Immobilization of manganese peroxidase fromLentinula edodes on alkylaminated emphazeTM AB 1 polymer for generation of Mn3+ as an oxidizing agent

Manganese peroxidase (MnP) is secreted by white-rot fungi and participates in the degradation of lignin by these organisms. MnP uses H₂O₂ as an oxidant to oxidize Mn^II to Mn^III as the manganic ion Mn³⁺. The Mn³⁺ stabilized by chelation, is a highly reactive nonspecific oxidant capable of oxidizing a variety of toxic organic compounds. Previous attempts at immobilization of MnP, purified fromLentinula edodes through reactive amino groups, have been hindered by the protein’s low lysine content of only 1% and its instability above pH 6.0. As an alternative to amine coupling, the enzyme has now been covalently immobilized through its carboxyl groups, using an azlactonefunctional copolymer derivatized with ethylenediamine and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) as a coupling reagent. The immobilization reaction was performed under acidic (pH 5.25) conditions, and 90% coupling efficiency was achieved within 2 h. Net immobilization efficiencies, expressed as the product of protein coupling efficiency and enzyme activity, have been measured at > 95% within 4 h. The MnP-NH-polymer and the free soluble protein were characterized and compared for their pH, temperature, and storage stabilities, as well as their H₂O₂ dependence and kinetics. The tethered MnP, employed in an immobilized enzyme bioreactor for generation of chelated Mn₃₊ may have industrial applications as a nonspecific oxidant of organopollutants.     

Mn2+ alters peroxidase profiles and lignin degradation by the white-rot fungus Pleurotus ostreatus under different nutritional and growth conditions

The white-rot fungus Pleurotus ostreatus produces two types of extracellular peroxidases: manganese-dependent peroxidase (MnP) and versatile peroxidase (VP). The effect of Mn²⁺ on fungal growth, peroxidase activity profiles, and lignin degradation by P. ostreatus was studied in liquid culture and under solid-state fermentation conditions on perlite, the latter resembling the natural growth conditions of this fungus. The fungus was grown in either a defined asparagine-containing basidiomycete selective medium (BSM) or in a rich peptone medium (PM). Biomass production, as determined by respiration experiments in solid-state fermentation and liquid cultures and fungal growth on Petri dishes, was higher in the PM than in the BSM. Mn²⁺ affected biomass production only in the PM on Petri dishes. In the nonamended PM, high levels of MnP and VP activity were detected relative to the nonamended BSM. Nevertheless, a higher rate of ¹⁴C-lignin mineralization was measured in the Mn²⁺-amended BSM, as determined during the course of 47 d of fermentation. Mn²⁺ amendment of the PM increased mineralization rate to that obtained in the Mn²⁺-amended BSM. The enzyme activity profiles of MnP and VP were studied in the BSM using anion-exchange chromatography. In the nonamended BSM, only minute levels of MnP and VP were detected. On Mn²⁺ amendment, two MnP isoenzymes (B1 and B2) appeared. Isoenzyme B2 was purified and showed 100% identity with the MnP isoenzyme purified in our previous study from PM-solid-state fermentation (P6). P6 was found to be the dominant isoenzyme in terms of activity level and gene expression compared with the VP isoenzymes. Based on these results, we concluded that Mn²⁺ plays a key role in lignin degradation under different nutritional and growth conditions, since it is required for the production of MnP in P. ostreatus.     

Transformation of Tetracycline by Manganese Peroxidase from Phanerochaete chrysosporium

The negative impacts on the ecosystem of antibiotic residues in the environment have become a global concern. However, little is known about the transformation mechanism of antibiotics by manganese peroxidase (MnP) from microorganisms. This work investigated the transformation characteristics, the antibacterial activity of byproducts, and the degradation mechanism of tetracycline (TC) by purified MnP from Phanerochaete chrysosporium. The results show that nitrogen-limited and high level of Mn²⁺ medium could obtain favorable MnP activity and inhibit the expression of lignin peroxidase by Phanerochaete chrysosporium. The purified MnP could transform 80% tetracycline in 3 h, and the threshold of reaction activator (H₂O₂) was about 0.045 mmol L⁻¹. After the 3rd cyclic run, the transformation rate was almost identical at the low initial concentration of TC (77.05–88.47%), while it decreased when the initial concentration was higher (49.36–60.00%). The antimicrobial potency of the TC transformation products by MnP decreased throughout reaction time. We identified seven possible degradation products and then proposed a potential TC transformation pathway, which included demethylation, oxidation of the dimethyl amino, decarbonylation, hydroxylation, and oxidative dehydrogenation. These findings provide a novel comprehension of the role of MnP on the fate of antibiotics in nature and may develop a potential technology for tetracycline removal.     

