Construction and application of fusion proteins of d-amino acid oxidase and glutaryl-7-aminocephalosporanic acid acylase for direct bioconversion of cephalosporin C to 7-aminocephalosporanic acid

Two fusion proteins of D-amino acid oxidase (DAAO) and glutaryl-7-aminocephalosporanic acid acylase (GLA) were designed to simplify the bioconversion process of cephalosporin C to 7-aminocephalosporanic acid (7-ACA), which is conventionally produced in a two-step enzymatic process. Two recombinant plasmids, pET-DLA and pET-ALD, were constructed to express fusion proteins of DAAO-linker-GLA (DLA) and GLA-linker-DAAO (ALD), respectively. When the recombinant plasmids were expressed in E. coli, the fusion protein DLA was not correctly folded and only DAAO activity could be detected. ALD, however, possessed activities of both DAAO and GLA, which directly catalyze the conversion of cephalosporin C into 7-ACA.     

Redox potentials and their pH dependence of D-amino-acid oxidase of Rhodotorula gracilis and Trigonopsis variabilis.

The redox potentials and pH characteristics of D-amino-acid oxidase (EC 1.4.3.3; DAAO) from the yeast Rhodotorula gracilis and Trigonopsis variabilis were measured in the pH range 6.5-8.5 at 15 degrees C. In the free enzyme form, the anionic red semiquinone is quantitatively formed in both DAAOs, indicating that a two single-electron transfer mechanism is active. The semiquinone species is also thermodynamically stable, as indicated by the large separation of the single-electron transfer potentials. The first electron potential is pH-independent, while the second electron transfer is pH-dependent exhibiting a approximately -60 mV/pH unit slope, consistent with a one-electron/one-proton transfer. In the presence of the substrate analogue benzoate, the two-electron transfer is the thermodynamically favoured process for both DAAOs, with only a quantitative difference in the stabilization of the anionic semiquinone. Clearly binding of the substrate (or substrate analogue) modulates the redox properties of the two enzymes. In both cases, in the presence and absence of benzoate, the slope of Em vs. pH (-30 mV/pH unit) corresponds to an overall two-electron/one-proton transfer in the reduction to yield the anionic reduced flavin. This behaviour is similar to that reported for DAAO from pig kidney. The differences in potentials and the stability of the semiquinone intermediate measured for the three DAAOs probably stem from different isoalloxazine environments. In the case of R. gracilis DAAO, the low stability of the semiquinone form in the DAAO-benzoate complex can be explained by the shift in position of the side chain of Arg285 following substrate analogue binding.     

Fusion protein of Vitreoscilla hemoglobin with D-amino acid oxidase enhances activity and stability of biocatalyst in the bioconversion process of cephalosporin C

In this study we constructed an artificial flavohemoprotein by fusing Vitreoscilla hemoglobin (VHb) with D-amino acid oxidase (DAO) of Rhodotorula gracilis to determine whether bacterial hemoglobin can be used as an oxygen donor to immobilized flavoenzyme. This chimeric enzyme significantly enhanced DAO activity and stability in the bioconversion process of cephalosporin C. In a 200-mL bioreactor, the catalytic efficiency of immobilized VHb-DAO against cephalosporin C was 12.5-fold higher than that of immobilized DAO, and the operational stability of the immobilized VHb-DAO was approximately threefold better than that of the immobilized DAO. In the scaled-up bioprocess with a 5-L bioreactor, immobilized VHb-DAO (2500 U/L) resulted in 99% bioconversion of 120 mM cephalosporin C within 60 min at an oxygen flow rate of 0.2 (v/v) x min. Ninety percent of the initial activity of immobilized VHb-DAO could be maintained at up to 50 cycles of the enzymatic reaction without exogenous addition of H(2)O(2) and flavin adenine dinucleotide (FAD). The purity of the final product, glutaryl-7-aminocephalosporanic acid, was confirmed to be 99.77% by high-performance liquid chromatography (HPLC) analysis. Relative specificity of immobilized VHb-DAO on D-alpha-aminoadipic acid, a precursor in cephalosporin C biosynthesis, increased twofold, compared with that of immobilized DAO, suggesting that conformational modification of the VHb-DAO fusion protein may be altered in favor of cephalosporin C.     

