Enhancing Endo-nitrilase production by a newly isolated Arthrobacter nitroguajacolicus ZJUTB06-99 through optimization of culture medium

The medium components of nitrilase production by Arthrobacter nitroguajacolicus ZJUTB06-99 were optimized in this study. Effects of factors such as carbon sources, nitrogen sources, and inducers on nitrilase production were investigated. Glucose, yeast extract, and ε-caprolactam were chosen as the suitable components. Moreover, experiments were carried out to fix the concentration of three factors for the zero coded level of variables in the subsequent optimization. Response surface methodology (RSM) and central composite design (CCD) were employed for further optimization. A quadratic model was found to fit the nitrilase activity and the variables. The results revealed that the optimized medium contained (%, w/v) 2.80, glucose; 0.57, yeast extract; and 0.42, ε-caprolactam. Validation experiments were carried out under the optimized conditions and nitrilase activity of 107.49 U/L was close to the predicted activity 110.82 U/L. After optimization, the nitrilase activity attained 2.86 fold of activity compared to the unoptimized conditions and the conversion of acrylonitrile was significantly improved. The strain growth curve and nitrilase activity alteration in the course of culture were tested. The cells were suitably harvested after cultured for 72∼78 h.     

Purification and characterization of a novel α-amylase from a newly isolated Bacillus methylotrophicus strain P11-2

An aerobic bacterial strain P11-2 with high amylolytic activity was isolated from soil sample collected from wheat field of Jiyuan, China. The strain was identified as Bacillus methylotrophicus by morphological and physiological characteristics as well as by analysis of the gene encoding the 16S rRNA. The α-amylase was purified to homogeneity by a combination of 80% (NH₄)₂SO₄ precipitation, DEAE FF anion exchange, and superdex 75 10/300 GL gel filtration chromatography. The purified α-amylase exhibited specific activity of 330.7 U/mg protein that corresponds to 13.1 fold purification. The relative molecular mass of the α-amylase was 44.0 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature for enzyme activity were 7.0 and 70 °C, respectively. The α-amylase activity was stimulated by Mg²⁺, Ba²⁺, Al³⁺ and dl-dithiothreitol (DTT), however, Ca²⁺ almost had no activation or inhibition on the α-amylase. After 4 h of reaction toward soluble starch, the end products were glucose, maltose and maltotriose. The 10 residues of the N-terminal sequence of the purified α-amylase were SVKNGQILHA, which showed no homology to other reported α-amylases from Bacillus strain.     

Characterization of a spore surface lipase from the biocontrol agent Metarhizium anisopliae

A Metarhizium anisopliae spore surface lipase (MASSL) strongly bound to the fungal spore surface has been purified by ion exchange chromatography on DEAE sepharose followed by ultrafiltration and hydrophobic interaction chromatography on phenyl sepharose. Electrophoretic analyses showed that the molecular weight of this lipase is ～66 kDa and pI is 5.6. Protein sequencing revealed that identified peptides in MASSL shared identity with several lipases or lipase-related sequences. The enzyme was able to hydrolyze triolein, the animal lipid cholesteryl stearate and all ρNP ester substrates tested with some preference for esters with a short acyl chain. The values of K_m and V_max for the substrates ρNP palmitate and ρNP laurate were respectively 0.474 mM and 1.093 mMol min^?1 mg^?1 and 0.712 mM and 5.696 mMol min^?1 mg^?1. The optimum temperature of the purified lipase was 30 °C and the enzyme was most stable within the most acid pH range (pH 3–6). Triton X-100 increased and SDS reduced enzyme lipolytic activity. MASSL activity was stimulated by Ca²⁺, Mg²⁺ and Co²⁺ and inhibited by Mn²⁺. The inhibitory effect on activity exerted by EDTA and EGTA was limited, while the lipase inhibitor Ebelactone B completely inhibited MASSL activity as well as PMSF. Methanol 0.5% apparently did not affect MASSL activity while β-mercaptoethanol activated the enzyme.     

Purification and characterization of a nitroreductase from the soil bacterium Streptomyces mirabilis

A NADH-dependent nitroreductase from an efficient nitro-reducing soil bacterium, Streptomyces mirabilis DUT001, was isolated and characterized. The enzyme was purified to near homogeneity using ammonium sulfate precipitation, ion exchange chromatography, and gel filtration chromatography. The native enzyme was estimated by gel filtration to have a molecular weight of 68 kDa, and its subunit molecular weight determined by SDS-PAGE was about 34 kDa, which indicated this enzyme was a dimer. Polycyclic nitroaromatic compounds were preferred substrates for this enzyme. The purified enzyme exhibited maximum activity at pH 7.5 and 40 °C. The addition of various chemicals such as reducing agents, metal ions, and chelating agents, had effects on enzyme activity. Mg²⁺, Ca²⁺, Sr²⁺, and 1% (w/v) Triton X-100 increased activity. However, Hg²⁺, Co²⁺, Ni²⁺, Cu²⁺, and SDS reduced activity. The maximum reaction rate (V_max) was 64 μM min^?1 mg^?1 enzyme and the apparent Michaelis–Menten constants (K_m) for 4-nitro-1,8-naphthalic anhydride and NADH were 276 and 29 μM, respectively. Menadione, bimethylenebis, sodium benzoate, and antimycin A were inhibitors of the purified nitroreductase with apparent inhibition constants (K_is) of 20, 36, 44 and 80 μM, respectively.     

Homologous expression,purification and characterization of a novel high-alkaline and thermal stable lipase from Burkholderia cepacia ATCC 25416

The lipase secreted by Burkholderia cepacia ATCC 25416 was particularly attractive in detergent and leather industry due to its specific characteristics of high alkaline and thermal stability. The lipase gene (lipA), lipase chaperone gene (lipB), and native promoter upstream of lipA were cloned. The lipA was composed of 1095 bp, corresponding to 364 amino acid residues. The lipB located immediately downstream of lipA was composed of 1035 bp, corresponding to 344 amino acid residues. The lipase operon was inserted into broad host vector pBBRMCS1 and electroporated into original strain. The homologous expression of recombinant strain showed a significant increase in the lipase activity. LipA was purified by three-step procedure of ammonium sulfate precipitation, phenyl-sepharose FF and DEAE-sepharose FF. SDS-PAGE showed the molecular mass of the lipase was 33 kDa. The enzyme optimal temperature and pH were 60 °C and 11.0, respectively. The enzyme was stable at 30–70 °C. After incubated in 70 °C for 1 h, enzyme remained 72% of its maximal activity. The enzyme exhibited a good stability at pH 9.0–11.5. The lipase preferentially hydrolyzed medium-chain fatty acid esters. The enzyme was strongly activated by Mg²⁺, Ca²⁺, Cu²⁺, Zn²⁺, Co²⁺, and apparently inhibited by PMSF, EDTA and also DTT with SDS. The enzyme was compatible with various ionic and non-ionic surfactants as well as oxidant H₂O₂. The enzyme had good stability in the low- and non-polar solvents.     

Arginase from Bacillus thuringiensis SK 20.001: Purification,characteristics, and implications for l-ornithine biosynthesis

An l-ornithine high producing strain Bacillus thuringiensis SK20.001 was screened by our laboratory. An intracellular arginase used to biosynthesize l-ornithine from the strain was purified and characterized. The final specific arginase activity was 589.2 units/mg, with 70.1 fold enrichment and 22.4% recovery. The molecular weight of the enzyme was approximately 33,000 Da as evaluated by SDS-PAGE and 191,000 Da as determined by gel filtration. The enzyme had an optimum pH of 10.0 and an optimum temperature of 40 °C. It was stable from pH 8.0–12.0 and <50 °C without Mn²⁺. The presence of Mn²⁺ and Ni²⁺ had strong effects on the enzyme activity, and Mn²⁺ significantly increased the thermal stability of the enzyme. The arginase was slightly inhibited by Ca²⁺, Fe²⁺ and Zn²⁺. Trp, Asp, Glu, Tyr, and Arg residues were directly involved in the arginase activity evaluated by chemical modifications. The K_m and V_max for l-arginine were estimated to be 15.6 mM and 538.9 μmol/min/mg. The biosynthesis yield of l-ornithine was 72.7 g/L with the enzyme.     

Purification and biochemical properties of an extracellular acid phytase produced by the Saccharomyces cerevisiae CY strain

An extracellular acid phytase was purified to homogeneity from the culture supernatant of the Saccharomyces cerevisiae CY strain by ultrafiltration, DEAE-Sepharose column chromatography, and Sephacryl S-300 gel filtration. The molecular weight of the purified enzyme was estimated to be 630 kDa by gel filtration. Removing the sugar chain by endoglycosidase H digestion revealed that the molecular mass of the protein decreased to 446 kDa by gel filtration and gave a band of 55 kDa by SDS-PAGE. The purified enzyme was most active at pH 3.6 and 40 °C and was fairly stable from pH 2.5 to 5.0. The phytase displayed broad substrate specificity and had a K_m value of 0.66 mM (sodium phytate, pH 3.6, 40 °C). The phytase activity was completely inhibited by Fe³⁺ and Hg²⁺, and strongly inhibited (maximum of 91%) by Ba²⁺, Co²⁺, Cu⁺, Cu²⁺, Fe²⁺, Mg²⁺, and Sn²⁺ at 5 mM concentrations.     

Characterization of a recombinant thermostable β-glucosidase from Putranjiva roxburghii expressed in Saccharomyces cerevisiae and its use for efficient biomass conversion

A β-glucosidase gene from Putranjiva roxburghii (PRGH1) was heterologously expressed in Saccharomyces cerevisiae to enable growth on cellobiose. The recombinant enzyme was secreted to the culture medium, purified and biochemically characterized. The enzyme is a glycoprotein with a molecular weight of ∼68 kDa and exhibited enzymatic activity with β‐linked aryl substrates like pNP-Fuc, pNP-Glc, pNP-Gal and pNP-Cel with catalytic efficiency in that order. Significant enzyme activity was observed for cellobiose, however the enzyme activity was decreased with increase in chain length of glycan substrates. Using cellobiose as substrate, the enzyme showed optimal activity at pH 5.0 and 65 °C. The enzyme was thermostable up to 75 °C for 60 min. The enzyme showed significant resistance towards both glucose and ethanol induced inhibition. The recombinant S. cerevisiae strain showed advantages in cell growth, glucose and bio-ethanol production over the native strain with cellobiose as sole carbon source. In simultaneous saccharification and fermentation (SSF) experiments, the recombinant strain was used for bio-ethanol production from two different cellulosic biomass sources. At the end of the SSF, we obtained 9.47 g L⁻¹ and 14.32 g L⁻¹ of bio-ethanol by using carboxymethyl cellulose and pre-treated rice straw respectively. This is first report where a β-glucosidase gene from plant origin has been expressed in S. cerevisiae and used in SSF.     

Optimization and immobilization of amylase obtained from halotolerant bacteria isolated from solar salterns

The objective of the present study was to isolate halotolerant bacteria from the sediment sample collected from Marakanam Solar Salterns, Tamil Nadu, India using NaCl supplemented media and screened for amylase production. Among the 22 isolates recovered, two strains that had immense potential were selected for amylase production and designated as P1 and P2. The phylogenetic analysis revealed that P1 and P2 have highest homology with Pontibacillus chungwhensis (99%) and Bacillus barbaricus (100%). Their amylase activity was optimized to obtain high yield under various temperature, pH and NaCl concentration. P1 and P2 strain showed respective, amylase activity maximum at 35 °C and 40 °C; pH 7.0 and 8.0; 1.5 M and 1.0 M NaCl concentration. Further under optimized conditions, the amylase activity of P1 strain (49.6 U mL^?1) was higher than P2 strain. Therefore, the amylase enzyme isolated from P. chungwhensis P1 was immobilized in sodium alginate beads. Compared to the free enzyme form (49.6 U mL^?1), the immobilized enzyme showed higher amylase activity as 90.3 U mL^?1. The enzyme was further purified partially and the molecular mass was determined as 40 kDa by SDS–PAGE. Thus, high activity of amylase even under increased NaCl concentration would render immense benefits in food processing industries.     

A novel fibrinolytic protease from Streptomyces sp. CS684

A fibrinolytic protease (FP84) was purified from Streptomyces sp. CS684, with the aim of isolating economically viable enzyme from a microbial source. SDS-PAGE and fibrin zymography of the purified enzyme showed a single protein band of approximately 35 kDa. Maximal activity was at 45 °C and pH 7–8, and the enzyme was stable between pH 6 and 9 and below 40 °C. It exhibited fibrinolytic activity, which is stronger than that of plasmin. FP84 hydrolyzed Bβ-chains of fibrinogen, but did not cleave Aα- and γ-chains. K_m, V_max and K_cat values for azocasein were 4.2 mg ml⁻¹, 305.8 μg min⁻¹ mg⁻¹ and 188.7 s⁻¹, respectively. The activity was suppressed by Co²⁺, Zn²⁺, Cu²⁺ and Fe²⁺, but slightly enhanced by Ca²⁺ and Mg⁺². Additionally, the activity was slightly inhibited by aprotinin and PMSF, but significantly inhibited by pefabloc, EDTA and EGTA. The first 15 amino acids of N-terminal sequence were GTQENPPSSGLDDID. They are highly similar to those of serine proteases from various Streptomyces strains, but different with known fibrinolytic enzymes. These results suggest that FP84 is a novel serine metalloprotease with potential application in thrombolytic therapy.     

Purification and characterization of SDG-β-d-glucosidase hydrolyzing secoisolariciresinol diglucoside to secoisolariciresinol from Aspergillus oryzae

The SDG-β-d-glucosidase that hydrolyzes the glucopyranoside bond of secoisolariciresinol diglucoside (SDG) to release secoisolariciresinol (SECO) was isolated from Aspergillus oryzae 39 strain and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 64.9 kDa. The optimum temperature of the SDG-β-d-glucosidase was 40 °C, and the optimum pH was 5.0. The SDG-β-d-glucosidase was stable at less than 65 °C, and pH 4.0–6.0. Ca²⁺, K⁺, Mg²⁺ and Na⁺ ions have no significant effect on enzyme activity, Zn²⁺ and Cu²⁺ ions have weakly effect on enzyme activity, but Fe³⁺ ion inhibits enzyme activity strongly. The K_m value of SDG-β-d-glucosidase was 0.14 mM for SDG.     

Purification and characterization of nitrilase from Fusarium solani IMI196840

Nitrilase activity in Fusarium solani IMI196840 (approx. 1500 U l⁻¹ of culture broth) was induced by 2-cyanopyridine. The enzyme was purified by a factor of 20.3 at a yield of 26.9%. According to gel filtration, the holoenzyme was an approx. 550-kDa homooligomer consisting of subunits with a molecular weight of approximately 40 kDa, as determined by SDS-PAGE. Mass spectrometry analysis of the tryptic fragments suggested a high similarity of this enzyme to the hypothetical CN hydrolases from Aspergillus oryzae, Gibberella zeae, Gibberella moniliformis and Nectria haematococca. Circular dichroism and fluorescence spectra indicated that secondary structure content and overall tertiary structure, respectively, were almost identical in nitrilases from F. solani IMI196840 and F. solani O1. The melting temperatures of the enzymes were 49.3 °C and 47.8 °C, respectively. The best substrates for the purified nitrilase from F. solani IMI196840 were benzonitrile and 4-cyanopyridine, which were hydrolyzed at the rates of 144 and 312 U mg⁻¹ protein, respectively, under the optimum conditions of pH 8 and 45 °C. The enzyme was highly chemoselective, producing ≤2% amides as by-products.     

Effects of galactomannan as carbon source on production of α-galactosidase by Thermomyces lanuginosus: Fermentation,purification and partial characterisation

Thermophilic fungus Thermomyces lanuginosus CBS 395.62/b strain is able to grow and synthesise extracellular α-galactosidase in media containing galactomannan such as locust bean gum (LBG) or guar gum (GG). Production of extracellular α-galactosidase was enhanced from 1.2 U/mL to 4–6 U/mL meaning about 3–5 times increase by optimisation of medium composition. This enzyme was purified to homogeneity by partial precipitation with 2-propanol and different liquid chromatographical steps. The developed purification protocol yielded 22% of enzyme activity with 900 purified fold. Molecular mass of the purified α-galactosidase enzyme was estimated to be 53 kDa. Maximal catalytic activity of the enzyme was obtained in the acidic pH range between pH 4.6 and 4.8 and in the temperature range 60–66 °C. More than 95% of enzyme activity was remaining after 1-day incubation at 70 °C and on pH in the range from 4.0 to 7.0. The enzyme activity was significantly stimulated by Mg²⁺, Mn²⁺ and K⁺ ions, while considerably inhibited by the presence of Ca²⁺, Ag⁺ and Hg²⁺.     

Extracellular cold-active lipase of Microbacterium luteolum isolated from Gangotri glacier,western Himalaya: Isolation,partial purification and characterization

A psychrophilic bacterium producing cold-active lipase upon growth at low temperature was isolated from the soil samples of Gangotri glacier and identified as Microbacterium luteolum. The bacterial strain produced maximum lipase at 15 °C, at a pH of 8.0. Beef extract served as the best organic nitrogen source and ammonium nitrate as inorganic for maximum lipase production. Castor oil served as an inducer and glucose served as an additional carbon source for production of cold-active lipase. Ferric chloride as additional mineral salt in the medium, highly influenced the lipase production with an activity of 8.01 U ml^?1. The cold-active lipase was purified to 35.64-fold by DEAE-cellulose column chromatography. It showed maximum activity at 5 °C and thermostability up to 35 °C. The purified lipase was stable between pH 5 and 9 and the optimal pH for enzymatic hydrolysis was 8.0. Lipase activity was stimulated in presence of all the solvents (5%) tested except with acetonitrile. Lipase activity was inhibited in presence of Mn²⁺, Cu²⁺, and Hg²⁺; whereas Fe⁺, Na⁺ did not have any inhibitory effect on the enzyme activity. The purified lipase was stable in the presence of SDS; however, EDTA and dithiothreitol inhibited enzyme activity. Presence of Ca²⁺ along with inhibitors stabilized lipase activity. The cold active lipase thus exhibiting activity and stability at a low temperature and alkaline pH appears to be practically useful in industrial applications especially in detergent formulations.     

Characterization of an extracellular thermophilic alkaline esterase produced by Bacillus subtilis DR8806

This work is a report of the characterization of an alkaline lipolytic enzyme isolated from Bacillus subtilis DR8806. The extracellular extract was concentrated using ammonium sulfate, and ultrafiltration. The active enzyme was purified by Q-sepharose ion exchange chromatography. The molecular mass of the enzyme was estimated to be 60.25 kDa based on SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). The optimum pH and temperature of this enzyme were observed to be 8.0 and 50 °C, respectively. The enzyme exhibited a half-life of 72 min at its optimum temperature. It was stable in the presence of metal ions (10 mM) such as Ca²⁺, K⁺ and Na⁺, whereas Cu²⁺, Fe²⁺, Zn²⁺, Mn²⁺, Co²⁺, Mg²⁺ and Hg²⁺ were found to have inhibitory effects. However, the enzyme activity was not affected significantly by 1% Triton X-100. The study of substrate specificity showed that the purified enzyme has a preferential specificity for small ester of p-nitrophenyl acetate (C₂), and it was the most efficiently hydrolyzed substrate as compared to the other esters. The kinetic parameters showed that the enzyme has K_m of 4.2 mM and V_max of 151 μmol min⁻¹ mg⁻¹ for p-nitrophenyl acetate. The hydrolysis rates of the fluorescence substrates were increased in the presence of the purified enzyme. Regarding the features of the enzyme, it may be utilized as a novel candidate for industrial applications.     

Purification and characterization of an extracellular cholesterol oxidase from a Bordetella species

A soil-isolated bacterium (strain B4) was identified as a species of Bordetella and deposited with the China General Microbiological Culture Collection (code, CGMCC 2229). The bacterium grew in a mineral medium, on cholesterol as a sole source of carbon and energy. Only one metabolite of cholesterol was accumulated in detectable amounts during the strain growth. It was identified as 4-cholesten-3-one. Cholesterol oxidase (COD) (EC 1.1.3.6), which catalyzes cholesterol into this metabolite, was evidenced from the strain. The conditions of the bacterium growth were optimized for extracellular enzyme production, which then reached around 1700 UL⁻¹ within 24 h culturing. The enzyme was purified from the spent medium of the strain to homogeneity on SDS-PAGE, and characterized. Its molecular mass, as estimated by this technique, was 55 kDa. COD showed an optimum activity at pH 7.0. It was completely stable at pH 5.0 and 4 °C for 48 h, and retained 80% at least of its initial activity at pH 4.0 or at a pH of 6.0–10.0. The optimum temperature for its reaction was 37 °C. The thermal stability of COD was appreciable, as 90% or 80% of its initial activity was recovered after 1 h or 2 h incubation at 50 °C. Ag⁺ or Hg⁺ at 1 mM, was inhibitor of COD activity, while Cu²⁺, at the same concentration, was activator. The COD K_m, determined at 37 °C and pH 7.0, was 0.556 mM. The enzyme was stable at pH 7.0 and 37 °C during 24 h mechanical shaking in the presence of 33% (v/v) of either of the solvents, dimethylsulfoxide, ethyl acetate, butanol, chloroform, benzene, xylene or cyclohexane.     

Production,characterization and application of a keratinase from Chryseobacterium L99 sp. nov.

Keratins are important bioresources for apparels and feedstuffs, but recalcitrant to common enzymes. Now, it is popular and essential to develop keratinolytic enzymes for environmental prevention and improvement of keratin product quality. In the study, the medium optimization, purification, characterization and application of the keratinase from a newly isolated Chryseobacterium L99 sp. nov. were conducted. Exogenous sucrose, malt sugar, glucose, starch, tryptone, Mg²⁺, Zn²⁺, Ca²⁺ and Cu²⁺ could promote the keratinase production, while exogenous urea, NH₄Cl and yeast extract exhibited strong inhibition effects. Response surface methodology predicted a maximum keratinase yield of 213.8 U mL⁻¹, at (g L⁻¹) sucrose 16.8, MgCl₂·6H₂O 1.9, feather keratin 40.0, NaH₂PO₄·2H₂O 6.0 and K₂HPO₄·6H₂O 1.0, where dry cell weight nearly had a minimum 8.58 g L⁻¹. Then, a serine keratinase about 33 kDa was purified, and its optimal activity was acquired at 40 °C and pH 8.0 with K⁺, Zn²⁺or Co²⁺. Compared with Savinase 16 L and transglutaminase, the L99 keratinase could efficient prevent shrinkage and eliminate directional frictional effect of wool, indicating it as a promising prospect in the biotreatment of wool fibres.     

Purification of a thermostable laccase from Leucaena leucocephala using a copper alginate entrapment approach and the application of the laccase in dye decolorization

Laccase from a tree legume, Leucaena leucocephala, was purified to homogeneity using a quick two-step procedure: alginate bead entrapment and celite adsorption chromatography. Laccase was purified 110.6-fold with an overall recovery of 51.0% and a specific activity of 58.5 units/mg. The purified laccase was found to be a heterodimer (∼220 kDa), containing two subunits of 100 and 120 kDa. The affinity of laccase was found to be highest for catechol and lowest for hydroquinone, however, highest K_cat and K_cat/K_m were obtained for hydroquinone. Purified laccase exhibited pH and temperature optima of 7.0 and 80 °C, respectively. Mn²⁺, Cd²⁺, Fe²⁺, