicillin amidase and select enzymes that efficiently hydrolyze substrates with alpha-aminophenylacetyl moieties at low pH, at which the alpha-amino group is nearly completely protonated. In this study, D-(-)-alpha-aminophenylacetyl-(L)-leucine (APAL) was used as a substrate analog of ampicillin and cephalexin. The gene for the penicillin amidase of E. coli ATCC 11105 was cloned and transferred to a leucine auxotroph of E. coli; numerous amidase mutants were selected by their ability to cleave APAL and provide leucine for growth in low-pH medium. The plasmid encoding one of the mutant amidases (pA135) was used to transform naive cells, and transformants that expressed the mutant amidase were shown to grow more rapidly in medium at pH 6.5 containing 0.1 mM APAL as the sole leucine source than did cells with the wild-type amidase. The mutant amidase was purified, and the second-order rate constant (kcat/Km) for APAL hydrolysis at pH 6.5 was found to be 10-fold greater than the rate observed with the wild-type enzyme. The difference between the rates of APAL hydrolysis by the mutant and wild-type amidases increased as the pH of the reactions decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration of the catalytic efficiency of penicillin amidase from Escherichia coli.

Ampicillin and cephalexin are beta-lactam antibiotics that are synthesized by the condensation of D-(-)-alpha-aminophenylacetic acid with 6-aminopenicillanic acid or 7-aminodeacetoxycephalosporanic acid, respectively. The rates at which the penicillin amidase of Escherichia coli catalyzes these reactions are too low to be of practical use. The objective of this study was to determine whether it is possible to alter the substrate specificity of penicillin amidase and select enzymes that efficiently hydrolyze substrates with alpha-aminophenylacetyl moieties at low pH, at which the alpha-amino group is nearly completely protonated. In this study, D-(-)-alpha-aminophenylacetyl-(L)-leucine (APAL) was used as a substrate analog of ampicillin and cephalexin. The gene for the penicillin amidase of E. coli ATCC 11105 was cloned and transferred to a leucine auxotroph of E. coli; numerous amidase mutants were selected by their ability to cleave APAL and provide leucine for growth in low-pH medium. The plasmid encoding one of the mutant amidases (pA135) was used to transform naive cells, and transformants that expressed the mutant amidase were shown to grow more rapidly in medium at pH 6.5 containing 0.1 mM APAL as the sole leucine source than did cells with the wild-type amidase. The mutant amidase was purified, and the second-order rate constant (kcat/Km) for APAL hydrolysis at pH 6.5 was found to be 10-fold greater than the rate observed with the wild-type enzyme. The difference between the rates of APAL hydrolysis by the mutant and wild-type amidases increased as the pH of the reactions decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stabilisation and immobilisation of penicillin amidase

Penicillin amidase was coupled to a periodate-oxidised dextran by reductive alkylation in the presence of sodium cyanoborohydride. A loss of activity (25%) was observed but the conjugate enzyme dextran was more thermostable than the native enzyme. Native and dextran-conjugated penicillin amidase were immobilised on amino activated silica (Promaxon, Spherosil, Aerosil) by a classical method using glutaraldehyde for the native enzyme and reductive alkylation for the modified enzyme. Good relative activity of the enzymes was obtained after insolubilisation. Immobilisation of both native and modified enzymes resulted in the thermostabilisation of the penicillin amidase.     

Positive regulation of amidase synthesis in Pseudomonas aeruginosa.

Mutants of Pseudomonas aeruginosa were isolated that were acetamide-negative in growth phenotype at 41 degrees C and constitutive for amidase synthesis at 28 degrees C. Two mutants were derived from the magno-constitutive amidase mutant PAC111 (C11), and a third from a mutant that had enhanced inducibility by formamide, PAC153 (F6). The three temperature-sensitive mutants produced amidases with the same thermal stabilities as the wild-type enzyme. Cultures growing exponentially at 28 degrees C, synthesizing amidase constitutively, ceased amidase synthesis almost immediately on transfer to 41 degrees C. Cultures growing at 41 degrees C were transferred to 28 degrees C and had a lag of about 0.5 of a generation before amidase synthesis became detectable. Pulse-heating for 10 min at 45 degrees C of a culture growing exponentially at 28 degrees C resulted in a lag of about 0.5 of a generation before amidase synthesis recommenced after returning to 28 degrees C. Acetamide-negative mutants that were unable to synthesize amidase at any growth temperature were isolated from an inducible strain producing the mutant B amidase PAC398 (IB10). Two mutants were examined that gave revertants producing B amidase but with novel regulatory phenotypes. It is suggested that amidase synthesis is regulated by positive control exerted by gene amiR.     

Purification,substrate specificity,and N-terminal amino acid sequence analysis of a β-lactamase-free penicillin amidase from <Emphasis Type="Italic">Alcaligenes</Emphasis> sp.