Yeast D-amino acid oxidase: structural basis of its catalytic properties

The 3D structure of the flavoprotein D-amino acid oxidase (DAAO) from the yeast Rhodotorula gracilis (RgDAAO) in complex with the competitive inhibitor anthranilate was solved (resolution 1.9A) and structural features relevant for the overall conformation and for catalytic activity are described. The FAD is bound in an elongated conformation in the core of the enzyme. Two anthranilate molecules are found within the active site cavity; one is located in a funnel forming the entrance, and the second is in contact with the flavin. The anchoring of the ligand carboxylate with Arg285 and Tyr223 is found for all complexes studied. However, while the active site group Tyr238-OH interacts with the carboxylate in the case of the substrate D-alanine, of D-CF(3)-alanine, or of L-lactate, in the anthranilate complex the phenol group rotates around the C2-C3 bond thus opening the entrance of the active site, and interacts there with the second bound anthranilate. This movement serves in channeling substrate to the bottom of the active site, the locus of chemical catalysis. The absence in RgDAAO of the "lid" covering the active site, as found in mammalian DAAO, is interpreted as being at the origin of the differences in kinetic mechanism between the two enzymes. This lid has been proposed to regulate product dissociation in the latter, while the side-chain of Tyr238 might exert a similar role in RgDAAO. The more open active site architecture of RgDAAO is the origin of its much broader substrate specificity. The RgDAAO enzyme forms a homodimer with C2 symmetry that is different from that reported for mammalian D-amino acid oxidase. This different mode of aggregation probably causes the differences in stability and tightness of FAD cofactor binding between the DAAOs from different sources.     

D-amino acid oxidase: structure,catalytic mechanism,and practical application

D-Amino acid oxidase (DAAO) is a FAD-dependent enzyme that plays an important role in microbial metabolism, utilization of endogenous D-amino acids, regulation of the nervous system, and aging in mammals. DAAO from yeasts Rhodotorula gracilis and Trigonopsis variabilis are used to convert cephalosporin C into 7-aminocephalosporanic acid, the precursor of other semi-synthetic cephalosporins. This review summarizes the recent data on the enzyme localization, physiological role, gene cloning and expression, and the studies on the enzyme structure, stability, catalytic mechanism, and practical applications.Translated from Biokhimiya, Vol. 70, No. 1, 2005, pp. 51–67.Original Russian Text Copyright © 2005 by Tishkov, Khoronenkova.     

Structure-based prediction of modifications in glutarylamidase to allow single-step enzymatic production of 7-aminocephalosporanic acid from cephalosporin C.

Glutarylamidase is an important enzyme employed in the commercial production of 7-aminocephalosporanic acid, a starting compound in the synthesis of cephalosporin antibiotics. 7-aminocephalosporanic acid is obtained from cephalosporin C, a natural antibiotic, either chemically or by a two-step enzymatic process utilizing the enzymes D-amino acid oxidase and glutarylamidase. We have investigated possibilities for redesigning glutarylamidase for the production of 7-aminocephalosporanic acid from cephalosporin C in a single enzymatic step. These studies are based on the structures of glutarylamidase, which we have solved with bound phosphate and ethylene glycol to 2.5 A resolution and with bound glycerol to 2.4 A. The phosphate binds near the catalytic serine in a way that mimics the hemiacetal that develops during catalysis, while the glycerol occupies the side-chain binding pocket. Our structures show that the enzyme is not only structurally similar to penicillin G acylase but also employs essentially the same mechanism in which the alpha-amino group of the catalytic serine acts as a base. A subtle difference is the presence of two catalytic dyads, His B23/Glu B455 and His B23/Ser B1, that are not seen in penicillin G acylase. In contrast to classical serine proteases, the central histidine of these dyads interacts indirectly with the O(gamma) through a hydrogen bond relay network involving the alpha-amino group of the serine and a bound water molecule. A plausible model of the enzyme-substrate complex is proposed that leads to the prediction of mutants of glutarylamidase that should enable the enzyme to deacylate cephalosporin C into 7-aminocephalosporanic acid.     

