Novel biocatalytic process of N-substituted acrylamide synthesis

The enzymatic synthesis of N-substituted acrylamides (N-isopropyl acrylamide and N, N-dimethylaminopropyl acrylamide) was demonstrated for the first time. The Rhodococcus erythropolis 37 strain, exhibiting acylamidase activity, was used as a source of enzyme, and water-dissolved acrylamide and isopropylamine/dimethylaminopropylamine served as substrates. The optimum conditions for the synthesis of acrylamide N-substitutes were determined using N-isopropyl acrylamide. The yield of the product was maximum at pH 9.5–10.5, substrate (acrylamide/isopropylamine) ratio within the range from 1.3: 1 to 2: 1, and absolute substrate concentrations of 8.0 (acrylamide) and 4.0% (isopropylamine). These conditions allowed for the synthesis of 22 g/L of N-isopropyl acrylamide.     

Purification and properties of asparagine synthetase from soybean root nodules

Asparagine synthetase was purified 240-fold from soybean (Glycine max (L.) Merr.) root nodules with a final recovery of 5% using Reactive Blue 2-crossed linked Agarose affinity gel chromatography. High levels of sulfhydryl protectants were required and the inclusion to glycerol and substrates in the extraction buffer helped to stabilize the enzyme. The final preparation had a specific activity of 3.77 mkat/kg protein when assayed at 30°C and was free of contaminating asparaginase activity. The enzyme had a broad pH maximum around pH 8.0 and apparent K_m values for the substrates aspartate, Mg · ATP, and glutamine were 1.24 mM, 0.076 mM and 0.16 mM, respectively. Ammonium ion could partially replace glutamine as the nitrogen donor. Initial velocity patterns yielded parallel inverse plots with all substrate pairs suggesting an overall ping-pong reaction mechanism. Product inhibition patterns provided evidence that glutamine was the first substrate to bind to the enzyme and asparagine was the last product released.     

Biosynthesis of 1,3-propanediol from recombinant E. coli by optimization process using pure and crude glycerol as a sole carbon source under two-phase fermentation system

The environmental and nutritional condition for 1,3-propanediol (1,3-PD) production by the novel recombinant E. coli BP41Y3 expressing fusion protein were first optimized using conventional approach. The optimum environmental conditions were: initial pH at 8.0, incubation at 37 °C without shaking and agitation. Among ten nutrient variables, fumarate, (NH₄)₂HPO₄ and peptone were selected to study on their interaction effect using the response surface methodology. The optimum medium contained modified Riesenberg medium (containing pure glycerol as a sole carbon source) supplemented with 63.65 mM fumarate, 3.80 g/L (NH₄)₂HPO₄ and 1.12 g/L peptone, giving the maximum 1,3-PD production of 2.43 g/L. This was 3.5-fold higher than the original medium (0.7 g/L). Two-phase cultivation system was conducted and the effect of pH control (at 6.5, 7.0 and 8.0) was investigated under anaerobic condition by comparing with the no pH control condition. The cultivation system without pH control (initial pH of 8.0) gave the maximum values of 1.65 g/L 1,3-PD, the 1,3-PD production rate of 0.13 g/L h and the yield of 0.31 mol 1,3-PD/mol crude glycerol. Hence, using crude glycerol as a sole carbon source resulted in 32 % lower 1,3-PD production from this recombinant strain that may be due to the presence of various impurities in the crude glycerol of biodiesel plant. In addition, succinic acid was found to be a major product during fermentation by giving the maximum concentration of 11.92 g/L after 24 h anaerobic cultivation.     

Purification of a hyperactive nitrile hydratase from resting cells of Rhodococcus rhodochrous PA-34

A propionitrile-induced nitrile hydratase (NHase), a promising biocatalyst for synthesis of organic amides has been purified from cell-free extract of Rhodococcus rhodochrous PA-34. About 11-fold purification of NHase was achieved with 52% yield. The SDS-PAGE of the purified enzyme revealed that it consisted of two subunits of 25.04 kD and 30.6 kD. However, the molecular weight of holoenzyme was speculated to be 86 kD by native-PAGE. This NHase exhibited maximum activity at pH 8.0 and temperature 40°C. Half-life was 2 h at 40°C and 0.5 h at 50°C. The Km and Vmax were 167 mM and 250 μmole/min/mg using 25 mM 3-cyanopyridine as substrate. AgNO₃, Pb(CH₃COO)₂ and HgCl₂ inhibited the NHase to extent of 89–100%.     

