Co-autodisplay of Z-domains and bovine caseins on the outer membrane of E. coli

In this work, two proteins, Z-domains and bovine casein, were autodisplayed on the outer membrane of the same Escherichia coli cells by co-transformation of two different autodisplay vectors. On the basis of SDS-PAGE densitometry, Z-domains and bovine casein were expressed at 3.12 × 10⁵ and 1.55 × 10⁵ proteins/E. coli cell, respectively. The co-autodisplayed Z-domains had antibody-binding activity and the bovine casein had adhesive properties. E. coli with co-autodisplayed proteins were analyzed by fluorescence assisted cell sorting (FACS). E. coli with co-autodisplayed Z-domains and bovine casein aggregated due to hydrophobic interaction. For application to immunoassays, the Z-domain activity was estimated after (1) immobilizing the E. coli and (2) forming an OM layer. E. coli with co-autodisplayed two proteins that were immobilized on a polystyrene microplate had the same antibody-binding activity as did E. coli with autodisplayed Z-domains only. The OM layer from the co-transformed E. coli had Z-domains and bovine casein expressed at a 1:2 ratio from antibody-binding activity measurements.     

Immobilization of lysine oxidase on a gold–platinum nanoparticles modified Au electrode for detection of lysine

A commercial lysine oxidase (LyOx) from Trichoderma viride was immobilized covalently onto gold nanoparticles (AuNPs) and platinum nanoparticles (PtNPs) electrodeposited onto Au electrode using 3-aminopropyltriethoxy silane (3-APTES) and glutaraldehyde cross linking chemistry. A lysine biosensor was fabricated using LyOx/3-APTES/AuNPs-PtNPs/Au electrode as a working electrode, Ag/AgCl (3 M KCl) as standard electrode and Pt wire as auxiliary electrode connected through a potentiostat. The enzyme electrode was characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The cumulative effect of AuNPs and PtNPs showed excellent electrocatalytic activity at low applied potential for detection of H₂O₂, a product of LyOx reaction. The sensor showed its optimum response within 4 s, when polarized at 0.2 V vs. Ag/AgCl in 0.1 M phosphate buffer, pH 7.5 at 30 °C. The linear range and detection limit of the sensor were 1.0–600 μM and 1.0 μM (S/N = 3), respectively. Biosensor measured lysine level in sera, milk and amino acid tablet, which correlated well with those by standard HPLC method. The enzyme electrode lost 50% of its initial activity after 200 uses over a period of 4 months.     

Determination of the genetic similarities of fingerprints from Escherichia coli O157:H7 isolated from different sources in the North West Province,South Africa using ISR,BOXAIR and REP-PCR analysis

The aim of this study was to determine the genetic relationships of Escherichia coli O157:H7 isolated from pigs, cattle, pork, beef, humans and water samples using REP, ISR and BOXAIR PCR analysis. A total of 94 isolates were subjected to the REP-PCR analysis while 95 were screened for ISR and BOXAIR PCR fingerprints. The band sizes for amplicons from the ISR-PCR analysis ranged from 0.173 kb to 0.878 kb. However, a large proportion of the isolates had four bands ranging from 0.447 kb to 0.878 kb. Cluster analysis of the BOXAIR PCR profiles based on banding patterns revealed seven main clusters. It was identified in the clusters III, IV and VII in the BOXAIR PCR that 17.9%, 16.8% and 18.9%, of E. coli O157:H7 isolates respectively were present from all the animal species, meat and water samples. REP-PCR analysis produced 9 different patterns with bands ranging from 0 to 12 per isolate. The band sizes ranged from 200 bp to 8000 bp. Nine major clusters (I–IX) were identified. From the three different species sampled cluster eight was the largest and a mixed cluster with 23.4% (22/94) of the E. coli O157:H7 isolates. These indicate that food products obtained from supermarkets in the study area are contaminated with E. coli O157:H7.     