A -lactamase-free penicillin amidase from Alcaligenes sp. active against various -lactams was purified to homogeneity. The enzyme can hydrolyze penicillin G to 6-amino penicillanic acid (6-APA) and furnish penicillin G from 6-APA and phenyl acetic acid by condensation. The penicillin amidase is a heterodimer of subunit masses of 63 kDa and 22 kDa, respectively. Its isoelectric point is at pH 8.5. Cephalothin was found to be the best substrate. This is a novel type II penicillin amidase which shares the properties of both type II and type III enzymes. It is thermostable and, unlike penicillin amidase from A. faecalis, its stability remains unperturbed even in presence of reductant. An inhibition study by 2-hydroxy-5-nitro benzylbromide indicated the involvement of tryptophan in catalysis by the enzyme.     

Study of E. coli penicillin amidase. The pH-dependence of the enzymatic inactivation kinetics]

The pH-dependence of the inactivation rate constant of penicillin amidase at a temperature of 40 degrees C was studied. It was shown that in all cases the enzyme inactivation corresponded to the kinetics of the reaction of the 1st order. The pH-dependence profile was found to be bell-shaped, the effect of transfer from the highest to the lowest values of the inactivation rate constants increasing more than 100 times. On the basis of the data obtained and published earlier it was concluded that the enzyme inactivation proceeded in accordance with the scheme in which out of 3 equilibrium ionic forms of penicillin amidase, i.e. "acid", "neutral" and "alkaline" the neutral form of the active enzyme was most stable. Kinetic analysis of the scheme was carried out and it was shown that the dependence found was in accordance with the theoretical curve in which the pK values of the ionogenic groups controlling the interconvertions between the penicillin amidase forms were equal to 2.4 and 10.1 at a temperature of 40 degrees C. The value of the inactivation rate constant of the "acid" or "alkaline" form was equal to 5.95 min-1, while the "neutral" form of the enzyme was characterized by the inactivation rate constant equal to 5.1.10(-4) min-1. A mechanism for the enzyme inactivation was proposed. According to this mechanism, destruction of the salt bridge in the native structure of penicillin amidase resulted in production of extremely labile forms of the enzyme as compared to the native form.     

Pulchellin, a highly toxic type 2 ribosome-inactivating protein from Abrus pulchellus. Cloning heterologous expression of A-chain and structural studies

Pulchellin is a type 2 ribosome-inactivating protein isolated from seeds of the Abrus pulchellus tenuiflorus plant. This study aims to obtain active and homogeneous protein for structural and biological studies that will clarify the functional aspects of this toxin. The DNA fragment encoding pulchellin A-chain was cloned and inserted into pGEX-5X to express the recombinant pulchellin A-chain (rPAC) as a fusion protein in Escherichia coli. The deduced amino acid sequence analyses of the rPAC presented a high sequential identity (> 86%) with the A-chain of abrin-c. The ability of the rPAC to depurinate rRNA in yeast ribosome was also demonstrated in vitro. In order to validate the toxic activity we promoted the in vitro association of the rPAC with the recombinant pulchellin binding chain (rPBC). Both chains were incubated in the presence of a reduced/oxidized system, yielding an active heterodimer (rPAB). The rPAB showed an apparent molecular mass of approximately 60 kDa, similar to the native pulchellin. The toxic activities of the rPAB and native pulchellin were compared by intraperitoneal injection of different dilutions into mice. The rPAB was able to kill 50% of the tested mice with doses of 45 microg x kg(-1). Our results indicated that the heterodimer showed toxic activity and a conformational pattern similar to pulchellin. In addition, rPAC produced in this heterologous system might be useful for the preparation of immunoconjugates with potential as a therapeutic agent.     

Penicillin amidase-catalysed transfer of low specific acyl moiety. Synthesis of 7-benzoxazolonylacetamido desacetoxycephalosporanic acid.

The methyl ester of 2-benzoxazolon-3-yl-acetic acid was used as an acyl donor in the penicillin amidase-catalysed transfer reaction to 7-aminodesacetoxycephalosporanic acid. The synthesis of 7-(2-benzoxazolon-3-yl-acetamido)-desacetoxycephalosporanic acid was carried out as a kinetically controlled reaction. A characteristic feature of this system is that the benzoxazolone derivatives are very low specific substrates for penicillin amidase (the kcat/Km values for their hydrolysis were shown to be 10(5)-fold lower compared to the corresponding values for phenylacetyl derivatives). Nevertheless, penicillin amidase proved to be an effective catalyst for the synthesis of these new cephem derivatives (50% yield for 6 h). The reason is the observed unusually high value for the transferase-hydrolase activity ratio. The determined value for (k3'/k3)app = 120,000 implies that in this case of low specific acyl moiety, penicillin amidase acts more like a transferase than a hydrolase. The maximum yield has been increased up to 70% by lowering the reaction temperature and stepwise feeding of the reaction medium with the acyl component. The results obtained extend the potential of the penicillin amidase as a catalyst for the synthesis of a new group of biologically active cephem derivatives.     