Biochemical and enzymatic properties of a novel marine fibrinolytic enzyme from <Emphasis Type="Italic">Urechis unicinctus</Emphasis>

A novel potent protease, Urechis unicinctus fibrinolytic enzyme (UFE), was first discovered by our laboratory. In this study, we further investigated the enzymatic properties and dynamic parameters of UFE. As a low molecular weight protein, UFE appeared to be very stable to heat and pH. When the temperature was <50°C, the remnant enzyme activity remained almost unchanged, but when the temperature was raised to 60#x00B0;C the remnant enzyme activity began to decrease rapidly. UFE was quite stable in a pH range of 3.0–12.0, especially at slightly alkaline pH values. Mn²⁺, Cu²⁺, and Fe²⁺ ions were activators of UFE, whereas Fe³⁺ and Ag⁺ ions were inhibitors. Fe²⁺ ion along with Fe³⁺ ion might regulate UFE activity in vivo. The optimum pH and temperature of UFE were about 8.0 and 50°C, respectively. When using casein as substrate and a substrate concentration <0.1% casein (w/v), the reaction velocity was increased with substrate concentration. Also when using casein as substrate, the determined K _m and V _max of UFE were 0.5298 mg/mL and 3.0845 mol of l-tyrosine equivalent, respectively. Our systematic research results are significant when UFE is applied for medical and industrial purposes.     

Single-Stage Extraction and Separation of Co2+ from Ni2+ Using Ionic Liquid of [C4H9NH3][Cyanex 272]

The purpose of this study was to optimize the extraction conditions for separating Co²⁺ from Ni²⁺ using N-butylamine phosphinate ionic liquid of [C₄H₉NH₃][Cyanex 272]. A Box–Behnken design of response surface methodology was used to analyze the effects of the initial pH, extraction time, and extraction temperature on the separation factor of Co²⁺ from sulfuric acid solution containing Ni²⁺. The concentrations of Co²⁺ and Ni²⁺ in an aqueous solution were determined using inductively coupled plasma-optical emission spectrometry. The optimized extraction conditions were as follows: an initial pH of 3.7, an extraction time of 55.8 min, and an extraction temperature of 330.4 K. The separation factor of Co²⁺ from Ni²⁺ under optimized extraction conditions was 66.1, which was very close to the predicted value of 67.2, and the error was 1.7%. The equation for single-stage extraction with high reliability can be used for optimizing the multi-stage extraction process of Co²⁺ from Ni²⁺. The stoichiometry of chemical reaction for ion-exchange extraction was also investigated using the slope method.     

Isomerization ofn-butane on the SO4/ZrO2 catalyst promoted by IV Period metals

The catalytic activity of superacidic systems based on SO₄/ZrO₂ and modified by IV Period metals in isomerization ofn-butane was studied. At low temperatures of the reaction, the introduction of Fe³⁺, Sc³⁺, Co²⁺, or Zn²⁺ ions (1%) increases the yield of isobutane by 1.5 times due to the activation ofn-butane on the sites created by the promoting ions. The addition of Cr³⁺, V⁴⁺, or Mn²⁺ (1%) decreases the catalytic activity because of a decrease in the catalyst acidity, most likely, due to the reduction of surface sulfur species. The influence of the nature of the support and surface additives of SiO₂, TiO₂, and ZrO₂ on the activity and selectivity of the catalytic system inn-butane isomerization was studied. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7 pp. 1276–1280, July, 1999     

Screening of laccase, manganese peroxidase, and versatile peroxidase activities of the genus Pleurotus in media with some raw plant materials as carbon sources