Development of electrochemical sulfite biosensor based on SOX/PBNPs/PPY modified Au electrode

A sulfite oxidase (SO_X) (EC 1.8.3.1) purified from Syzygium cumini leaves was immobilized onto Prussian blue nanoparticles/polypyrrole (PBNPs/PPY) nanocomposite film electrodeposited onto the surface of gold (Au) electrode. An electrochemical sulfite biosensor was fabricated using SO_X/PBNPs/PPY/Au electrode as working electrode, Ag/AgCl as standard electrode and Pt wire as auxiliary electrode connected through a potentiostat. The working electrode was characterized by Fourier Transform infrared (FTIR) spectroscopy, cyclic voltammetry (CV), scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS) at different stages of its construction. The biosensor showed optimum response within 2 s, when operated at 20 mV s⁻¹ in 0.1 M Tris–HCl buffer, pH 8.0 and at 30 °C. Linear range and minimum detection limit were 0.5–1000 μM and 0.1 μM (S/N = 3) respectively. The sensor was evaluated with 95.0% recovery of added sulfite in red wine samples and 1.9% and 3.3% within and between batch coefficients of variation respectively. There was a good correlation (r = 0.96) between red wine samples sulfite value by standard DTNB method and the present method. The sensor was employed for determination of sulfite level in red, white and rose wine samples. The enzyme electrode was used 300 times over a period of 4 months, when stored at 4 °C.     

An amperometric H2O2 biosensor based on cytochrome c immobilized onto nickel oxide nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline modified gold electrode

Cytochrome c was immobilized covalently onto nickel oxide nanoparticles/carboxylated multiwalled carbon nanotubes/polyaniline composite (NiO-NPs/cMWCNT/PANI) electrodeposited on gold (Au) electrode. An amperometric H₂O₂ biosensor was constructed by connecting this modified Au electrode along Ag/AgCl as reference and Pt wire as counter electrode to the galvanostat. The modified Au electrode was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), scanning electron microscopy (SEM) and Fourier transform infra-red spectroscopy (FTIR). Cyclic voltammetric (CV) studies of the electrode at different stages demonstrated that the modified Au electrode had enhanced electrochemical oxidation of H₂O₂, which offered a number of attractive features to develop an amperometric biosensor based on split of H₂O₂. There was a good linear relationship between the current (mA) and H₂O₂ concentration in the range 3–700 μM. The sensor had a detection limit of 0.2 μM (S/N = 3) with a high sensitivity of 3.3 mA μM^?1 cm^?2. The sensor gave accurate and satisfactory results, when employed for determination of H₂O₂ in different fruit juices.     

Biochemical characteristics of phytases from fungi and the transformed microorganism

Five sources of phytases were used to study their biochemical characteristics. Phytase E was from an original Escherichia coli (E. coli), phytase PI and PG from the transformed Pichia pastoris (P. pastoris) with phytase gene of E. coli, phytase B and R from Aspergillus niger (A. niger). The results showed that the relative phytase activities had no significant changes when temperature was below 60 °C (P>0.05), and then decreased significantly with temperature increasing (P<0.01). The fungal phytase with the phytase gene from A. niger had the higher thermostability than the bacterial phytase with the phytase gene from E. coli; i.e. at 70 °C, 27–58% of phytase activity (compared with 30 °C) was retained for the bacterial phytase, and 73–96% for the fungal phytase; at 90 °C, 20–47% was retained for the bacterial phytase, and 41–52% for the fungal phytase, especially for the most thermostable phytase R (P<0.01). The optimum pH ranges were 3.0–4.5 for the bacterial phytases and 5.0–5.5 for the fungal phytases (P<0.01). When pH levels were 1, 7 and 8, only 3–7% of phytase activity (compared with the maximum phytase activity at a pH point) was retained for both bacterial and fungal phytases. The amount of inorganic P released from soybean meal was significantly increased when the levels of phytase activity in the soybean meal increased from 0 to 1.0 U/g soybean meal (P<0.01), except for phytase PI. The maximum P released was obtained at 1 U/g soybean meal for all five kinds of phytases (P<0.01). The most economical phytase concentration for P released was 0.25 U/g for phytase PI and B, and 0.50–1.0 U/g for phytase PG, E and R. In addition, the linear and non-linear regression models were established to estimate phytase activity and its characteristics very easily and economically.     