Entrapment of a Trigonopsis variabilis D‐amino acid oxidase variant F54Y for oxidative deamination of cephalosporin C

Trigonopsis variabilis D ‐amino acid oxidase (TvDAAO) is an enzyme used in the industrial bioconversion of cephalosporin C (CPC) into 7‐aminocephalosporanic acid, a crucial biosynthetic nucleus for a wide spectrum of semi‐synthetic cephem antibiotics. Using homology modeling and site‐directed mutagenesis, we have previously shown that the TvDAAO variant F54Y possesses improved catalytic activity and thermostability. To further explore its industrial application, the conditions for immobilization of the enzyme were examined in the present investigation. The results showed that entrapment in a calcium alginate (Ca‐alginate) matrix using 2% alginate, 500 mM CaCl₂, and 15 min stabilization appeared to be optimal for the immobilization of F54Y. The entrapped enzyme allowed complete CPC conversion. The entrapped enzyme also showed good operational stability and retained at least 90% of its original activity after 20 reaction cycles. To conclude, the entrapment of F54Y in Ca‐alginate appeared to be a simple and efficient biocatalysis system with potential application in the antibiotics industry.     

Properties and applications of microbial D-amino acid oxidases: current state and perspectives

D-Amino acid oxidase (DAAO) is a biotechnologically relevant enzyme that is used in a variety of applications. DAAO is a flavine adenine dinucleotide-containing flavoenzyme that catalyzes the oxidative deamination of D-isomer of uncharged aliphatic, aromatic, and polar amino acids yielding the corresponding imino acid (which hydrolyzes spontaneously to the α-keto acid and ammonia) and hydrogen peroxide. This enzymatic activity is produced by few bacteria and by most eukaryotic organisms. In the past few years, DAAO from mammals has been the subject of a large number of investigations, becoming a model for the dehydrogenase-oxidase class of flavoproteins. However, DAAO from microorganisms show properties that render them more suitable for the biotechnological applications, such as a high level of protein expression (as native and recombinant protein), a high turnover number, and a tight binding of the coenzyme. Some important DAAO-producing microorganisms include Trigonopsis variabilis, Rhodotorula gracilis, and Fusarium solani. The aim of this paper is to provide an overview of the main biotechnological applications of DAAO (ranging from biocatalysis to convert cephalosporin C into 7-amino cephalosporanic acid to gene therapy for tumor treatment) and to illustrate the advantages of using the microbial DAAOs, employing both the native and the improved DAAO variants obtained by enzyme engineering.      

The role of a conserved histidine residue, His324, in Trigonopsis variabilisd-amino acid oxidase

To investigate the functional role of an invariant histidine residue in Trigonopsis variabilis D-amino acid oxidase (DAAO), a set of mutant enzymes with replacement of the histidine residue at position 324 was constructed and their enzymatic properties were examined. Wild-type and mutant enzymes have been purified to homogeneity using the His-bound column and the molecular masses were determined to be 39.2 kDa. Western blot analysis revealed that the in vivo synthesized mutant enzymes are immuno-identical with that of the wild-type DAAO. The His324Asn and His324Gln mutants displayed comparable enzymatic activity to that of the wild-type enzyme, while the other mutant DAAOs showed markedly decreased or no detectable activity. The mutants, His324/Asn/Gln/Ala/Tyr/Glu, exhibited 38-181% increase in Km and a 2-10-fold reduction in kcat/Km. Based on the crystal structure of a homologous protein, pig kidney DAAO, it is suggested that His324 might play a structural role for proper catalytic function of T. variabilis DAAO.     

