Glucose biosensor based on nano-SiO2 and "unprotected" Pt nanoclusters

The “unprotected” Pt nanoclusters (average size 2 nm) mixed with the nanoscale SiO₂ particles (average size 13 nm) were used as a glucose oxidase immobilization carrier to fabricate the amperometric glucose biosensor. The bioactivity of glucose oxidase (GOx) immobilized on the composite was maintained and the as-prepared biosensor demonstrated high sensitivity (3.85 μA mM⁻¹) and good stability in glucose solution. The Pt–SiO₂ biosensor showed a detection limit of 1.5 μM with a linear range from 0.27 to 4.08 mM. In addition, the biosensor can be operated under wide pH range (pH 4.9–7.5) without great changes in its sensitivity. Cyclic voltammetry measurements showed a mixed controlled electrode reaction.     

Immobilization and hybridization by single sub-millisecond electric field pulses, for pixel-addressed DNA microarrays

Single square voltage pulses applied to buried electrodes result in dramatic rate increases for (1) selective covalent bonding (immobilization) of single-stranded DNA (ssDNA) probes to a functionalized thin film SiO₂ surface on a plastic substrate and (2) hybridization of ssDNA to the immobilized probe. DNA immobilization and hybridization times are 100 ns and 10 μs, respectively, about 10⁹ times faster than the corresponding passive reactions without electric field. Surface coverage is comparable. Duration, magnitude and slew rate of the voltage pulse are all key factors controlling the rates of ssDNA immobilization and hybridization. With rise times of 4.5 ns, pulses shorter than 1 ms and voltages below 1 V are effective. The ssDNA adsorbed on the surface is reoriented by the rapidly changing electric field. This reduces steric barriers and speeds the immobilization and hybridization reactions. These results open the way for pixel-addressed microarrays driven by silicon microelectronics circuits.     

Covalent immobilization of pure isoenzymes from lipase of Candida rugosa

Covalent immobilization of pure lipases A and B from Candida rugosa on agarose and silica is described. The immobilization increases the half-life of the biocatalysts ( ) with respect to the native pure lipases ( ). The percentage immobilization of lipases A and B is similar in both supports (33–40%). The remaining activity of the biocatalysts immobilized on agarose (70–75%) is greater than that of the enzymatic derivatives immobilized on SiO₂ (40–50%). The surface area and the hydrophobic/hydrophilic properties of the support control the lipase activity of these derivatives. The thermal stability of the immobilized lipase A derivatives is greater than that of lipase B derivatives. The nature of the support influences the thermal deactivation profile of the immobilized derivatives. The immobilization in agarose (hydrophilic support) gives biocatalysts that show a greater initial specific reaction rate than the biocatalysts immobilized in SiO₂ (hydrophobic support) using the hydrolysis of the esters of (R) or (S) 2-chloropropanoic and of (R,S) 2-phenylpropanoic acids as the reaction test. The enzymatic derivatives are active for at least 196 h under hydrolysis conditions. The stereospecificity of the native and the immobilized enzymes is the same.     

Formaldehyde-sensitive sensor based on recombinant formaldehyde dehydrogenase using capacitance versus voltage measurements

A new formaldehyde-selective biosensor was constructed using NAD⁺- and glutathione-dependent recombinant formaldehyde dehydrogenase as a bio-recognition element immobilised on the surface of Si/SiO₂/Si₃N₄ structure. Sensor's response to formaldehyde was evaluated by capacitance measurements. The calibration curves obtained for formaldehyde concentration range from 10 μM to 20 mM showed a broad linear response with a sensitivity of 31 mV/decade and a detection limit about 10 μM. It has been shown that the output signal decreases with the increase of borate buffer concentration and the best sensitivity is observed in 2.5 mM borate buffer, pH 8.40. The response of the created formaldehyde-sensitive biosensor has also been examined in 2.5 mM Tris–HCl buffer, and the shift to the positive bias of the C(V) curves along with the potential axis has been observed, but the sensitivity of the biosensor in this buffer is decreased dramatically to the value of 2.4 mV/decade.     

