Immobilization Of D-amino Acid Oxidase Research Articles

Effects of His-Tag Length on the Soluble Expression and Selective Immobilization of D-Amino Acid Oxidase from Trigonopsis variabilis: A Preliminary Study

His-tags are widely used for the purification of recombinant proteins. High-cost carriers functionalized with nickel ions are commonly required for the selective immobilization of His-tagged enzymes. In this study, His-tags of varying lengths were fused to the N-terminus of D-amino acid oxidase (DAO) from Trigonopsis variabilis. The attachment of a His6 tag significantly improved the solubility of the recombinant DAO expressed in Escherichia coli. By modulating the tag lengths, a better balance between cell growth and protein solubility was achieved, resulting in a higher volume activity (His3). Furthermore, the fusion of longer tags (His6 and His9) facilitated the rapid immobilization of DAOs onto a commercial epoxy carrier without metal bearing, resulting in more selective immobilization. In conclusion, the modulation of His-tag length was preliminarily demonstrated as a simple and cost-effective approach to achieve efficient expression, as well as fast and selective immobilization of DAO.

Vitreoscilla hemoglobin enhances the catalytic performance of industrial oxidases in vitro.

Oxidases are a group of oxidoreductases and need molecular oxygen in the catalytic process. Vitreoscilla hemoglobin (VHb) can improve the growth and productivity of host cells under hypoxic conditions, rendering it attractive for industrial application. In this work, we demonstrated the addition of immobilized VHb increased the catalytic activity of immobilized D-amino acid oxidase of Trigonopsis variabilis by two-fold when catalyzing cephalosporin C under oxygen-limited conditions. A similar increase of activities was observed in glucose oxidase, alcohol oxidase, and p-hydroxymandelate synthase by adding free VHb or immobilized VHb under hypoxic conditions. When L-glutamate oxidase was used to catalyze L-glutamate to produce α-ketoglutarate, the yield increased from 80.6 to 96.9% by fusing VHb with L-glutamate oxidase. Results demonstrated that the addition of free VHb, immobilized VHb, or fused VHb could increase the catalytic efficiency of oxidases, which was considered by increasing the concentration of the microenvironmental oxygen. Thus, VHb may become a potential additive agent to promote the efficiency of oxidases on industrial scale . KEY POINTS: • First time confirmation of facilitation of VHb on several industrial oxidases in vitro • VHb functions under hypoxic conditions rather than oxygen-enriched conditions • VHb functions in vitro in the form of free, immobilized protein and fusion enzyme.

Polythiophene supported MnO2 nanoparticles as nano-stabilizer for simultaneously electrostatically immobilization of d-amino acid oxidase and hemoglobin as efficient bio-nanocomposite in fabrication of dopamine bi-enzyme biosensor

A novel, highly selective and sensitive voltammetric bi-enzyme biosensor for sensing dopamine (DA), in presence of H2O2 which resulted as d-alanine enzymatic oxidation, was fabricated on the basis of simultaneously electrostatically immobilization of d-amino acid oxidase (DAAO) and hemoglobin (Hb) on MnO2 nanoparticles (MnO2 NPs) enriched poly thiophene (PTh). Cyclic voltammetry (CV), technique was applied for electropolymerization of thiophene on glassy carbon electrode (GCE) surface and MnO2 NPs dispersed on PTh network by soaking PTh/GCE in potassium permanganate (KMnO4) solution. Excellent catalytic properties and large surface area of MnO2 NPs/PTh composite caused it was used as an enzymes immobilization host in this developed bi-enzyme biosensor. The developed biosensor was characterized by scanning electron microscopy (SEM), energy dispersive X-ray analysis (EDX), and CV. The performance of the modified bi-enzyme biosensor was investigated in terms of its response time, detection limit, sensitivity, stability and selectivity in a lab environment. The composite of DAAO-Hb/MnO2 NPs/PTh to construct a bi-enzyme biosensor in this study showed a linear response with DA in the concentration range of 0.04–9.0μM with R-squared value of 0.994 (for S/N=3) and its sensitivity and detection limite were about 12.801μA/μM and 41nM respectively. Also this bi-enzyme biosensors exhibited high selectivity, rapid response (5s) and long-term stability (42days). At the end, the proposed biosensor was applied successfully in human serum as real sample.