A process for bioconversion of cephalosporin C by Rhodotorula gracilis D-amino acid oxidase

Summary A process for the production (in a stirred tank reactor) of glutaryl-7-ACA from cephalosporin C using immobilized D-amino acid oxidase is described. Results so obtained under optimal conditions (1.2 mg coupled enzyme/L, pH 8.5, 2 mM cephalosporin C) point to a system which shows high conversion efficiency and a remarkable operational stability. No exogenous H₂O₂ is requested to shift the reaction equilibrium toward glutaryl-7-ACA production, nor any side product is detected. The immobilized system productivity was 54 g/day/mg of enzyme. This process represents the first reported case of a reactor successfully developed with a DAAO for bioconversion of cephalosporin C.     

Characterization of human D-amino acid oxidase

D-Amino acid oxidase (DAAO) has been proposed to be involved in the oxidation of D-serine, an allosteric activator of the NMDA-type glutamate receptor in the brain, and to be associated with the onset of schizophrenia. The recombinant human DAAO was expressed in Escherichia coli and was isolated as an active homodimeric flavoenzyme. It shows the properties of the dehydrogenase-oxidase class of flavoproteins, possesses a low kinetic efficiency, and follows a ternary complex (sequential) kinetic mechanism. In contrast to the other known DAAOs, the human enzyme is a stable homodimer even in the apoprotein form and weakly binds the cofactor in the free form.     

Single-pot conversion of cephalosporin C to 7-aminocephalosporanic acid in the absence of hydrogen peroxide

In this study, d-amino acid oxidase (DAAO) and catalase (CAT) in the permeabilized recombinant Pichia pastori cells were well investigated. It appeared that their thermal stability was negatively correlated with the apparent enzymatic activities. The frozen-melted cells presented the best stability and the lowest apparent activities of DAAO and CAT, whereas the cetyltrimethylammonium bromide (CTAB) permeabilized cells displayed the weakest stability and the highest apparent activities of the two enzymes. Simultaneous action of DAAO and CAT in the CTAB-permeabilized cells and glutaryl-7-aminocephalosporanic acid acylase (GA) immobilized on carrier contributed to the conversion of cephalosporin C (CPC) to 7-aminocephalosporanic acid (7-ACA) with a yield of 76.2%. During such a reaction cycle, no visible activity loss occurred at the immobilized GA, whereas the loss rates of DAAO and CAT activities were about 0.029 and 1.13 U min⁻¹, respectively. Nevertheless, this problem could be easily solved by continuous feeding of the new permeabilized cell suspension at the rate of 6 ml h⁻¹ to the reactor. Following such a fed-batch strategy, these permeabilized cells and the immobilized GA could be efficiently reused for 6 and 15 reaction cycles, respectively, yielding around 76% 7-ACA at each reaction cycle.     

Construction of recombinant Escherichia coli D11/pMSTO and its use in enzymatic preparation of 7-aminocephalosporanic acid in one pot

The main drawback in the industrial production of 7-aminocephalosporanic acid is the accumulation of intermediate (AKA-7-ACA) and destruction of substrate (cephalosporin C) catalyzed by catalase and beta-lactamase. To overcome the adverse effect of these enzymes on the conversion process, Escherichia coli D11 with mutation of katG, katE and ampC genes was constructed by P1 phage transduction, which enabled it not to produce catalase and beta-lactamase, respectively. At the same time, recA mutation in D11 increased the stability of foreign plasmid. With D11 used as host, both d-amino acid oxidase and GL-7-ACA acylase were cloned and expressed by the recombinant plasmids of pMSS or pMSTO, and the production of two enzymes could be increased by addition of 1.0% glucose. Cells of recombinant strain D11/pMSTO could directly convert cephalosporin C into 7-aminocephalosporanic acid at 25 degrees C, with the yield of more than 74%. The data suggested that the constructed D11/pMSTO could be an alternative catalyst for production of 7-aminocephalosporanic acid in one pot.     