Post-mortem degradation of brain glutamate decarboxylase

The post-mortem stability of the GABA synthesizing enzyme glutamate decarboxylase (GAD) was studied by using SDS–PAGE and quantitative immunoblotting to measure the rates of degradation of GAD in the cerebral cortex, hippocampus, and cerebellum of rats and mice as a function of time after death. The intact 65- and 67-kDa isoforms of GAD (GAD₆₅ and GAD₆₇) disappeared gradually over a 24-h period. In both rats and mice, the degraded GAD appeared as a band with an apparent molecular mass of 55–57 kDa; no significant amounts of smaller forms were observed. The 55–57 kDa band reacted with antiserum W887, which recognizes a shared epitope at the carboxyl-terminal end of both GADs, indicating that GAD was cleaved near the amino-terminal end of the molecule. GAD₆₇ was cleaved at a site between the amino-terminus and the epitope for antiserum W883 (located within residues 79–93 of GAD₆₇), as antiserum W883 stained a 56-kDa band on the blots. The appearance of degraded GAD paralleled the loss of total GAD (GAD₆₅+GAD₆₇), and after 24 h the 55–57 kDa band accounted for 97, 88, and 59% of the intact GAD lost from rat cerebellum, cerebral cortex and hippocampus. On a percentage basis, GAD₆₇ was degraded more rapidly than was GAD₆₅ in all brain regions studied. The loss of GAD activity was greater in rat than mouse brain, even though the percent loss of intact GAD protein was similar.     

Affinity separation of immunoglobulin G subclasses on dye attached poly(hydroxypropyl methacrylate) beads

Poly(hydroxypropyl methacrylate) [poly(HPMA)] gel beads with an average size of 150–200 μm were prepared by suspension polymerization of hydroxypropyl methacrylate (HPMA). The poly(HPMA) gel beads were characterized by swelling studies, surface area measurements, scanning electron microscopy (SEM) and elemental analysis. Poly(HPMA) gel beads had a specific surface area of 88.6 m²/g. The dye Reactive Green HE 4BD was chemically attached to yield dye-poly(HPMA) gel beads at an average concentration of 44.3 μmol dye/g bead with a swelling ratio of 75%. These dye attached gel beads were used in the separation of immunoglobulin-G (IgG) through adsorption–elution studies. The non-specific adsorption of IgG on the poly(HPMA) gel beads was 0.5 mg/g. The attachment of Reactive Green HE 4BD significantly increased the adsorption of IgG up to 71 mg/g. The Langmuir adsorption model was found to be applicable in interpretation of data pertaining to the adsorption studies of IgG with Reactive Green HE 4BD attached to the poly(HPMA) gel beads. The adsorption of IgG was found to be optimal at pH 7.0. The adsorption of IgG was observed to decrease by about 76% as the NaCl concentration was increased from 0.001 to 0.1 M. The IgG adsorption capacity of the dye attached poly(HPMA) gel beads was determined for a commercially available IgG solution to be 4.2 mg/g for IgG₁, 64.5 mg/g for IgG₂, 7.1 mg/g for IgG₃ and 10.8 mg/g for IgG₄. The Reactive Green HE 4BD attached poly(HPMA) gel beads have a significant adsorption capacity for IgG₂. The quantity of adsorbed IgG₂ is three times higher than the quantity of the other subclasses, IgG₁, IgG₃ and IgG₄. A similar adsorption behaviour was observed when the albumin free human plasma was used. The quantity of adsorbed IgG₂ is higher than the quantity of the other subclasses, IgG₁, IgG₃ and IgG₄. Adsorption capacities for albumin free human plasma were obtained as 6.4 mg/g for IgG₁, 67.8 mg/g for IgG₂, 5.2 mg/g for IgG₃ and 8.6 mg/g for IgG₄. Significant amount of the adsorbed IgG (up to 95%) was eluted in 1 h in the elution medium containing 2.0 M NaCl. Repeated adsorption/elution processes showed that these dye attached gel beads are suitable for IgG adsorption.     