Species of the genus Pleurotus are among the most efficient natural species in lignin degradation belonging to the subclass of ligninolytic organisms that produce laccase (Lac), Mn-dependent peroxidase (MnP), versatile peroxidase (VP), and the H₂O₂-generating enzyme aryl-alcohol oxidase, but not lignin peroxidases. Production of Lac and oxidation of 2,6-dimethoxyphenol (DMP) in the presence and absence of Mn²⁺ were detected both in submerged fermentation (SF) of dry ground mandarine peels and in solid-state fermentation (SSF) of grapevine sawdust in all investigated Pleurotus species and strains. Evidence of cultivation methods having a distinct influence on the level of enzyme activities has been demonstrated. Most of the species and strains had higher Lac activity under SSF conditions than under SF conditions. DMP oxidation in the presence and absence of Mn²⁺ was detected in all investigated species and strains, but was lower under SF conditions than under SSF conditions for most of them. However, relative activities of DMP oxidation in the absence of Mn²⁺, as percentages of activity agasint DMP in the presence of Mn²⁺, were higher under conditions of SF than in SSF cultures in most of the investigated species and strains. The obtained results showed that strains of different origins have different efficiently ligninolytic systems and that conditions of SSF are more favorable for ligninolytic activity than those in SF owing to their similarity to natural conditions on wood substrates.     

Flowerlike hybrid horseradish peroxidase nanobiocatalyst for the polymerization of guaiacol

In this study, the catalytic activity and stability of flowerlike hybrid horseradish peroxidase (HRP) nanobiocatalyst (HRP-Cu ²⁺ ) obtained from Cu ²⁺ ions and HRP enzyme in the polymerization reaction of guaiacol were analyzed. We demonstrated that HRP-Cu ²⁺ and hydrogen peroxide (H ₂ O ₂ ) initiator showed significantly increased catalytic activity and stability on the polymerization of guaiacol compared to that of free HRP enzyme. Poly(guaiacol) was observed with quite high yields (88%) and molecular weights (38,000 g/mol) under pH 7.4 phosphate-buffered saline (PBS) conditions at 60 °C with 5 weight% of HRP-Cu ²⁺ loading. HRP-Cu ²⁺ also shows very high thermal stability and works even at 70 °C reaction temperature; free HRP enzyme denatures at that temperature. Furthermore, HRP-Cu ²⁺ provided considerable repeated use and showed some degree of catalytic activity, even after the fourth recycle, in the polymerization of guaiacol.     

Screening and Characteristics of Marine Bacillus velezensis Z-1 Protease and Its Application of Enzymatic Hydrolysis of Mussels to Prepare Antioxidant Active Substances

Bacillus velezensis is a type of microorganism that is beneficial to humans and animals. In this work, a protease-producing B. velezensis strain Z-1 was screened from sludge in the sea area near Qingdao (deposit number CGMCC No. 25059). The response surface methodology was used to analyze protease production, and the optimal temperature was 37.09 °C and pH 7.73 with the addition of 0.42% NaCl, resulting in maximum protease production of 17.64 U/mL. The optimum reaction temperature and pH of the protease of strain Z-1 were 60 °C and 9.0, respectively. The protease had good temperature and pH stability, and good stability in solvents such as methanol, ethanol and Tween 80. Ammonium, NH₄⁺,and Mn²⁺ significantly promoted enzyme activity, while Zn²⁺ significantly inhibited the enzyme activity. The protease produced by strain Z-1 was used for the enzymolysis of mussel meat. The mussel hydrolysate exhibited good antioxidant function, with a DPPH free radical removal rate of 75.3%, a hydroxyl free radical removal rate of 75.9%, and a superoxide anion removal rate of 84.4%. This study provides a reference for the application of B. velez protease and the diverse processing applications of mussel meat.     

Back cover: Mn2+ or Mn3+? Investigating transition metal dissolution of manganese species in lithium ion battery electrolytes by capillary electrophoresis

A new CE method with ultraviolet–visible detection was developed in this study to investigate manganese dissolution in lithium ion battery electrolytes. The aqueous running buffer based on diphosphate showed excellent stabilization of labile Mn³⁺, even under electrophoretic conditions. The method was optimized regarding the concentration of diphosphate and modifier to obtain suitable signals for quantification. Additionally, the finally obtained method was applied on carbonate-based electrolytes samples. Dissolution experiments of the cathode material LiNi_0.5Mn_1.5O₄ (lithium nickel manganese oxide [LNMO]) in aqueous diphosphate buffer at defined pH were performed to investigate the effect of a transition metal-ion-scavenger on the oxidation state of dissolved manganese. Quantification of both Mn species revealed the formation of mainly Mn³⁺, which can be attributed to a comproportionation reaction of dissolved and complexed Mn²⁺ with Mn⁴⁺ at the surface of the LNMO structure. It was also shown that the formation of Mn³⁺ increased with lower pH. In contrast, dissolution experiments of LNMO in carbonate-based electrolytes containing LIPF₆ showed only dissolution of Mn²⁺.     