Improvement of the glutaryl-7-aminocephalosporanic acid acylase activity of a bacterial gamma-glutamyltranspeptidase

7-Aminocephalosporanic acid (7-ACA) is an important material in the production of semisynthetic cephalosporins, which are the best-selling antibiotics worldwide. 7-ACA is produced from cephalosporin C via glutaryl-7-ACA (GL-7-ACA) by a bioconversion process using d-amino acid oxidase and cephalosporin acylase (or GL-7-ACA acylase). Previous studies demonstrated that a single amino acid substitution, D433N, provided GL-7-ACA acylase activity for gamma-glutamyltranspeptidase (GGT) of Escherichia coli K-12. In this study, based on its three-dimensional structure, residues involved in substrate recognition of E. coli GGT were rationally mutagenized, and effective mutations were then combined. A novel screening method, activity staining followed by a GL-7-ACA acylase assay with whole cells, was developed, and it enabled us to obtain mutant enzymes with enhanced GL-7-ACA acylase activity. The best mutant enzyme for catalytic efficiency, with a k(cat)/K(m) value for GL-7-ACA almost 50-fold higher than that of the D433N enzyme, has three amino acid substitutions: D433N, Y444A, and G484A. We also suggest that GGT from Bacillus subtilis 168 can be another source of GL-7-ACA acylase for industrial applications.     

Role of arginine 285 in the active site of Rhodotorula gracilis D-amino acid oxidase. A site-directed mutagenesis study

Arg(285), one of the very few conserved residues in the active site of d-amino acid oxidases, has been mutated to lysine, glutamine, aspartate, and alanine in the enzyme from the yeast Rhodotorula gracilis (RgDAAO). The mutated proteins are all catalytically competent. Mutations of Arg(285) result in an increase ( approximately 300-fold) of K(m) for the d-amino acid and in a large decrease ( approximately 500-fold) of turnover number. Stopped-flow analysis shows that the decrease in turnover is paralleled by a similar decrease in the rate of flavin reduction (k(2)), the latter still being the rate-limiting step of the reaction. In agreement with data from the protein crystal structure, loss of the guanidinium group of Arg(285) in the mutated DAAOs drastically reduces the binding of several carboxylic acids (e.g. benzoate). These results highlight the importance of this active site residue in the precise substrate orientation, a main factor in this redox reaction. Furthermore, Arg(285) DAAO mutants have spectral properties similar to those of the wild-type enzyme, but show a low degree of stabilization of the flavin semiquinone and a change in the redox properties of the free enzyme. From this, we can unexpectedly conclude that Arg(285) in the free enzyme form is involved in the stabilization of the negative charge on the N(1)-C(2)=O locus of the isoalloxazine ring of the flavin. We also suggest that the residue undergoes a conformational change in order to bind the carboxylate portion of the substrate/ligand in the complexed enzyme.     

Unfolding intermediate in the peroxisomal flavoprotein D-amino acid oxidase

The flavoenzyme d-amino acid oxidase (DAAO) from Rhodotorula gracilis is a peroxisomal enzyme and a prototypical member of the glutathione reductase family of flavoproteins. DAAO is a stable homodimer with a FAD molecule tightly bound to each 40-kDa subunit. In this work, the urea-induced unfolding of dimeric DAAO was compared with that of a monomeric form of the same protein, a deleted dimerization loop mutant. By using circular dichroism spectroscopy, protein and flavin fluorescence, 1,8-anilinonaphtalene sulfonic acid binding and activity assays, we demonstrated that the urea-induced unfolding of DAAO is a three-state process, yielding an intermediate, and that this process is reversible. The intermediate species lacks the catalytic activity and the characteristic tertiary structure of native DAAO but has significant secondary structure and retains flavin binding. Unfolding of DAAO proceeds through formation of an expanded, partially unfolded inactive intermediate, characterized by low solubility, by increased exposure of hydrophobic surfaces, and by increased sensitivity to trypsin of the beta-strand F5 belonging to the FAD binding domain. The oligomeric state does not modify the inferred folding process. The strand F5 is in contact with the C-terminal alpha-helix containing the Ser-Lys-Leu sequence corresponding to the type 1 peroxisomal targeting signal, and this structural element interacts with the N-terminal betaalphabeta flavin binding motif (Rossmann fold). The expanded conformation of the folding intermediate (and in particular the higher disorder of the mentioned secondary structure elements) could match the structure of the inactive holoenzyme required for in vivo trafficking of DAAO through the peroxisomal membrane.     