Immobilization of D-Amino Acid Oxidase (DAAO) Enzyme on Hybrid Mesoporous MCF, SBA-15 and MCM-41 Nanomaterial.

Hybrid mesoporous materials as carriers for immobilization of D-amino acid oxidase (DAAO) were prepared via three steps: (i) hydrothermal synthesis of nanoporous MCF, SBA-15 and MCM-41 powders, (ii) functionalization with 3-aminopropyltriethoxysilane (APTES) by post-synthesis grafting; and (iii) activation with glutardialdehyde. The resulting mesostructured solids were characterized by various techniques: XRD, IR, TGA-DTA and N2 adsorption–desorption (BET). The characterization results indicated that these materials still maintained their structure after functionalization. IR data and TGA-DTA analysis demonstrated the existence of amine functional groups on the surface of APTES-functionalized samples. The DAAO immobilized on these materials exhibited higher catalytic activity and stability of enzyme for conversion of cephalosporin C (CPC) as compared to those of the non-functionalized ones. The catalytic activity and stability of enzyme decreased in the order MCF > SBA-15 > MCM-41.

Let the substrate flow, not the enzyme: Practical immobilization of d-amino acid oxidase in a glass microreactor for effective biocatalytic conversions.

Exploiting enzymes for chemical synthesis in flow microreactors necessitates their reuse for multiple rounds of conversion. To achieve this goal, immobilizing the enzymes on microchannel walls is a promising approach, but practical methods for it are lacking. Using fusion to a silica-binding module to engineer enzyme adsorption to glass surfaces, we show convenient immobilization of d-amino acid oxidase on borosilicate microchannel plates. In confocal laser scanning microscopy, channel walls appeared uniformly coated with target protein. The immobilized enzyme activity was in the range expected for monolayer coverage of the plain surface with oxidase (2.37 × 10(-5) nmol/mm(2) ). Surface attachment of the enzyme was completely stable under flow. The operational half-life of the immobilized oxidase (25°C, pH 8.0; soluble catalase added) was 40 h. Enzymatic oxidation of d-Met into α-keto-γ-(methylthio)butyric acid was characterized in single-pass and recycle reactor configurations, employing in-line measurement of dissolved O2 , and off-line determination of the keto-acid product. Reaction-diffusion time-scale analysis for different flow conditions showed that the heterogeneously catalyzed reaction was always slower than diffusion of O2 to the solid surface (DaII ≤ 0.3). Potential of the microreactor for intensifying O2 -dependent biotransformations restricted by mass transfer in conventional reactors is thus revealed. Biotechnol. Bioeng. 2016;113: 2342-2349. © 2016 Wiley Periodicals, Inc.

English

Porcine kidney D-amino acid oxidase was stabilized by covalent immobilization on spherical particles of Eupergit C because of its low stability in soluble form. The main focus of this work was put on evaluation of operational stability of the immobilized enzyme. To evaluate D-amino acid oxidase’s operational stability during process conditions, repetitive batch reactor experiments of D-methionine oxidation reaction were carried out with continuous aeration for oxygen supply at air-flow rates: of 5 and 10 dm3 h-1. Kinetic analysis of the immobilized enzyme was done as well. The mathematical model of D-methionine oxidative deamination catalyzed by the immobilized D-amino acid oxidase was developed and it described the data well. It enabled the estimation of operational stability decay rate constant. It was possible to achieve 100 % substrate conversion in all batch experiments.