Characterization of Cyclodextrin Glucanotransferase Produced by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Cyclodextrin glucanotransferase, produced by Bacillus megaterium, was characterized, and the biochemical properties of the purified enzyme were determined. The substrate specificity of the enzyme was tested with different α-1,4-glucans. Cyclodextrin glucanotransferase displayed maximum activity in the case of soluble starch, with a K _m value of 3.4 g/L. The optimal pH and temperature values for the cyclization reaction were 7.2 and 60 °C, respectively. The enzyme was stable at pH 6.0–10.5 and 30 °C. The enzyme activity was activated by Sr²⁺, Mg²⁺, Co²⁺, Mn²⁺, and Cu²⁺, and it was inhibited by Zn²⁺and Ag⁺. The molecular mass of cyclodextrin glucanotransferase was established to be 73,400 Da by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, 68,200 Da by gel chromatography, and 75,000 Da by mass spectrometry. The monomer form of the enzyme was confirmed by the analysis of the N-terminal amino acid sequence. Cyclodextrin glucanotransferase formed all three types of cyclodextrins, but the predominant product was β-cyclodextrin.     

基于TiO2的绿色多相催化剂催化CO2与环氧化合物偶联反应

金属离子掺杂纳米TiO2(M-TiO2,M=Zn2+,Cu2+,Co2+,Mn2+,Ni2+)在CO2与环氧化合物的偶联反应中表现出较高的催化活性.反应以四正丁基碘化铵(TBAI)为共催化剂,在无溶剂条件下进行.考察了反应温度、反应时间和CO2压力在Zn-TiO2/TBAI体系中对反应性能的影响.作为无毒的多相催化剂,Zn-TiO2可循环使用5次,其催化活性没有明显降低.     

Thermokinetic studies on the activation of bovine liver arginase by manganese ions

The activation of bovine liver arginase, which catalyzes the hydrolysis of l-arginine to l-ornithine and urea, by manganese ions was studied by thermokinetic methods at 37 °C in 40 mM sodium barbiturate-HCl buffer solution (pH 9.4). Full activation of arginase, by incubation with 0.1 mM Mn²⁺, resulted in increased of V_max, and a higher sensitivity of the enzyme to product and l-lysine inhibition, with no change in the K_m for arginine. Upon addition of 0.1 mM Mn²⁺ to the reaction, the inhibitory constants of product (K_P) and l-lysine (K_I) decreased from 1.18 to 0.70 mM and from 5.60 to 3.10 mM, respectively. We suggest that the exogenous manganese ions in reaction recovered the activity of arginase, which was lost in dissolving and dilution, without effecting on the mechanism of the reaction.     

Highly dispersed Mn2O3−Co3O4 nanostructures on carbon matrix as heterogeneous Fenton-like catalyst

This work reports the synthesis of various carbon (Vulcan XC-72 R) supported metal oxide nanostructures, such as Mn₂O₃, Co₃O₄ and Mn₂O₃−Co₃O₄ as heterogeneous Fenton-like catalysts for the degradation of organic dye pollutants, namely Rhodamine B (RB) and Congo Red (CR) in wastewater. The activity results showed that the bimetallic Mn₂O₃−Co₃O₄/C catalyst exhibits much higher activity than the monometallic Mn₂O₃/C and Co₃O₄/C catalysts for the degradation of both RB and CR pollutants, due to the synergistic properties induced by the Mn−Co and/or Mn (Co)−support interactions. The degradation efficiency of RB and CR was considerably increased with an increase of reaction temperature from 25 to 45°C. Importantly, the bimetallic Mn₂O₃−Co₃O₄/C catalyst could maintain its catalytic activity up to five successive cycles, revealing its catalytic durability for wastewater purification. The structure–activity correlations demonstrated a probable mechanism for the degradation of organic dye pollutants in wastewater, involving •OH radical as well as Mn²⁺/Mn³⁺ or Co²⁺/Co³⁺ redox couple of the Mn₂O₃−Co₃O₄/C catalyst.     