AhpC (alkyl hydroperoxide reductase) from Anabaena sp. PCC 7120 protects Escherichia coli from multiple abiotic stresses

Alkyl hydroperoxide reductase (AhpC) is known to detoxify peroxides and reactive sulfur species (RSS). However, the relationship between its expression and combating of abiotic stresses is still not clear. To investigate this relationship, the genes encoding the alkyl hydroperoxide reductase (ahpC) from Anabaena sp. PCC 7120 were introduced into E. coli using pGEX-5X-2 vector and their possible functions against heat, salt, carbofuron, cadmium, copper and UV-B were analyzed. The transformed E. coli cells registered significantly increase in growth than the control cells under temperature (47 °C), NaCl (6% w/v), carbofuron (0.025 mg ml^?1), CdCl₂ (4 mM), CuCl₂ (1 mM), and UV-B (10 min) exposure. Enhanced expression of ahpC gene as measured by semi-quantitative RT-PCR under aforementioned stresses at different time points demonstrated its role in offering tolerance against multiple abiotic stresses.     

Microbial surface displaying formate dehydrogenase and its application in optical detection of formate

In the present work, NAD⁺-dependent formate dehydrogenase (FDH), encoded by fdh gene from Candida boidinii was successfully displayed on Escherichia coli cell surface using ice nucleation protein (INP) from Pseudomonas borealis DL7 as an anchoring protein. Localization of matlose binding protein (MBP)-INP-FDH fusion protein on the E. coli cell surface was characterized by SDS-PAGE and enzymatic activity assay. FDH activity was monitored through the oxidation of formate catalyzed by cell-surface-displayed FDH with its cofactor NAD⁺, and the production of NADH can be detected spectrometrically at 340 nm. After induction for 24 h in Luria-Bertani medium containing isopropyl-β-d-thiogalactopyranoside, over 80% of MBP-INP-FDH fusion protein present on the surface of E. coli cells. The cell-surface-displayed FDH showed optimal temperature of 50 °C and optimal pH of 9.0. Additionally, the cell-surface-displayed FDH retained its original enzymatic activity after incubation at 4 °C for one month with the half-life of 17 days at 40 °C and 38 h at 50 °C. The FDH activity could be inhibited to different extents by some transition metal ions and anions. Moreover, the E. coli cells expressing FDH showed different tolerance to solvents. The recombinant whole cell exhibited high formate specificity. Finally, the E. coli cell expressing FDH was used to assay formate with a wide linear range of 5–700 μM and a low limit of detection of 2 μM. It is anticipated that the genetically engineered cells may have a broad application in biosensors, biofuels and cofactor regeneration system.     

Glucose biosensor based on glucose oxidase immobilized at gold nanoparticles decorated graphene-carbon nanotubes

Biopolymer pectin stabilized gold nanoparticles were prepared at graphene and multiwalled carbon nanotubes (GR-MWNTs/AuNPs) and employed for the determination of glucose. The formation of GR-MWNTs/AuNPs was confirmed by scanning electron microscopy, X-ray diffraction, UV–vis and FTIR spectroscopy methods. Glucose oxidase (GOx) was successfully immobilized on GR-MWNTs/AuNPs film and direct electron transfer of GOx was investigated. GOx exhibits highly enhanced redox peaks with formal potential of −0.40 V (vs. Ag/AgCl). The amount of electroactive GOx and electron transfer rate constant were found to be 10.5 × 10⁻¹⁰ mol cm⁻² and 3.36 s⁻¹, respectively, which were significantly larger than the previous reports. The fabricated amperometric glucose biosensor sensitively detects glucose and showed two linear ranges: (1) 10 μM – 2 mM with LOD of 4.1 μM, (2) 2 mM – 5.2 mM with LOD of 0.95 mM. The comparison of the biosensor performance with reported sensors reveals the significant improvement in overall sensor performance. Moreover, the biosensor exhibited appreciable stability, repeatability, reproducibility and practicality. The other advantages of the fabricated biosensor are simple and green fabrication approach, roughed and stable electrode surface, fast in sensing and highly reproducible.     