Immobilization of horseradish peroxidase on chitosan/silica sol-gel hybrid membranes for the preparation of hydrogen peroxide biosensor

A simple and effective strategy for fabrication of hydrogen peroxide (H₂O₂) biosensor has been developed by entrapping horseradish peroxidase (HRP) in chitosan/silica sol–gel hybrid membranes (CSHMs) doped with potassium ferricyanide (K₃Fe(CN)₆) and gold nanoparticles (GNPs) on platinum electrode surface. The hybrid membranes are prepared by cross-linking chitosan (CS) with 3-aminopropyltriethoxysilane (APTES), while the presence of GNPs improved the conductivity of CSHMs, and the Fe(CN)₆^3−/4− was used as a mediator to transfer electrons between the electrode and HRP due to its excellent electrochemistry activity. UV–Vis absorption spectroscopy was employed to characterize the different components in the CSHMs and their interaction. The parameters influencing the performance of the resulting biosensor were optimized and the characteristic of the resulting biosensor was characterized by cyclic voltammetry and chronoamperometry. Linear calibration for hydrogen peroxide was obtained in the range of 3.5 × 10^− 6 to 1.4 × 10^− 3 M under the optimized conditions with the detection limit (S/N = 3) of 8.0 × 10^− 7 M. The apparent Michaelis–Menten constant of the enzyme electrode was 0.93 mM. The enzyme electrode retained about 78% of its response sensitivity after 30 days. The system was applied for the determination of the samples, and the results obtained were satisfactory.     

Electrochemical impedance spectroscopy for study of aptamer-thrombin interfacial interactions

A simple and highly sensitive electrochemical impedance spectroscopy (EIS) biosensor based on a thrombin-binding aptamer as molecular recognition element was developed for the determination of thrombin. The signal enhancement was achieved by using gold nanoparticles (GNPs), which was electrodeposited onto a glassy carbon electrode (GCE), as a platform for the immobilization of the thiolated aptamer. In the measurement of thrombin, the change in interfacial electron transfer resistance of the biosensor using a redox couple of [Fe(CN)₆]^3−/4− as the probe was monitored. The increase of the electron transfer resistance of the biosensor is linear with the concentration of thrombin in the range from 0.12 nM to 30 nM. The association and dissociation rate constants of the immobilized aptamer–thrombin complex were 6.7 × 10³ M⁻¹ s⁻¹ and 1.0 × 10⁻⁴ s⁻¹, respectively. The association and dissociation constants of three different immobilized aptamers binding with thrombin were measured and the difference of the dissociation constants obtained was discussed. This work demonstrates that GNPs electrodeposited on GCE used as a platform for the immobilization of the thiolated aptamer can improve the sensitivity of an EIS biosensor for the determination of protein. This work also demonstrates that EIS method is an efficient method for the determination of association and dissociation constants on GNPs modified GCE.     

Amperometric glucose biosensor based on self-assembly hydrophobin with high efficiency of enzyme utilization