Development and other characteristics of α-methyl-d-glucoside transport by rat kidney cortex slices

α-Methyl-d-glucoside has been shown to be a non-metabolizable sugar which is accumulated against a concentration gradient by a Na⁺-dependent and phlorizin inhibited process by adult rat renal cortical slices incubatedin vitro at 37 °C. (2) The velocity of accumulation increased linearly with substrate concentrations up to 1.5 mM, but at higher concentrations obeyed saturable kinetics with an apparentK_m of about 6 mM. (3) Uptake was enhanced as Na⁺ was increased from 0 to 100 mequiv/l. Higher Na⁺ concentrations caused no further effect. (4) A pH maximum of transport occurred between 7.35 and 8.0. (5) Glucoside uptake was inhibited byd-glucose,d-galactose,d-fructose,d-mannose andd-ribose. The inhibition byd-glucose andd-galactose was competitive with apparentK_t of 24 and 53 mM, respectively. (6) Bothd-glucose andd-galactose accelerated the efflux of α-methyl-d-glucoside from preloaded cells. (7) Kidney cortex slices from 1-day-old rats were unable to accumulate α-methyl-d-glucoside to form a concentration gradient. The ability to concentrate the glucoside increased progressively after birth, reaching near normal in tissue from 15-day-old animals. The data indicate that the transport process in the newborn is rudimentary, failing also to display accelerated efflux phenomenon. (8) α-Methyl-d-glucoside is transported in rat kidney cortex by a mechanism similar in many ways to that ofd-galactose.     

Inversion of sucrose with β-d-fructofuranosidase (invertase) immobilized on bead DEAHP-cellulose: Batch process

The inversion of sucrose with β-d-fructofuranosidase (EC 3.2.1.26) immobilized by an ionic bond on bead cellulose containing weak basic N,N-diethylamino-2-hydroxypropyl groups has been investigated. The immobilized enzyme is strongly bound at an ionic strength up to 0.1 M in the pH range 3–6. The amount adsorbed is proportional to porosity and to the exchange capacity of the ion exchange cellulose, reaching values up to 200 mg/g dry carrier, with an activity in 10% sucrose solution at 30°C, pH 5, >8000 μmol min^?1 g^?1. The inversion of sucrose with immobilized β-d-fructofuranosidase was carried out in a stirred reactor. The dependence of activity on pH (3–7), temperature (0–70°C) and concentration of the substrate (2–64 wt%) were determined, and the inversion was compared with that obtained using non-immobilized enzyme under similar conditions. The rate of inversion at low substrate concentration (2–19 wt%) was described by Michaelis-Menten kinetics.     

Kinetic and Structural Properties of NADP-Malic Enzyme from Sugarcane Leaves

Oligomeric structure and kinetic properties of NADP-malic enzyme, purified from sugarcane (Saccharam officinarum L.) leaves, were determined at either pH 7.0 and 8.0. Size exclusion chromatography showed the existence of an equilibrium between the dimeric and the tetrameric forms. At pH 7.0 the enzyme was found preferentially as a 125 kilodalton homodimer, whereas the tetramer was the major form found at pH 8.0. Although free forms of l-malate, NADP⁺, and Mg²⁺ were determined as the true substrates and cofactors for the enzyme at the two conditions, the kinetic properties of the malic enzyme were quite different depending on pH. Higher affinity for l-malate (K_m = 58 micromolar), but also inhibition by high substrate (K_i = 4.95 millimolar) were observed at pH 7.0. l-Malate saturation isotherms at pH 8.0 followed hyperbolic kinetics (K_m = 120 micromolar). At both pH conditions, activity response to NADP⁺ exhibited Michaelis-Menten behavior with K_m values of 7.1 and 4.6 micromolar at pH 7.0 and 8.0, respectively. Negative cooperativity detected in the binding of Mg²⁺ suggested the presence of at least two Mg²⁺ - binding sites with different affinity. The K_a values for Mg²⁺ obtained at pH 7.0 (9 and 750 micromolar) were significantly higher than those calculated at pH 8.0 (1 and 84 micromolar). The results suggest that changes in pH and Mg²⁺ levels could be important for the physiological regulation of NADP-malic enzyme.     