A variant conferring cofactor-dependent assembly of Escherichia coli dimethylsulfoxide reductase

We have investigated the final steps of complex iron–sulfur molybdoenzyme (CISM) maturation using Escherichia coli DMSO reductase (DmsABC) as a model system. The catalytic subunit of this enzyme, DmsA, contains an iron–sulfur cluster (FS0) and a molybdo-bis(pyranopterin guanine dinucleotide) cofactor (Mo-bisPGD). We have identified a variant of DmsA (Cys59Ser) that renders enzyme maturation sensitive to molybdenum cofactor availability. DmsA-Cys59 is a ligand to the FS0 [4Fe–4S] cluster. In the presence of trace amounts of molybdate, the Cys59Ser variant assembles normally to the cytoplasmic membrane and supports respiratory growth on DMSO, although the ground state of FS0 as determined by EPR is converted from high-spin (S = 3/2) to low-spin (S = 1/2). In the presence of the molybdenum antagonist tungstate, wild-type DmsABC lacks Mo-bisPGD, but is translocated via the Tat translocon and assembles on the periplasmic side of the membrane as an apoenzyme. The Cys59Ser variant cannot overcome the dual insults of amino acid substitution plus lack of Mo-bisPGD, leading to degradation of the DmsABC subunits. This indicates that the cofactor can serve as a chemical chaperone to mitigate the destabilizing effects of alteration of the FS0 cluster. These results provide insights into the role of the Mo–bisPGD–protein interaction in stabilizing the tertiary structure of DmsA during enzyme maturation.     

Rational design,synthesis and biological evaluation of 1,3,4-oxadiazole pyrimidine derivatives as novel pyruvate dehydrogenase complex E1 inhibitors

On the basis of previous study on 2-methylpyrimidine-4-ylamine derivatives I, further synthetic optimization was done to find potent PDHc-E1 inhibitors with antibacterial activity. Three series of novel pyrimidine derivatives 6, 11 and 14 were designed and synthesized as potential Escherichia coli PDHc-E1 inhibitors by introducing 1,3,4-oxadiazole-thioether, 2,4-disubstituted-1,3-thiazole or 1,2,4-triazol-4-amine-thioether moiety into lead structure I, respectively. Most of 6, 11 and 14 exhibited good inhibitory activity against E. coli PHDc-E1 (IC₅₀ 0.97–19.21 μM) and obvious inhibitory activity against cyanobacteria (EC₅₀ 0.83–9.86 μM). Their inhibitory activities were much higher than that of lead structure I. 11 showed more potent inhibitory activity against both E. coli PDHc-E1 (IC₅₀ < 6.62 μM) and cyanobacteria (EC₅₀ < 1.63 μM) than that of 6, 14 or lead compound I. The most effective compound 11d with good enzyme-selectivity exhibited most powerful inhibitory potency against E. coli PDHc-E1 (IC₅₀ = 0.97 μM) and cyanobacteria (EC₅₀ = 0.83 μM). The possible interactions of the important residues of PDHc-E1 with title compounds were studied by molecular docking, site-directed mutagenesis, and enzymatic assays. The results indicated that 11d had more potent inhibitory activity than that of 14d or I due to its 1,3,4-oxadiazole moiety with more binding position and stronger interaction with Lsy392 and His106 at active site of E. coli PDHc-E1.     