One-pot enzymatic production of deacetyl-7-aminocephalosporanic acid from cephalosporin C via immobilized cephalosporin C acylase and deacetylase

The three-enzyme three-pot using immobilized d-amino acid oxidase (DAAO), glutaryl-7-ACA acylase (GLA), and cephalosporin C deacetylase (CAH) was the main strategy for industrial conversion of cephalosporin C (CPC) to deacetyl-7-aminocephalosporanic acid (D-7-ACA), a key pharmaceutical intermediate that is used for the production of 3-vinyl substituted β-lactams antibiotics. In order to simplify this traditional process, we developed an one-pot bi-enzyme catalyzed procedure by coupling immobilized cephalosporin C acylase (CPCA) with immobilized cephalosporin C deacetylase (CAH), which contained two parallel cascade reactions: firstly, hydrolysis of CPC to 7-ACA by CPCA, then deacetylation of 7-ACA to D-7-ACA by CAH (route 1); or firstly, hydrolysis of CPC to deacetyl-CPC (D-CPC) by CAH, then de-α-aminoadipoylation of D-CPC to D-7-ACA by CPCA (route 2). A D-7-ACA yield of 78.39% was achieved in 30min in a single reactor under the optimized enzyme amount. The specific productivity of D-7-ACA reached 10.85gg−1h−1L−1, which increased approximately 4-fold compared to that obtained from the two-pot enzymatic process. The half-time of immobilized CPCA was approximately 18h while immobilized CAH was approximately 534h under the operational conditions. These results demonstrated the industrial feasibility of this one-pot process and its clear advantages towards the traditional three-enzyme three-pot process.

A rapid in situ immobilization of d-amino acid oxidase based on immobilized metal affinity chromatography

A rapid in situ immobilization process was developed based on conventional separation technique of immobilized metal affinity chromatography (IMAC) and was studied in the case of D-amino acid oxidase (DAAO) with binding-enhancing Heli-tag (His-Arg-Asn-Tyr-Gly-Gly-Cys-Cys-Gly). A recombinant Escherichia coli strain JM105 (Δase)/pGEMK-R-DAAO-Heli was successfully constructed to synthesize chimeric protein DAAO-Heli. Without additional purification procedure, the tagged enzyme DAAO-Heli could be directly immobilized to EP-IDA-Ni(2+) support with purity of 90% and DAAO activity of over 70U/g support. Experimental results showed that the immobilized DAAO-Heli was 73 times more thermally stable than free enzyme. Besides, it remained 67% of initial activity after 100 cycles of batch catalysis and its operational stability was improved 36 times than that of the previously IMAC-immobilized DAAO-His. Furthermore, the epoxy (EP) support could be easily recovered and repeatedly used with simple steps, which could reduce the immobilization costs significantly.

Construction of an amperometric d-amino acid biosensor based on d-amino acid oxidase/carboxylated mutliwalled carbon nanotube/copper nanoparticles/polyalinine modified gold electrode

An improved d-amino acid biosensor was constructed based on covalent immobilization of d-amino acid oxidase onto carboxylated mutliwalled carbon nanotube/copper nanoparticles/polyalinine hybrid film electrodeposited on gold electrode. The biosensor exhibited an optimal response within 2s at pH 8.0 and 30°C when polarized at 0.09V. There was a linear relationship between biosensor response (μA) and d-alanine concentration ranging from 0.001 to 0.7mM. The sensitivity of the biosensor was 54.85μAcm−2mM−1 with a lower limit of detection of 0.2μM (signal/noise=3). The enzyme electrode was used 150 times over a period of 4months. The biosensor measured the d-amino acid level in fruit juices.

Properties of Rhodotorula gracilis d-Amino Acid Oxidase Immobilized on Magnetic Beads through His-Tag