Some glucosinolates of Farsetia aegyptia and Farsetia ramosissima

Air-dried leaves of Farsetia aegyptia and F. ramosissima have been analysed for their glucosinolates; the former was shown to contain at least six but chiefly allylglucosinolate, whilst the latter contains at least five but mainly but-3-enylglucosinolate with some 4-(methylthio)butylglucosinolate. Without the addition of extraneous thioglucosidase enzyme, both species gave predominantly nitrile degradation products of glucosinolates; but if extra enzyme were added, corresponding isothiocyanates became the major products instead. Varying the pH from the natural level for the plant also considerably affected the ratios of glucosinolate products.     

Optimization of extracellular xylanase production by Dictyoglomus sp. B1 in continuous culture

The thermophilic, xylanolytic, anaerobic organism, Dictyoglomus sp. B1, was cultivated in batch and continuous cultures in media containing insoluble beech-wood xylan. The extracellular xylanase activity levels obtained for the two cultivation methods were compared. Experiments were performed separately to determine the optimum substrate concentration, dilution rate, pH and temperature for xylanase production. Maximum xylanase activity was found at a substrate concentration of 1.5 g xylan/l, a dilution rate of 0.112 h^–1, pH 8.0 and at 7°C. Different combinations of these optimum values were used in a 2³ factorial experiment to investigate whether an increase in the xylanase production/activity could be achieved. A maximum xylanase activity of 2312 U/l was found when fermentors were operated at 73°C with a substrate concentration of 1.5 g xylan/l, pH 8.0, and a dilution rate of 0.112 h^–1. Thus, the optimum xylanase activity in the factorial experiment was obtained when the conditions that gave the maximum xylanase activities in the individual experiments were combined. Optimum xylanase activity obtained in the 2³ factorial experiment was 6.2 times higher than the activity found in the initial batch culture (373 U/l) and 3.0 times higher than the activity of a batch culture (783 U/l) grown at the same optimum conditions as the factorial experiment. The higher specific xylanase activity (217 U/mg protein) found in the 2³ factorial experiment was 4.1 times higher than the specific activity in the initial batch culture (53 U/mg protein).     

Production and purification of high levels of cellulase-free bacterial xylanase by Bacillus sp. SV-34S using agro-residue

Xylanase produced from the isolated bacterial strain Bacillus sp. SV-34S showed a 8.74-fold increase in enzyme activity under optimized submerged fermentation conditions. Cultivation using wheat bran as the carbon source and beef extract and (NH₄)H₂PO₄ as the nitrogen source resulted in productivity of 3,454.01 IU/mL xylanase. Xylanase was purified by 12.94-fold, with a recovery of 13.4 % and a specific activity of 3417.2 IU/mg protein, employing ammonium sulphate fractionation followed by cation-exchange chromatography using CM-Sephadex C-50 column chromatography, with a product of 27 kDa. The purified xylanase showed an optimum temperature and pH of 50 °C and 6.5, respectively although it was active even at pH 11.0. The thermostability study revealed that Bacillus sp. SV-34S was thermotolerant, being stable up to 50 °C; the residual activity at 55 and 60 °C was 96 and 93 %, respectively. The enzyme was stable between pH 6.0 and 8.0, although it retained >100 % activity at pH 8.0 and 9.0, respectively, following pre-incubation for 24 h. Xylanase activity was inhibited by various metal ions added to the assay mixture, with maximum inhibition observed in the presence of HgCl₂. The K_m and V_max values of the purified xylanase using birch wood xylan as substrate were 3.7 mg/mL and 133.33 IU/mL, respectively. The isolated bacterial strain produced high levels of extremophilic cellulase-free xylanase. The fact that it can be used in crude form and that it can be produced cheaply with renewable carbon sources make the process economically feasible. The characteristics of the purified enzyme suggest its potential application in industries such as the paper and pulp industry.     

Oxidation of trans- and cis-1,2-cyclohexanediol by Gluconobacter oxydans

Summary The enzymatic oxidation of 1,2-cyclohexanediol and related substrates by Gluconobacter oxydans (ATCC 621) was investigated. At low pH, membrane-bound enzymes were active and at high pH, NAD-dependent, soluble enzymes showed activity. Whole bacterial cells were used to catalyze some bioconversions. Racemic trans-1,2-cyclohexanediol was oxidized at pH 3.5 to give (R)-2-hydroxycyclohexanone (96% e.e.) and at pH 8.0 the same substrate was oxidized to (S)-2-hydroxycyclohexanone (97% e.e.). The latter conversion was severely inhibited by the reaction product while the former was not significantly product inhibited. (S)-2-hydroxycyclohexanone (97% e.e.) was also prepared from cis-1,2-cyclohexanediol by oxidation with G. oxydans cells at pH 3.5 in a reaction which continued to 100% conversion.     