A comparative study of different dyes for the detection of proteomes derived from Escherichia coli and MDCK cells: Sensitivity and selectivity

We compared sensitivity and selectivity of five dyes for detection of 2D PAGE-resolved proteins derived from Escherichia coli and MDCK cells. The sensitivity of these dyes was in the following order: SYPRO Ruby > Deep Purple > CBB-G250 > CBB-R250 > Colloidal Gold. Also, we report herein for the first time the application of Colloidal Gold (which is commonly used for staining proteins on blotted membranes) for in-gel staining of proteins. For E. coli, most of the dyes preferably detected proteins with pI range of 4.0–6.9, whereas Deep Purple preferably detected proteins with less acidic range (pI 5.0–7.9). For MDCK cells, while other dyes preferably stained proteins at pI range of 5.0–7.9, Colloidal Gold preferably stained more basic proteins (pI 7.0–9.9). This preferential staining property of Colloidal Gold to basic proteins was confirmed in SDS-PAGE-separated lysozyme (pI 9.4), compared to calmodulin (pI 4.0) and albumin (pI 6.0). These data provide useful information to select appropriate dyes for gel-based proteomic analysis of individual samples.     

Were there any “misassignments” among iron–sulfur clusters N4, N5 and N6b in NADH-quinone oxidoreductase (complex I)?

NADH-quinone oxidoreductase (complex I) in bovine heart mitochondria has a molecular weight of approximately 1 million Da composed of 45 distinct subunits. It is the largest energy transducing complex so far known. Bacterial complex I is simpler and smaller, but the essential redox components and the basic mechanisms of electron and proton translocation are the same. Over the past three decades, Ohnishi et al. have pursued extensive EPR studies near liquid helium temperatures and characterized most of the iron–sulfur clusters in complex I. Recently, Yakovlev et al. [G. Yakovlev, T. Reda, J. Hirst, Reevaluating the relationship between EPR spectra and enzyme structure for the iron-sulfur clusters in NADH:quinone oxidoreductase, Proc. Natl. Acad. Sci. U. S. A. 104 (2007) 12720–12725] challenged Ohnishi's group by claiming that there were EPR “misassignments” among clusters N4, N5 and N6b (in order to prevent confusion, we used current consensus nomenclature, as the nickname). They claimed that we misassigned EPR signals arising from cluster N5 to cluster N4, and signals from cluster N6b to cluster N4. They also proposed that cluster N5 has (4Cys)-ligands. Based on the accumulated historical data and recent results of our site-specific mutagenesis experiments, we confirmed that cluster N5 has (1His + 3Cys)-ligands as we had predicted. We revealed that E. coli cluster N5 signals could be clearly detected at the sample temperature around 3 K with microwave power higher than 5 mW. Thus Hirst's group could not detect N5 signals under any of their EPR conditions, reported in their PNAS paper. It seems that they misassigned the signals from cluster N4 to N5. As to the claim of “misassignment” between clusters N4 and N6b, that was not a possibility because our mutagenesis systems did not contain cluster N6b. Therefore, we believe that we have not made any “misassignment” in our work.     

Differences in RNA processing underlie the tissue specific phenotype of ISCU myopathy

Hereditary myopathy with lactic acidosis, or myopathy with exercise intolerance, Swedish type (OMIM #255125) is caused by mutations in the iron–sulfur cluster scaffold (ISCU) gene. The g.7044G > C ISCU mutation induces a splicing error in the pre-mRNA that strengthens a weak intronic splice site leading to inclusion of a new exon and subsequent loss of mRNA and protein. While ISCU is widely expressed, homozygosity for this particular intronic mutation gives rise to a pure myopathy. In order to investigate tissue specificity and disease mechanism, we studied muscle, myoblasts, fibroblasts and blood cells from the first non-Swedish case of this disease. Consistent with the recognised role of ISCU, we found abnormal activities of respiratory chain complexes containing iron–sulfur clusters in patient muscle. We confirmed that, in the presence of the g.7044G > C mutation, splicing produces both abnormally and normally spliced mRNA in all tissues. The ratio of these products varies dramatically between tissues, being most abnormal in mature skeletal muscle that also has the lowest relative starting levels of ISCU mRNA compared with other tissues. Myoblasts and fibroblasts have more of the normally spliced variant as well as higher starting levels of ISCU mRNA. Up-regulation of mtDNA copy number was found in skeletal muscle and myoblasts, but not fibroblasts, and is thought to represent a compensatory response. Tissue specificity in this disorder appears therefore to be dependent on the mRNA starting level, the amount of remaining normally spliced RNA, and the degree to which compensatory mechanisms can respond.     