D-amino acid oxidase catalyzes one of the key steps in the production of semisynthetic cephalosporins. We expressed and purified recombinant Rhodotorula gracilis D-amino acid oxidase with C-terminal his-tags. This engineered enzyme was immobilized onto Ni(2+)-chelated nitrilotriacetic acid magnetic beads through the interaction between his-tag and Ni(2+). The kinetic constants, storage properties, and the reusability of the immobilized d-amino acid oxidase were determined. The effects of temperature, pH, and hydrogen peroxide on the activity of immobilized d-amino acid oxidase were also studied. The highest activity recovery was 75%. Thermal stability was improved after immobilization; the relative activity of the immobilized enzyme was 56% whereas the free enzyme was completely inactivated after incubation at 50 degrees C for 1 h. In the presence of 10 mM hydrogen peroxide, the immobilized enzyme did not show a rapid loss of activity during the first 2 h of incubation, which was observed in the case of the free enzyme; the residual activity of the immobilized enzyme after 9 h was 72% compared with 22% of the free form. The long-term storage stability was improved; the residual activity of the immobilized enzyme was 74% compared with 20% of the free enzyme when stored at room temperature for 10 d. The immobilized form retained 37% of its initial activity after 20 consecutive reaction cycles.

Characterization of a Yeastd-Amino Acid Oxidase Microbiosensor ford-Serine Detection in the Central Nervous System

d-Serine is an endogenous ligand for N-methyl-d-aspartate (NMDA) receptors, and alterations in its concentration have been related to several brain disorders, especially schizophrenia. It is therefore an important target neuromodulator for the pharmaceutical industry. To monitor d-serine levels in vivo, we have developed a microbiosensor based on cylindrical platinum microelectrodes, covered with a membrane of poly-m-phenylenediamine (PPD) and a layer of immobilized d-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO). By detecting the hydrogen peroxide produced by enzymatic degradation of d-serine, this microbiosensor shows a detection limit of 16 nM and a mean response time of 2 s. Interferences by ascorbic acid, uric acid, l-cysteine, and by biogenic amines and their metabolites are rejected at more than 97% by the PPD layer. Although several d-amino acids are potential substrates for RgDAAO, d-serine was the only endogenous substrate present in sufficient concentration to be detected by our microbiosensor in the central nervous system. When implanted in the cortex of anesthetized rats, this microbiosensor detected the increase in concentration of d-serine resulting from its diffusion across the blood-brain barrier after an intraperitoneal injection. This new device will make it possible to investigate in vivo the variations in d-serine concentrations occurring under normal and pathological conditions and to assess the pharmacological potency of new drugs designed to impact d-serine metabolism.

Enantioselective screen-printed amperometric biosensor for the determination of d-amino acids

d-amino acids are generally considered to be important markers of bacterial contamination of food products. A screen-printed amperometric biosensor for the detection of d-amino acids has been constructed by the immobilization of d-amino acid oxidase on a graphite working electrode of a screen-printed strip modified with Prussian Blue and Nafion layers. Enzyme immobilization was then carried out by cross-linking of a mixture of the enzyme and bovine serum albumin with glutaraldehyde. As a result of the mediator addition and because of the multi-layer construction of the biosensor, including a polymer layer to avoid the interferences, the limit of the detection of the developed biosensor was two orders of magnitude improved in comparison to other screen-printed biosensors, as far as the determination of amino acids is concerned. Additional modification of the graphite electrode with carbon nanotubes led to a significant enhancement of the signal magnitude. A fast linear response of the developed biosensor was subsequently observed in static measurements for d-alanine in the concentration range from 5 to 200 μM. Excellent enantioselectivity towards d-amino acids was discovered. During the experiment, d-amino acids were detected in fruit juices and some milk samples. The complex matrix of natural milk samples had no influence on the response of the biosensor. The results were in good agreement with those obtained by capillary electrophoresis measurements.

Performance of d-amino acid oxidase in presence of ionic liquids

The activity and stability of free and immobilized D-amino acid oxidase (DAAO, EC 1.4.3.3) from Trigonopsis variabilis CBS 4095 in different water-soluble and water-insoluble ionic liquids (ILs) as well as in organic solvents were studied for comparison. The most promising ILs ([BMIM][BF(4)] and [MMIM][MMPO(4)]) were investigated in detail. The kinetic parameters (v(max) = 187 nkat/g dry weight, K(M) = 1.38 mM) with D-phenylalanine as substrate were calculated in 40% [BMIM][BF(4)]. Bioconversions of D/L-phenylalanine in 40% [BMIM][BF(4)] and 20% [MMIM][MMPO(4)] on a 3 ml scale using immobilized DAAO were performed by addition of free catalase from Micrococcus lysodeikticus. After total conversion of substrate in presence of 20% [MMIM][MMPO(4)] the residual activity of the immobilized DAAO was 79% and 100% of the free catalase.