β-Lactamase-Free Penicillin Amidase from Alcaligenes sp.: Isolation Strategy, Strain Characteristics, and Enzyme Immobilization

Isolation and characterization of a β-lactamase (EC 3.5.2.6)-free, penicillin amidase (penicillin amidohydrolase, EC 3.5.1.11)-producing organism is reported. The test strain was isolated by an enrichment technique with a substrate other than penicillins. The isolated strain belongs to the genus Alcaligenes. Phenylacetic acid was found to be the inducer of penicillin amidase. The amidase has a broad substrate spectrum. It is very active against penicillin G and semisynthetic cephalosporins, whereas penicillin V and semisynthetic penicillins acted moderately as a substrate. Immobilized cells of Alcaligenes sp. were shown to act as a reversible enzyme. Received: 28 April 1999 / Accepted: 18 May 1999     

Development and characterization of a potent producer of penicillin G amidase by mutagenization

Summary The penicillin G amidase (PGA) activity of a parent strain of E. coli (PCSIR-102) was enhanced by chemical mutagenization with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). After screening and optimization, a penicillinase deficient mutant (MNNG-37) was isolated and found effective for the production of penicillin G amidase as compared to the parent strain of E. coli (PCSIR-102). Penicillin G amidase activity of MNNG-37 appeared during an early stage of growth, whereas PCSIR-102 did not exhibit PGA activity due to the presence of penicillinase enzyme which inhibits the activity of enzyme PGA. However, MNNG-37 gave a three-fold increase in enzyme activity (231 IU mg⁻¹) as compared to PCSIR-102 (77 IU mg⁻¹) in medium containing 0.15 and 0.1% concentrations of phenylacetic acid, respectively which was added after 6 h of cultivation. The difference in K _m values of the enzyme produced by parent strain PCSIR-102 (0.26 mM) and mutant strain MNNG-37 (0.20 mM) is significant (1.3-fold increase in K _m value) which may show the superiority of the latter in terms of better enzyme properties.     

Characterization of a new (R)-hydroxynitrile lyase from the Japanese apricot Prunus mume and cDNA cloning and secretory expression of one of the isozymes in Pichia pastoris

PmHNL, a hydroxynitrile lyase from Japanese apricot ume (Prunus mume) seed was purified to homogeneity by ammonium sulfate fractionation and chromatographic steps. The purified enzyme was a monomer with molecular mass of 58 kDa. It was a flavoprotein similar to other hydroxynitrile lyases of the Rosaceae family. It was active over a broad temperature, and pH range. The N-terminal amino acid sequence (20 amino acids) was identical with that of the enzyme from almond (Prunus dulcis). Based on the N-terminal sequence of the purified enzyme and the conserved amino acid sequences of the enzymes from Pr. dulcis, inverse PCR method was used for cloning of a putative PmHNL (PmHNL2) gene from a Pr. mume seedling. Then the cDNA for the enzyme was cloned. The deduced amino acid sequence was found to be highly similar (95%) to that of an enzyme from Pr. serotina, isozyme 2. The recombinant Pichia pastoris transformed with the PmHNL2 gene secreted an active enzyme in glycosylated form.     

粪产碱杆菌青霉素G酰化酶在枯草芽孢杆菌中的表达、纯化及其性质

利用PCR技术克隆了粪产碱杆菌 (Alcaligenesfaecalis,CICCAS1.76 7)青霉素G酰化酶 (pencillinGacylase ,PGA)基因 (GenBank登录号AF4 5 5 35 6 )。通过构建工程菌E .coli(pETAPGA) ,该酶在大肠杆菌中获得了表达 ,表达产物分泌到周质空间。进一步构建的工程菌B .subtilis (pMAPGA)和B .subtilis(pBAPGA)实现了该酶的胞外分泌表达。分泌表达的最高表达量为 6 5 3u/L ,比野生型A .faecalis表达量高 10 9倍。表达产物经硫酸铵分级沉淀和DEAE SepharoseCL 6B两步纯化 ,纯度提高 86倍 ,活力回收率达到 81% ,纯化后的PGA活力为 1.4 6 9u/mg。研究表明 ,PGA家族成员中只有粪产碱杆菌PGA和巨大芽孢杆菌PGA可以在枯草芽孢杆菌中分泌表达。与巨大芽孢杆菌PGA相比 ,粪产碱杆菌PGA的最适pH值为 8.0 ,最适温度为 6 0°C ,而且在有机溶剂中具有更强的稳定性。该酶在水相中具有较低的头孢氨苄合成活力。本研究为粪产碱杆菌PGA的获得提供了新的途径。