Evaluation and enhanced operational performance of microbial fuel cells under alternating anodic open circuit and closed circuit modes with different substrates

The present study evaluates the performance of air-cathode microbial fuel cells (MFCs) under alternating open circuit/closed circuit (OC/CC) modes and its effect on independent-electrode and full-cell potentials, power output (at different external resistances) and the polarization behaviour of the electrodes. Three different types of feeds were evaluated using this approach: (1) phosphorus buffer solution (PBS) with acetate as carbon source, (2) glucose-rich synthetic wastewater, and (3) sewage from wastewater treatment plant enriched with fermented molasses. When MFCs were suddenly switched to CC from OC and then again back to OC from CC, the behaviour of the anodes vs reference electrode (Ag/AgCl, 3 M KCl) was monitored. When electric circuit of the MFCs was switched from open to closed circuit, for all cases: (a) the anode potential-shift (vs Ag/AgCl) reallocated in the positive direction in about 200–400 mV, (b) the air-cathode potential-shift (vs Ag/AgCl) reallocated in the negative direction in about 10–25 mV, and (c) the cell-potential difference started at around 0 mV and progressively increased as the MFC reached stability. This behaviour was consistently reproduced during different OC/CC cycles. The systems studied delivered good performance with both controlled media and industrial wastewater. Additionally, this study provides insightful characterization of the independent-electrode behaviours.     

Activity of recombinant GST in Escherichia coli grown on glucose and glycerol

We have investigated production, solubility and activity of recombinant glutathione-S-transferase (GST) expressed in Escherichia coli BL21 grown in defined media with glucose or glycerol as carbon source. GST was predominantly expressed as a soluble protein on both carbon sources, and 83–84% was found in the supernatant after cell lysis. In cultures grown on glucose, only 32 ± 9% of the GST was active, while 76 ± 13% of the GST was active in cultures grown on glycerol. This shows that glycerol has the potential to increase the activity of soluble GST in E. coli cultures in vivo.     

Cloning,expression, purification and characterization of an oligomeric His-tagged thermophilic esterase from Thermus thermophilus HB27

The esterase E34Tt (YP_004875.1) from Thermus thermophilus HB27 was cloned, expressed in Escherichia coli as a His-tagged protein, purified and characterized. The gene sequence was subcloned into a T-vector, released with the restriction enzymes BamHI and HindIII, ligated to a pET-21d(+) vector, and transferred to E. coli BL21 (DE3) cells. Inducer concentration (isopropyl β-d-1-thiogalactopyranoside, IPTG) and cultivation time before and after induction were optimized. Best results were obtained by adding 0.25 mM IPTG after 8 h of cultivation and maintaining the induction during 4 extra hours. Most of the enzyme (94%) remained membrane-associated and had to be extracted with a detergent. From the membrane crude extract, the His-tagged E34Tt was purified as a dimer (71.8 kDa) in a single purification step by using metal affinity chromatography. The Rosso's model was used to optimize the reaction conditions. E34Tt-His₆ was active in a wide temperature (19.7–79.4 °C) and pH range (4.0–9.3), and maximal activity was determined at pH 6.3 and 58.2 °C, which is 10–18 °C higher than the optimal reaction temperature of the previously reported variants expressed in mesophilic yeasts. E34Tt-His₆ preferentially hydrolyzed esters with ten carbon atoms, and was highly thermostable (half-life of 107.9 min at 85 °C), suggesting that E34Tt-His₆ has potential for industrial applications.     