Preparation of a robust biocatalyst of d-amino acid oxidase on sepabeads supports using the glutaraldehyde crosslinking method

In this work different protocols to immobilize d-amino acid oxidase (DAAO) on sepabeads were assayed (ionic adsorption on different supports and covalent attachment using glutaraldehyde), studying the stability of the final preparations. The highest stability was achieved by the treatment with glutaraldehyde of DAAO adsorbed on Sepabeads EA (a commercial aminated support having ethylendiamine groups). In fact, this derivative was six times more stable than the enzyme adsorbed only by ionic interaction and much more stable than the soluble enzyme. The effect of the nature of the amino groups in the support was then analyzed. DAAO adsorbed on sepabeads coated with polyethylenimine (PEI) yielded a higher stability than the preparation on Sepabeads EA. The treatment with glutaraldehyde of DAAO adsorbed on Sepabeads PEI yielded the best results in terms of stability, being 200 times more stable than DAAO adsorbed onto Sepabeads EA. The effects of polyethylenimine size and glutaraldehyde concentration were also studied. sepabeads coated with 25 kDa polyethylenimine and treatment with 0.5% glutaraldehyde solution were the optimal parameters regarding the stability (the half life time was 9 h at 56° C, 600 times more stable than the soluble enzyme). Moreover, the optimal derivative showed a maximum load capacity of 15 mg/g of support. This derivative seems to fulfill the requirements for industrial applications.

Simultaneous purification and reversible immobilization of D-amino acid oxidase from Trigonopsis variabilis on a hydrophobic support.

Purification and reversible immobilization of D-amino acid oxidase from Trigonopsis variabilis could be simultaneously accomplished by hydrophobic interaction on Phenyl Sepharose CL-4B in the presence of 50 mM pyrophosphate buffer (pH 8.5). The presence of a high salt concentration of 2 M, which is generally required for the hydrophobic interactions, was not essential for the hydrophobic immobilization. The enzyme in free as well as immobilized form was optimally active between pH 7.0 and 9.0. The immobilized preparation could be reused in a batch process for the conversion of D-amino acids to alpha-keto acids. When the activity of the preparation dropped below practical limits, the gel could be regenerated by water wash and recharged with fresh crude extract from yeast.

One-step separation-free amperometric biosensor for the detection of protein

A novel enzyme biosensor for the detection of protein is presented. The biosensor was made from a screen-printed three-electrode configuration. Amino acid oxidase was immobilized with glutaraldehyde and polyethylenimine on a working electrode made of rodinised carbon. A protease was immobilized on an immunodyne membrane and was placed on the electrode. A protein sample was deposited on the membrane, and was subsequently hydrolyzed to amino acids in the presence of the protease. This in turn produced hydrogen peroxide by the immobilized amino acid oxidase. The oxidation of hydrogen peroxide was then detected at +400 mV vs. an Ag/AgCl reference electrode. The method was very effective at detecting a very low level of protein. The sensor does not require any washing step. The sensor works with only 40 μl of sample per detection, and may be used on-site as a disposable sensor using a hand-held meter. The electrodes are also stable for more than 6 weeks.