Production and properties of 3-cyanopyridine hydratase inRhodococcus equi SHB-121

A strain ofRhodococcus equi SHB-121 forming 3-cyanopyridine hydratase was screened from nitrile-polluted soil. The optimum conditions for the formation of 3-cyanopyridine hydratase by the strain SHB-121 have been studied. Under the optimum conditions, the specific activity of the enzyme reached 5.32 U/mg of dry cell, 95 times higher than that cultured in screening medium. In addition, the activity of coexistent amidase was very low. 3-Cyanopyridine hydratase was purified from methylacrylamide-induced cells ofRh. equi SHB-121 by procedures including ultrasonic oscillation, ammonium sulfate precipitation, and column chromatographies on DEAE-cellulose DE52, hydroxyapatite, and Sephadex G-25. The overall purification was 31-fold. The molecular weight of the enzyme was about 30 kDA by SDS-PAGE. The pI value was 4.1. The transition temperature and pH were 7.0°C and 6.0, respectively, resulting from the differential spectra. The optimum pH and temperature for the enzyme reaction were 8.0 and 30°C. The enzyme activity was strongly inhibited by Ag⁺, Hg²⁺, Cu²⁺, and NH₄ ⁺, whereas it was enhanced by Fe³⁺ slightly. The enzyme catalyzed the hydration of 3-cyanopyridine to nicotinamide, and itsKm value was 0.1 mol/L. Uncompetitive inhibitor sodium cyanide has a K, value of 5 mmol/L.     

小麦秸秆发酵产生的真菌Pleurotus ostreatus IBL-02锰过氧化物酶的溶胶-凝胶固定化及其催化污水脱色(英文)

Solid state bio-processing of wheat straw was carried out through an indigenous fungal strain Pleurotus ostreatus IBL-02 under pre-optimized fermentation conditions. The maximum activity, 692±12 U/mL, of the industrially important manganese peroxidase (MnP) enzyme was recorded after five days of still culture incubation. The crude MnP was 2.1-fold purified with a specific activity of 860 U/mg after purification on a Sephadex-G-100 gel column. On native and SDS-PAGE electrophoresis gels, the purified MnP fraction was a single homogenous band of 45 kDa. An active fraction of MnP was immobilized using hydrophobic sol-gel entrapment comprising tetramethoxysilane (T) and propyltrimethoxysilane (P) at different T:P molar ratios. Characterization revealed that after 24 h incubation at varying pH and temperatures, the MnP fraction immobilized at a T:P ratio of 1:2 in the sol-gel retained 82% and 75% of its original activity at pH4 and 70 ℃, respectively. The optimally active fraction at a 1:2 T:P ratio was tested against MnSO4 as a substrate to determine the kinetic catalytic constants KM and Vmax . To explore the industrial applicability of P. ostreatus IBL-02 MnP, both the free and immobilized MnP were used for the decolorization of four different textile industrial effluents. A maximum of 100% decolorization was achieved for the different textile effluents within the shortest time period. A lower KM , higher Vmax , hyper-activation, and enhanced acidic and thermal resistance up to 70 ℃ were the novel catalytic features of the sol-gel immobilized MnP, suggesting that it may be a potential candidate for biotechnological applications particularly for textile bioremediation purposes.     

Study of urease inhibitory activity by medicinal plants extract based on new catalyst for Berthelot reaction and Taguchi experimental design

The Berthelot reaction is a well-established colorimetric method for determination of ammonia. In this work, the effects of different bivalent ions (Ba²⁺, Cd²⁺, Co²⁺, Cu²⁺, Fe²⁺, Hg²⁺, Mg²⁺, Mn²⁺, Ni²⁺, and Pb²⁺) were studied as catalyst on the Berthelot reaction efficiency. CuCl₂ was generally found as the best catalyst that provides a rapid and stable blue indophenol color. The Taguchi experimental design methodology has been applied to find optimum conditions. Four factors including temperature, pH, reaction time, and CuCl₂ concentration at five levels were considered to achieve optimum conditions. Blue indophenol color stability for 40 min, and linearity response up to 20 mM of ammonium sulfate were achieved by further validation experiments. Limit of detection and quantification for this approach was 0.15 and 0.5 mM, respectively. Inhibitory activity of three traditional medicinal plants extract (Citrus aurantifolia, Laurus nobilis, and Zingiber officinale) was evaluated against jack bean urease activity by Berthelot reaction in the presence of CuCl₂ as catalyst, and results were compared with traditional Berthelot reaction.     