Development of a recombinant Escherichia coli-based biocatalyst to enable high styrene epoxidation activity with high product yield on energy source

A highly active recombinant whole-cell biocatalyst, Escherichia coli pETAB2/pG-KJE1, was developed for the efficient production of (S)-styrene oxide from styrene. The recombinant E. coli overexpressed styAB the genes of styrene monooxygenase of Pseudomonas putida SN1 and coexpressed the genes encoding chaperones (i.e., GroEL–GroES and DnaK–DnaJ–GrpE). The styrene monooxygenases were produced to ca. 40% of the total soluble proteins, enabling the whole-cell activity of the recombinant of 180 U/g CDW. The high StyAB activity in turn appeared to direct cofactors and molecular oxygen to styrene epoxidation. The product yield on energy source (i.e., glucose) reached ca. 40%. In addition, biotransformation in an organic/aqueous two-liquid phase system allowed the product to accumulate to 400 mM in the organic phase within 6 h, resulting in an average specific and volumetric productivity of 6.4 mmol/g dry cells/h (106 U/g dry cells) and 67 mM/h (1110 U/L_aq), respectively, under mild reaction conditions. These results indicated that the high productivity and the high product yield on energy source were driven by the high enzyme activity. Therefore, it was concluded that oxygenase activity of whole-cell biocatalysts is one of the critical factors to determine their catalytic performance.     

Construction of d-amino acid biosensor based on d-amino acid oxidase immobilized onto poly (indole-5-carboxylic acid)/zinc sulfide nanoparticles hybrid film

d-Amino acid oxidase (DAAO) purified from goat kidney was immobilized covalently via N-ethyl-N-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxy succinimide (NHS) chemistry onto poly indole 5-carboxylic acid (Pin5-COOH)/zinc sulfide nanoparticles (ZnSNPs) hybrid film electrodeposited on surface of an Au electrode. A highly sensitive d-amino acid biosensor was constructed using this enzyme electrode as working electrode, Ag/AgCl as reference electrode, and Pt wire as auxiliary electrode connected through potentiostat. The biosensor showed optimum response within 3 s at pH 7.5 and 35 °C, when polarized at 0.15 V vs. Ag/AgCl. There was a linear relationship between biosensor response (mA) and d-alanine concentration in the range 0.001–2.0 mM. The sensitivity of the biosensor was 58.85 μA cm^?2 mM^?1 with a detection limit of 0.001 mM (S/N = 3). The enzyme electrode was used 120 times over a period of 2 months when stored at 4 °C. The biosensor has an advantage over earlier enzyme sensors that it has no leakage of enzyme during reuse and is unaffected by the external environment due to the protective layer of poly indole-5-carboxylic acid film. The biosensor was evaluated and employed for measurement of d-amino acid level in fruits and vegetables.     

Stereoselective synthesis of l-homophenylalanine using the carbamoylase method with in situ racemization via N-acylamino acid racemase

N-Acylamino acid racemase (NAAAR) gene of Deinococcus radiodurans BCRC12827 was cloned into expression vector pQE30 to generate pQE-naaar and expressed in recombinant Escherichia coli JM109. The expressed enzyme purified from the crude cell extract of IPTG-induced E. coli JM109 (pQE-naaar) exhibited high racemization activity to N-carbamoyl-l-homophenylalanine (NCa-l-HPA) and N-carbamoyl-d-homophenylalanine (NCa-d-HPA) with specific activities of 1.91 U/mg protein and 1.31 U/mg protein, respectively. To develop a recombinant E. coli whole cell system for the conversion of racemic NCa-HPA to l-homophenylalanine (l-HPA), naaar gene from D. radiodurans and l-N-carbamoylase (LNCA) gene from Bacillus kaustophilus BCRC11223 were cloned and coexpressed in E. coli cells. Recombinant cells treated with 0.5% toluene at 30 °C for 30 min exhibited enhanced NAAAR and LNCA activities, which are about 20- and 60-fold, respectively, higher than those of untreated cells. Using toluene-permeabilized recombinant E. coli cells, a maximal productivity of 7.5 mmol l-HPA/l h with more than 99% yield could be obtained from 150 mmol racemic NCa-HPA. Permeabilized cells also showed considerable stability in the bioconversion process using 10 mmol racemic NCa-HPA as substrate, no significantly decrease in conversion yield for l-HPA was found in the eight cycles.