Production and characterization of a solvent-tolerant protease from a novel marine isolate Bacillus tequilensis P15

Thirty-six proteolytic bacteria were isolated from the Jakhau coast, Kutch, India, amongst which isolate P15 identified as Bacillus tequilensis (JQ904626) was found to produce an extracellular solvent-- and detergent-tolerant protease (116.69?±?0.48 U/ml) and was selected for further investigation. Deoiled Jatropha seedcake (JSC) was found to be a suitable substrate for protease production under submerged condition. Upon optimization of process parameters following one-factor-at-a-time approach, an overall 6.4-fold (860.27?±?18.48 U/ml) increase in protease production was achieved. The maximum protease yield was obtained using a medium containing 2 % (w/v) deoiled JSC as substrate (pH of 8.0) upon 36 h of fermentation at 30 °C. The optimum temperature and pH for activity of B. tequilensis P15 protease was found to be 50 °C and 8.0, respectively. The enzyme exhibited a half-life of 190 min at 50 °C, which was enhanced to 270 min in presence of 5 mM Ca²⁺. The enzyme exhibited significant stability in almost all the solvents tested in the range of log P _ow varying from 8.8 to ?0.76. The enzyme activity was strongly inhibited by PMSF at 5 mM concentration, whereas the presence of EDTA (5 mM) and pCMB (5 mM) enhanced enzyme activity by 20.9 and 13.7 %, respectively. The enzyme was also found to be stable in the presence of surfactants, commercial detergents and bleach-oxidant (H₂O₂). This protease was demonstrated to be effective in removal of blood stains from fabrics, dehairing of hide, and stripping off the gelatin from used photographic films.     

Purification and characterization of an intracellular esterase from a Fusarium species capable of degrading dimethyl terephthalate

Esterase is the key enzyme involved in microbial degradation of phthalate esters (PAEs). In this study, an intracellular esterase was purified from a coastal sediment fungus Fusarium sp. DMT-5-3 capable of utilizing dimethyl terephthalate (DMT) as a substrate. The purified enzyme is a polymeric protein consisting of two identical subunits with a molecular mass of about 84 kDa. The enzyme showed a maximum esterase activity at 50 °C and was stable below 30 °C. The optimal pH was 8.0 and the enzyme was stable between pH 6.0 and 10.0. The esterase activity was inhibited by Cr³⁺, Hg²⁺, Cu²⁺, Zn²⁺, Ni²⁺, and Cd²⁺. Substrate specificity analysis showed that the enzyme was specific to DMT hydrolysis, but had no effect on other isomers of dimethyl phthalate esters (DMPEs) or monomethyl phthalate esters (MMPEs). These findings suggest that the phthalate esterase produced by Fusarium sp. DMT-5-3 is inducible and distinctive esterases involved in hydrolysis of the two carboxylic ester linkages of DMPEs.     

Combined effect of salinity and pH on lipid content and fatty acid composition of Tisochrysis lutea

The haptophyte microalga Tisochrysis lutea was heterotrophically grown in F2 medium with different combinations of pH and salinity. Growth, oil content and fatty acids (FAs) profile were determined under each set of conditions. The salinity was adjusted using NaCl at concentrations of 0.4, 0.6, 0.8, or 1.0 M, while pH was adjusted at 7, 8, or 9, and heterotrophic growth was performed using organic carbon in the form of sugar cane industry waste (CM). Fatty acid methyl esters (FAMEs) were identified by gas chromatography. The results showed that pH of 8.0 was the optimal for dry weight and oil production, regardless of the salinity level. At pH 8.0, growth at a salinity of 0.4 M NaCl was optimal for biomass accumulation (1.185 g L^-1). Under these conditions, the maximum growth rate was 0.055 g L^-1 d^-1, with a doubling time of 17.5 h and a degree of multiplication of 2.198. Oil content was maximal (34.87%) when the salinity was 0.4 M and the pH was 9.0. The ratio of saturated to unsaturated FAs was affected by the pH value and salinity, in that unsaturated FAs increased to 58.09% of the total FAs, considerably greater than the value of 40.59% obtained for the control (0.4 M NaCl and pH 8.0).     

Purification and Characterization of Phosphoenolpyruvate Carboxylase from Developing Seeds of Brassica

Phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31) was purified to apparent homogeneity with about 29% recovery from developing seeds of Brassica using ammonium sulfate fractionation, DEAE-cellulose chromatography, and gel filtration through Sepharose CL-6S. The purified enzyme with mol wt of about 400 kD exhibited maximum activity at pH 8.0. The enzyme had an absolute requirement for a divalent cation which was satisfied by Mg²⁺. The enzyme showed typical hyperbolic kinetics with PEP and HCO^?3 with Km of 0.125 and 0.104 mM, respectively. Glu-6-P could activate the enzyme, whereas other phosphate esters such as fru-1, 6-P₂, L-glycerophosphate and 3-PGA did not have any effect on the enzyme activity. Noneof the amino acids at 5 mM concentration had any significant effect on the enzyme activity. Nucleotide monophosphates and diphosphates did not inhibit the enzyme significantly, whereas ATP inhibited the enzyme activity. Oxaloacetate and malate inhibited the enzyme non-competitively with respect to PEP with Ki values of 0.127 and 1.25 mM, respectively. The enzyme activity in vivo seems to be regulated ’Tlainly by availability of its substrate and activation by glu-6-P, both of which are supplied through glycolysis.     

Oxidation-reduction potentials of respiratory chain components in ThiobacillusA2

(1) Cells of ThiobacillusA₂ grown chemoautotrophically on thiosulfate or heterotrophically on succinate with oxygen contained b-, c-, o-, a- and a₃-type cytochromes. The amount of cytochrome per mg of cell protein was much greater in thiosulfate-grown cells and differences in the relative concentrations of cytochromes were observed for the different growth conditions. (2) The half-reduction potentials at pH 7.0 (E_m,7.0) and spectral maxima of c-, b-, a- and a₃-type cytochromes were similar in cells grown aerobically with thiosulfate or with succinate as the growth substrate. (3) The half-reduction potential of the ‘invisible’, or high-potential copper, as determined from the potentiometric behavior of the carbon monoxide-reduced cytochrome a₃ complex at pH 8.0, was 365 mV. (4) Reducing equivalents from thiosulfate appear to enter the respiratory chain at the cytochrome c level; however, studies in cell-free extracts were limited due to a loss in respiratory activity with thiosulfate as a substrate upon cell disruption.     

Lysophosphatidate Acyltransferase in the Microsomes from Maturing Seeds of Meadowfoam (Limnanthes alba)

Lysophosphatidate (LPA) acyltransferase (EC 2.3. 1.51) in the microsomes from the maturing seeds of meadowfoam (Limnanthes alba), nasturtium (Tropaeolum majus), palm (Syagrus cocoides), castor bean (Ricinus communis), soybean (Glycine max), maize (Zea mays), and rapeseed (Brassica napus) were tested for their specificities toward 1-oleoyl-LPA or 1-erucoyl-LPA, and oleoyl coenzyme A (CoA) or erucoyl CoA. All the enzymes could use either of the two acyl acceptors and oleoyl CoA, but only the meadowfoam enzyme could use erucoyl CoA as the acyl donor to produce dierucoyl phosphatidic acid (PA). The meadowfoam enzyme was studied further. It had an optimal activity at pH 7 to 8, and its activity was inhibited by 1 millimolar MnCl₂, ZnCl₂, or p-chloromercuribenzoate. In a test of substrate specificity using increasing concentrations of either 1-oleoyl-LPA or 1-erucoyl-LPA, and either oleoyl CoA or erucoyl CoA, the enzyme activity in producing PA was highest for dioleoyl-PA, followed successively by 1-oleoyl-2-erucoyl-PA, dierucoyl-PA, and 1-erucoyl-2-oleoyl-PA. In a test of substrate selectivity using a fixed combined concentration, but varying proportions, of 1-oleoyl-LPA and 1-erucoyl-LPA, and of oleoyl CoA and erucoyl CoA, the enzyme showed a pattern of acyl preference similar to that observed in the test of substrate specificity, but the preference toward oleoyl moiety in the substrates was slightly stronger. The meadowfoam microsomes could convert [¹⁴C]glycerol-3-phosphate to diacylglycerols and triacylglycerols in the presence of erucoyl CoA. The meadowfoam LPA acyltransferase is unique in its ability to produce dierucoyl-PA, and should be a prime candidate for use in the production of trierucin oils in rapeseed via genetic engineering.     

A continuously monitored spectrophotometric assay of glycosidases with nitrophenyl glycosides

A general method for a continuously monitored spectrophotometric assay of glycosidases at all values of pH using p-nitrophenyl glycosides is presented. The method is demonstrated specifically by the development of a routine assay for α-galactosidase from fig and Mortierella vinacea using p-nitrophenyl galactopyranoside (NPG) at pH 3.9 and 5.8, respectively, and also for jack bean meal β-N-acetylhexosaminidase using p-nitrophenyl-β-2-acetamido-2-deoxy-d-glucopyranoside (NPADG) at pH 5.0. A number of different wavelengths may be used for the assay depending upon the criterion of the user; maximum sensitivity at a selected pH, determination of enzyme pH optima with a pH-independent difference extinction coefficient, or the reduction of background absorbance for kinetic studies at high substrate concentrations.     

Characterization of an Allylic/Benzyl Alcohol Dehydrogenase from Yokenella sp. Strain WZY002, an Organism Potentially Useful for the Synthesis of α,β-Unsaturated Alcohols from Allylic Aldehydes and Ketones

A novel whole-cell biocatalyst with high allylic alcohol-oxidizing activities was screened and identified as Yokenella sp. WZY002, which chemoselectively reduced the C=O bond of allylic aldehydes/ketones to the corresponding α,β-unsaturated alcohols at 30°C and pH 8.0. The strain also had the capacity of stereoselectively reducing aromatic ketones to (S)-enantioselective alcohols. The enzyme responsible for the predominant allylic/benzyl alcohol dehydrogenase activity was purified to homogeneity and designated YsADH (alcohol dehydrogenase from Yokenella sp.), which had a calculated subunit molecular mass of 36,411 Da. The gene encoding YsADH was subsequently expressed in Escherichia coli, and the purified recombinant YsADH protein was characterized. The enzyme strictly required NADP(H) as a coenzyme and was putatively zinc dependent. The optimal pH and temperature for crotonaldehyde reduction were pH 6.5 and 65°C, whereas those for crotyl alcohol oxidation were pH 8.0 and 55°C. The enzyme showed moderate thermostability, with a half-life of 6.2 h at 55°C. It was robust in the presence of organic solvents and retained 87.5% of the initial activity after 24 h of incubation with 20% (vol/vol) dimethyl sulfoxide. The enzyme preferentially catalyzed allylic/benzyl aldehydes as the substrate in the reduction of aldehydes/ketones and yielded the highest activity of 427 U mg⁻¹ for benzaldehyde reduction, while the alcohol oxidation reaction demonstrated the maximum activity of 79.9 U mg⁻¹ using crotyl alcohol as the substrate. Moreover, kinetic parameters of the enzyme showed lower K_m values and higher catalytic efficiency for crotonaldehyde/benzaldehyde and NADPH than for crotyl alcohol/benzyl alcohol and NADP⁺, suggesting the nature of being an aldehyde reductase.     

pH-induced quaternary assembly of Vitreoscilla hemoglobin: The monomer exhibits better peroxidase activity

pH-dependent (pH 6.0–8.0) quaternary structural changes of ferric Vitreoscilla hemoglobin (VHb) have been investigated using dynamic light scattering. The VHb exhibits a monomeric state under neutral conditions at pH 7.0, while the protein forms distinct homodimeric species at pH 6.0 and 8.0, respectively. The dissociation constant obtained using the Bio-Layer Interferometry technology indicates that, at pH 7.0, the monomer–monomer dissociation of VHb is about 6-fold or 5-fold higher (K_D = 6.34 μM) compared with that at slightly acidic pH (K_D = 1.05 μM) or slightly alkaline pH (K_D = 1.22 μM). The pH-dependent absorption spectra demonstrate that the heme microenvironment of VHb is sensitive to the changes of pH value. The maximum absorption band of heme group of VHb shifts from 402 nm to 407 nm when pH changes from 6.0 to 8.0. In addition, the fluorescence emission spectra of VHb, taken at excitation wavelength of 295 nm, suggest that the single Trp122 fluorescence quantum yields in VHb are decreased due to the formation of the homodimeric species. However, the circular dichroism spectra data display that the secondary structures of VHb are little affected by pH transitions. The pH-dependent peroxidase activity of VHb was also investigated in this study. The optimum pH for VHb using 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) as substrate is 7.0, which implies that the monomer state of VHb would exhibit better peroxidase activity than the homodimeric species of VHb at pH 6.0 and 8.0.