Purification and Characterization of Cell Suspensions Peroxidase from Cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis> L.)

Two peroxidases, cPOD-I and rPOD-II, have been isolated and purified from cotton cell suspension and their biochemical characteristics studied. rPOD-II from R405-2000, a non-embryogenic cultivar, has higher activity than cPOD-I derived from Coker 312, which developed an embryogenic structure. The cPOD-I and rPOD-II had molecular mass of 39.1 and 64 kDa respectively, as determined by SDS-PAGE. Both enzymes showed high efficiency of interaction with the guaiacol at 25 mM. The optimal pH for cPOD-I and rPOD-II activity was 5.0 and 6.0, respectively. The enzyme had an optimum temperature of 25 °C and was relatively stable at 20–30 °C. The isoenzymes were highly inhibited by ascorbic acid, dithiothreitol, sodium metabisulfite, and β-mercaptoethanol. Their activities were highly enhanced by Al³⁺, Fe³⁺, Ca²⁺, and Ni²⁺, but they were moderately inhibited by Mn²⁺ and K⁺. The enzyme lost 50% to 62% of its activity in the presence of Zn²⁺ and Hg²⁺.     

Properties and Structure of Spinel Li‐Mn‐O‐F Compounds for Cathode Materials of Secondary Lithium‐ion Battery

The spinel Li‐Mn‐O‐F compound cathode materials were synthesized by solid‐state reaction from calculated amounts LiOH‐H₂O, MnO₂(EMD) and LiF. The results of the electrochemical test demonstrated that these materials exhibited excellent electrochemical properties. It's initial capacity is ‐ 115 mAh.g¹ and reversible efficiency is about 100%. After 60 cycles, its capacity is still around 110 mAh.g¹ with nearly 100% reversible efficiency. The spinel Li‐Mn‐O‐F compound possibly has two structure models: interstitial model [Li]‐[Mn³⁺_xMn⁴⁺_2‐x]O₄F_δ, in which the fluorine is located on the interstice of crystal lattice, and substituted model [Li]‐[Mn³⁺_xMn⁴⁺_2‐x]O_4‐δF_δ, which the fluorine atom substituted the oxygen atom. The electrochemical result supports the interstitial model [Li][Mn³⁺_xMn⁴⁺_2‐x]O₄F_δ.     

Investigation of metal activation of a partially purified polyphenol oxidase enzyme electrode

Biosensors can be developed using different biological materials and immobilization technologies. Enzymes are generally used in biosensor construction, and some enzymes need metal ions or small organic molecules as a cofactor for their activation. Polyphenol oxidases can be activated by several metal ions such as Cu²⁺, Mg²⁺, Zn²⁺, Mn²⁺, and Ni²⁺. In this study, a new measurement method has been developed that is based on the metal ion activation of the polyphenol oxidase enzyme used in the biosensor preparation, especially to determine the concentration of Mg²⁺ ions. Polyphenol oxidase (PPO) (EC 1.10.3.1) was partially purified from potato (Solanum tuberosum) by using (NH₄)₂SO₄ precipitation, dialysis, and lyophylization processes. As a result of this processes, approximately 30-fold purification was achieved for PPO. For construction of the biosensor, the enzyme was immobilized on the dissolved oxygen probe membrane using gelatin and glutaraldehyde (2.5%). Using the biosensor, we obtained responses for catechol in the absence and presence of Mg²⁺ ions. Differences between the biosensor responses were related to the concentration of Mg²⁺ ions. The biosensor response depends linearly on concentration of Mg²⁺ ions between 0.05 and 7.5?mM. In the optimization studies, phosphate buffer (pH 7.0, 50?mM) and 35°C were determined to be the optimum conditions. This project will be a novel biosensor study and it might bring a new term, ‘activation based biosensor’ into the biosensor area.