Characterization of an industrial biocatalyst: Immobilized glutaryl-7-ACA acylase

A batch of the immobilized industrial biocatalyst glutaryl-7-ACA acylase (GA), one of the two enzymes involved in the biotransformation of cephalosporin C (CefC) into 7-aminocephalosporanic acid (7-ACA), was characterized. K(m) value for glutaryl-7-ACA was 5 mM. Enzyme activity was found to be optimal at pH between 7 and 9.5 and to increase with temperature and in buffered solutions. To avoid product degradation, optimal reaction conditions were obtained working at 25 degrees C using a 50-mM phosphate buffer, pH 8.0. Immobilized GA showed good stability at pH value below 9 and at temperature up to 30 degrees C. The inactivation of immobilized GA in the presence of different amounts of H(2)O(2), a side product that might be present in the plant-scale industrial solutions of glutaryl-7-ACA, was also investigated, but the deactivation rates were negligible at H(2)O(2) concentration that might be reached under operative conditions. Finally, biocatalyst performance in the complete two-step enzymatic conversion process from CefC to 7-ACA was determined on a laboratory scale. Following the complete conversion of a 75 mM solution of CefC into glutaryl-7-ACA catalyzed by an immobilized D-amino acid oxidase (DAAO), immobilized GA was used for the transformation of this intermediate into the final product 7-ACA. This reaction was repeated for 42 cycles. An estimation of the residual activity of the biocatalyst showed that 50% inactivation of immobilized GA was reached after approximately 300 cycles, corresponding to an enzyme consumption of 0.4 kU per kg of isolated 7-ACA.

Development of tailor-made glycidyl methacrylate-divinyl benzene copolymer for immobilization of d-amino acid oxidase from Aspergillus species strain 020 and its application in the bioconversion of cephalosporin C

A tailor-made glycidyl methacrylate-divinyl benzene (GMA-DVB) copolymer PC-3 was evolved by studying the effect of synthesis variables on binding and expression of d-amino acid oxidase (DAAO) from Aspergillus species strain 020. Almost quantitative binding (100%) and a high yield of immobilization per unit of enzyme loaded was achieved. Optimum pH, optimum temperature and K m for soluble and IME-DAAO preparations were 8.0–8.5 and 8.0; 30 and 37°C; and 0.73 and 2.78 mM for ceph C, respectively. The developed immobilized DAAO was used for the production of glutaryl-7-aminocephalosporanic acid from cephalosporin C (ceph C). A high conversion of >95% was achieved by using 3% (w/v) solution of ceph C, 48 U of DAAO per g of ceph C, keeping dissolved oxygen level above 50%, maintaining the pH between 7.6 and 7.8 and temperature at 24°C. The immobilized DAAO was used for 60 cycles in a stirred tank reactor.

Stabilization of enzymes ( d-amino acid oxidase) against hydrogen peroxide via immobilization and post-immobilization techniques

Three different approaches are proposed to increase the resistance of enzymes against hydrogen peroxide. (a) Multipoint covalent immobilization. Through this technique, enzyme rigidity would be greatly increased and hence, any conformational change on the enzyme structure involved before or after oxidation with hydrogen peroxide becomes greatly prevented. (b) Oriented immobilization on supports having large internal surfaces. The immobilization of enzymes, through different areas of their surface on solid supports with internal morphology composed by large surfaces, promotes a certain masking of the enzyme areas that are very close to the support surface. In this way, the accessibility of hydrogen peroxide to such protein areas becomes greatly restricted. (c) Additional chemical modification of immobilized enzyme derivatives with polymers. By adding thick barriers surrounding the whole enzyme molecule, the effective concentration of hydrogen peroxide in the proximity of the most sensitive residues may be strongly reduced. Multipoint covalently immobilized d-amino acid oxidase (DAAO) from Rhodotorula gracilis on glyoxyl-agarose is 11-fold more stable than native enzyme against the deleterious effect of hydrogen peroxide. On the other hand, DAAO from Trigonopsis variabilis was not stabilized by rigidification but it could be highly stabilized by an adequate combination of the best orientation on the support plus an additional modification with poly-aldehyde polymers.

Benzoquinone-Mediated Biosensor for Amperometric Determination ofD-Lysine

An amperometric enzyme sensor for the analysis of D-lysine is described. The sensor uses benzoquinone as mediator for electron transfer between immobilized D-amino acid oxidase and a glassy carbon electrode. A linear current response, proportional to D-lysine concentration in the range 1–10mM, was observed. The influence of oxygen, pH, and operating potential on the sensor performance were examined.