Reversible enzyme immobilization via a very strong and nondistorting ionic adsorption on support-polyethylenimine composites

New tailor-made anionic exchange resins have been prepared, based on films of large polyethylenimine polymers (e.g., MW 25,000) completely coating, via covalent immobilization, the surface of different porous supports (agarose, silica, polymeric resins). Most proteins contained in crude extracts from different sources have been very strongly adsorbed on them. Ionic exchange properties of such composites strongly depend on the size of polyethylenimine polymers as well as on the exact conditions of the covalent coating of the solids with the polymer. On the contrary, similar coating protocols yield similar matrices by using different porous supports as starting material. For example, 77% of all proteins contained in crude extracts from Escherichia coli were adsorbed, at low ionic strength, on the best matrices, and less than 15% of the adsorbed proteins were eluted from the support in the presence of 0.3 M NaCl. Under these conditions, 100% of the adsorbed proteins were eluted from conventional DEAE supports. Such polyethylenimine-support composites were also very suitable to perform very strong and nondistorting reversible immobilization of industrial enzymes. For example, lipase from Candida rugosa (CRL), beta-galactosidase from Aspergillus oryzae and D-amino acid oxidase (DAAO) from Rhodotorula gracilis, were adsorbed on such matrices in a few minutes at pH 7.0 and 4 degrees C. Immobilized enzymes preserved 100% of catalytic activity and remained fully immobilized in 0.2 M NaCl. In addition to that, CRL and DAAO were highly stabilized upon immobilization. Stabilization of DAAO, a dimeric enzyme, seems to be due to the involvement of both enzyme subunits in the ionic adsorption.     

A highly active adipyl-cephalosporin acylase obtained via rational randomization

There is strong interest in creating an enzyme that can deacylate natural cephalosporins such as cephalosporin C in order to efficiently acquire the starting compound for the industrial production of semisynthetic cephalosporin antibiotics. In this study, the active site of the glutaryl acylase from Pseudomonas SY-77 was randomized rationally. Several mutations that were found in previous studies to enhance the activity of the enzyme towards adipyl-7-aminodesacetoxycephalosporanic acid (ADCA) and cephalosporin C have now been combined, and libraries have been made in which random amino acid substitutions at these positions are joined. The mutants were expressed in a leucine-deficient Escherichia coli strain and subjected to growth selection with adipyl-leucine or amino-adipyl-leucine as sole leucine source. The mutants growing on these media were selected and purified, and their hydrolysis activities towards adipyl-7-ADCA and cephalosporin C were tested. Several mutants with highly improved activities towards the desired substrates were found in these rationally randomized libraries. The best mutant was selected from a library of totally randomized residues: 178, 266, and 375. This mutant comprises two mutations, Y178F + F375H, which synergistically improve the catalytic efficiency towards adipyl-7-ADCA 36-fold. The activity of this mutant towards adipyl-7-ADCA is 50% of the activity of the wild-type enzyme towards the preferred substrate glutaryl-7-aminocephalosporanic acid, and therefore the characteristics of this mutant approach those needed for industrial application.     

Enzymatic transformation of cephalosporin C to 7-amino-cephalosporanic acid. Part II: Single-step procedure using a coimmobilized enzyme system

The enzymatic transformation of cephalosporin C to 7-amino-cephalosporanic acid (7-ACA) using coimmobilized -aminoacid oxidase (DAAO) and 7-β-(4-carboxybutanamido)cephalosporanic acid acylase (Gl-7-ACA acylase) is reported. The results from the coimmobilization of the two enzymes on different carriers and at different ratios of enzyme activities are described. When an inhibitor of catalase activity, such as NaN₃ or H₂O₂, is present, the conversion rate to 7-ACA is higher, but more by-products are obtained. An optimum ratio of 60:1 between the enzymatic activities of DAAO and Gl-7-ACA acylase in the coimmobilized sample at 0.21 Ug⁻¹ Gl-7-ACA acylase activity was determined. The results of using coimmobilized enzymes and of using a mixture of separately immobilized enzymes in the same process are compared.