Hydrophobins are a family of natural self-assembling proteins with high biocompability, which are apt to form strong and ordered assembly onto many kinds of surfaces. These physical-chemical and biological properties make hydrophobins suitable for surface modification and biomolecule immobilization purposes. A class II hydrophobin HFBI was used as enzyme immobilization matrix on platinum electrode to construct amperometric glucose biosensor. Permeability of HFBI self-assembling film was optimized by selecting the proper HFBI concentration for electrode modification, in order to allow H₂O₂ permeating while prevent interfering compounds accessing. HFBI self-assembly and glucose oxidase (GOx) immobilization was monitored by quartz crystal microbalance (QCM), and characterization of the modified electrode surface was obtained by scanning electron microscope (SEM). The resulting glucose biosensors showed rapid response time within 6 s, limits of detection of 0.09 mM glucose (signal-to-noise ratio = 3), wide linear range from 0.5 to 20 mM, high sensitivity of 4.214 × 10⁻³ A M⁻¹ cm⁻², also well selectivity, reproducibility and lifetime. The all-protein modified biosensor exhibited especially high efficiency of enzyme utilization, producing at most 712 μA responsive current for per unit activity of GOx. This work provided a promising new immobilization matrix with high biocompatibility and adequate electroactivity for further research in biosensing and other surface functionalizing.     

Reversible immobilization of tyrosinase onto polyethyleneimine-grafted and Cu(II) chelated poly(HEMA-co-GMA) reactive membranes

The present study describes the preparation of poly(HEMA-co-GMA) reactive membranes that were grafted with polyethylenimine (PEI) following UV photo-polymerization. The immobilization of tyrosinase was carried out via multi-point ionic interactions based on ---NH₂ groups of PEI and Cu(II) ions. Tyrosinase is a copper-dependent enzyme, which should show a binding affinity for the chelated Cu(II) ions on the membrane surfaces. The tyrosinase immobilization was positively correlated with the input enzyme amount in the immobilization medium. The maximum tyrosinase immobilization capacities of the poly(HEMA-co-GMA)–PEI and poly(HEMA-co-GMA)–PEI–Cu(II) membranes were 19.3 and 24.6 mg/m², respectively. The enzyme activity when assessed at various pH and temperatures gave broader range for immobilized preparations when compared to free enzyme. The poly(HEMA-co-GMA)–PEI–Cu(II) tyrosinase membranes retained 82% of their initial activity at the end of 120 h of continuous reaction. Moreover, upon storage for 3 months the activity of the immobilized membranes retained 46% of their initial levels. After deactivation of the enzyme, the poly(HEMA-co-GMA)–PEI membrane was easily regenerated, re-chelated with the Cu(II) ions and reloaded with the enzyme for repeated use. The mild immobilization conditions, easy and rapid membrane preparation, one-step enzyme adsorption at substantially higher levels and membrane reusability are the beneficial properties of such systems and offers promising potential in several biochemical processes.     

Highly sensitive measurements of PNA-DNA hybridization using oxide-etched silicon nanowire biosensors

The highly sensitive and sequence-specific detection of single-stranded oligonucleotides using nonoxidized silicon nanowires (SiNWs) is demonstrated. To maximize device sensitivity, the surface of the SiNWs was functionalized with a densely packed organic monolayer via hydrosilylation, subsequently immobilized with peptide nucleic acid (PNA) capable of recognizing the label-free complementary target DNA. Because of the selective functionalization of the SiNWs, binding competition between the nanowire and the underlying oxide is avoided. Transmission electron microscopy was conducted to clearly differentiate the SiNW surface before and after removal of SiO₂. Fluorescence microscopy was used to further realize the selectivity of the oxide-etched chemistry on the SiNWs and sequence specificity of PNA-DNA hybridization. The concentration-dependent resistance change measurements upon hybridization of PNA-DNA show that detection limit down to 10 fM can be obtained. The SiNW devices also reveal the capability of an obvious discrimination against mismatched sequences. Among several efforts being made to improve detection sensitivity, this work addresses one significant issue regarding surface functionalization which enables highly sensitive biomolecular sensing with SiNWs.     

Immobilization of invertase onto poly(3-methylthienyl methacrylate)/poly(3-thiopheneacetic acid) matrix

A novel immobilization matrix, poly(3-methylthienyl methacrylate)–poly(3-thiopheneacetic acid) (PMTM–PTAA), was synthesized and used for the covalent immobilization of Saccharomyces cerevisiae invertase to produce invert sugar. The immobilization resulted in 87% immobilization efficiency. Optimum conditions for activity were not affected by immobilization and the optimum pH and temperature for both free and immobilized enzyme were found to be 4.5 and 55 °C, respectively. However, immobilized invertase was more stable at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined using the Lineweaver–Burk plot. The K_m values were 35 and 38 mM for free and immobilized enzyme, respectively. The V_max values were 29 and 24 mg glucose/mg enzyme min for free and immobilized enzyme, respectively. Immobilized enzyme could be used for the production of glucose and fructose from sucrose since it retained almost all the initial activity for a month in storage and retained the whole activity in repeated 50 batch reactions.     

Sol-gel powders and supported sol-gel polymers for immobilization of lipase in ester synthesis

Candida rugosa lipase was entrapped in hybrid organic–inorganic sol-gel powder prepared by acid-catalyzed polymerization of tetramethoxysilane (TMOS) and alkyltrimethoxysilanes, and used in catalyzing esterification reactions between ethanol and butyric acid in hexane. Optimum preparation conditions were studied, which are gels made from propyltrimethoxysilane (PTMS)/TMOS molar ratio=4:1, hydrolysis time of silane precursor=30 min, water/silane molar ratio=24, enzyme loading=6.25% (w/w) of gel, and 1 mg PVA/mg lipase. The percentage of protein immobilization was 95% and the resulting lipase specific activity was 59 times higher than that of a non-immobilized lyophilized lipase. To prepare magnetic lipase-immobilized sol-gel powder (MLSP) for easier recovery of the biocatalyst, Fe₃O₄ nanoparticles were prepared and co-entrapped with lipase during gel formation. This procedure induced surface morphological change of the sol-gel powder and showed adverse effect on enzyme activity. Hence, although only 9% decrease in protein immobilization efficiency was observed, the corresponding reduction in enzyme activity could be up to 45% when sol-gel powder was doped with 25% (v/v) Fe₃O₄ magnetic nanoparticles solution. Lipase-immobilized sol-gel polymer was also formed within the pores of different porous supports to improve its mechanical stability. Non-woven fabric, with a medium pore size of all the supports tested, was found to be the best support for this purpose. The thermal stability of lipase increased 55-fold upon entrapment in sol-gel materials. The half-lives of all forms of sol-gel-immobilized lipase were 4 months at 40 °C in hexane.     

Behavioral effects of low dissolved oxygen on the bivalve Macoma balthica

Hypoxia, a dissolved oxygen concentration (DO) below 2 mg l^– 1, is a significant stressor in many estuarine ecosystems. Many sedentary organisms, unable to move to avoid hypoxic areas, have metabolic and behavioral adaptations to hypoxic stress. We tested the effects of hypoxia on the behavior and mortality of the clam Macoma balthica, using four levels of dissolved oxygen in flow-through tanks. We used five replicates of each of four treatments: (1) Hypoxic (DO mean ± SE = 1.1 ± 0.06 mg O₂ l^– 1), (2) Moderately hypoxic (DO 2.6 ± 0.05 mg O₂ l^– 1), (3) Nearly normoxic (DO 3.2 ± 0.04 mg O₂ l^– 1), (4) Normoxic (DO = 4.9 ± 0.13 mg O₂ l^– 1). We lowered the dissolved oxygen with a novel fluidized mud-bed, designed to mimic field conditions more closely than the common practice of solely bubbling nitrogen or other gasses. This method for lowering the DO concentrations for a laboratory setup was effective, producing 1.4 l min^–1 of water with a DO of 0.8 mg O₂ l^– 1 throughout the experiment. The setup greatly reduced the use of compressed nitrogen and could easily be scaled up to produce more low-DO water if necessary. The lethal concentration for 50% of the M. balthica population (LC₅₀) was 1.7 mg O₂ l^– 1 for the 28-day experimental period. M. balthica decreased its burial depth under hypoxic and moderately hypoxic (~2.5 mg O₂ l^– 1) conditions within 72 hours of the onset of hypoxia. By the sixth day of hypoxia the burial depth had been reduced by 26 mm in the hypoxic tanks and 10 mm in the moderately hypoxic tanks. Because reduced burial depth makes the clams more vulnerable to predators, these results indicate that the sub-lethal effects of hypoxia could change the rate of predation on M. balthica in the field.     

Invertase reversibly immobilized onto polyethylenimine-grafted poly(GMA–MMA) beads for sucrose hydrolysis

The epoxy group containing poly(glycidyl methacrylate-co-methylmethacrylate) poly(GMA–MMA) beads were prepared by suspension polymerisation and the beads surface were grafted with polyethylenimine (PEI). The PEI-grafted beads were then used for invertase immobilization via adsorption. The immobilization of enzyme onto the poly(GMA–MMA)–PEI beads from aqueous solutions containing different amounts of invertase at different pH was investigated in a batch system. The maximum invertase immobilization capacity of the poly(GMA–MMA)–PEI beads was about 52 mg/g. It was shown that the relative activity of immobilized invertase was higher then that of the free enzyme over broader pH and temperature ranges. The Michaelis constant (K_m) and the maximum rate of reaction (V_max) were calculated from the Lineweaver–Burk plot. The K_m and V_max values of the immobilized invertase were larger than those of the free enzyme. The immobilized enzyme had a long-storage stability (only 6% activity decrease in 2 months) when the immobilized enzyme preparation was dried and stored at 4 °C while under wet condition 43% activity decrease was observed in the same period. After inactivation of enzyme, the poly(GMA–MMA)–PEI beads can be easily regenerated and reloaded with the enzyme for repeated use.     

Dynamic changes in sweat rates and evaporation rates through clothing during hot exposure

Dynamic changes in local sweat rates (S_w) and local evaporation rates from clothing (E_cl) have been observed during hot exposure. Four young male subjects wearing a cotton T-shirt and half shorts were exposed to 40 °C/50% for 1 h following exposure to 28 °C/50% for 30 min. Amount of water absorbed in clothing (M_sw), clothing surface temperatures (T_cl), local heat flow rates, skin temperatures, body weight, rectal temperature, S_w and E_cl were continuously measured. Upon exposure to the heat, decrease in heat gain to the skin was observed as opposed to increase in S_w, E_cl, M_sw and heat gain to the clothing surface.     

Methylene bridged binuclear bis(imino)pyridyl iron(II) complexes and their use as catalysts together with Al(i-Bu)3 for ethylene polymerization

Three novel methylene bridged binuclear iron(II) complexes: (R,R′ = i-C₃H₇ (6); R = i-C₃H₇, R′ = CH₃ (7); R,R′ = CH₃ (8))} have been synthesized. Activated by Al(i-Bu)₃, complex 6 shows very poor activity for the polymerization of ethylene at one bar ethylene pressure, whereas, 7 and 8 exhibit much higher activity than mononuclear iron catalysts {[ArNC(Me)C₅H₃N(Me)CNAr′]FeCl₂ (Ar,Ar′ = 2,6-C₆H₃-i-Pr (9); Ar = 2,6-C₆H₃-i-Pr₂, Ar′ = 2,6-C₆H₃–Me₂ (10); Ar,Ar′ = 2,6-C₆H₃–Me₂ (11))}. The molecular weight (M_w) of PE produced by 7 and 8 are in the range 13.2–46.0 × 10⁴ and much higher than those produced by mononuclear iron catalysts 9 and 10. GPC results demonstrate that 7 and 8 yield PE with a broad/bimodal molecular weight distribution (MWD). In contrast, 9 and 10 yield PE with relatively narrow and unimodal MWD (4.26 and 3.55). Elevating the temperature and Al/Fe molar ratio will narrow the MWD of PE.     

Spin-crossover iron(II) complexes [Fe(Medpq)(py)2(NCS)2] and [Fe(Medpq)(py)2(NCSe)2]: syntheses, characterization and magnetic properties

Two new spin-crossover complexes, [Fe(Medpq)(py)₂(NCS)₂] · py · 0.5H₂O (1) and [Fe(Medpq)(py)₂(NCSe)₂] · py (2) (Medpq = 2-methyldipyrido[3,2-f:2′,3′-h]-quinoxaline, py = pyridine), have been synthesized. The crystal structures were determined at both room temperature (298 K) and low temperature (110 K). Complexes 1 and 2 crystallize in the orthorhombic space group Pbca and monoclinic space group P2₁/n, respectively. In both complexes, the distorted [FeN₆] octahedron is formed by six nitrogen atoms from Medpq, the trans pyridine molecules and the cis NCX⁻ groups. The thermal spin transition is accompanied by the shortening of the mean Fe–N distances by 0.194 Å for 2. The mononuclear [Fe(Medpq)(py)₂(NCS)₂] and [Fe(Medpq)(py)₂(NCSe)₂] neutral species interact each other via π-stacking, resulting in a one-dimensional extended structure for both 1 and 2. There exist C–HX (X = S, Se) hydrogen bonds for both complexes. Variable-temperature magnetic susceptibility measurements and Mössbauer spectroscopy reveal the occurrence of a gradual spin transition. The transitions are centered at T_1/2 = 120 K for 1 and T_1/2 = 180 K for 2, respectively.     

Optimization of culture conditions for production of cis-epoxysuccinic acid hydrolase using response surface methodology

Response surface methodology, which allows for rapid identification of important factors and optimization of them to enhance enzyme production, was employed here to optimize culture conditions for the production of cis-epoxysuccinic acid hydrolase from Bordetella sp. strain 1–3. In the first step, a Plackett–Burman design was used to evaluate the effects of nine variables (yeast extract, cis-epoxysuccinic acid, KH₂PO₄, K₂HPO₄ · 3H₂O, MgSO₄ · 7H₂O, trace minerals solution, culture volume, initial pH and incubation time) on the enzyme production. Yeast extract, cis-epoxysuccinic acid and KH₂PO₄ had significant influences on cis-epoxysuccinic acid hydrolase production and their concentrations were further optimized using central composite design and response surface analysis. A combination of adjusting the concentration of yeast extract to 7.8 g/l, cis-epoxysuccinic acid to 9.8 g/l, and KH₂PO₄ to 1.12 g/l would favor maximum cis-epoxysuccinic acid hydrolase production. An enhancement of cis-epoxysuccinic acid hydrolase production from 5.6 U/ml to 9.27 U/ml was gained after optimization.     

Production of cyclodextrin by poly-lysine fused Bacillus macerans cyclodextrin glycosyltransferase immobilized on cation exchanger

Bacillus macerans cyclodextrin glycosyltransferase (CGTase) fused with 10 lysine residues at its C-terminus (CGTK10ase) was immobilized onto a cation exchanger by ionic interaction and used to produce -cyclodextrin (CD) from soluble starch. Poly-lysine fused immobilization increased the V_m of the immobilized CGTase by 40% without a change in K_m. The activation energies of thermal deactivation (E_a) were 41.4, 28.1, and 25.9 kcal mol⁻¹, respectively, for soluble wild-type (WT) CGTase, soluble CGTK10ase, and immobilized CGTK10ase, suggesting destabilization of CGTase by poly-lysine fusion and immobilization onto a cation exchanger. Maximum -CD productivity of 539.4 g l⁻¹ h⁻¹ was obtained with 2% soluble starch solution which was constantly fed at a flow rate of 4.0 ml min⁻¹ (D = 240 h⁻¹) in a continuous operation mode of a packed-bed reactor. The operational half-life of the packed-bed enzyme reactor was estimated 12 days at 25 °C and pH 6.0.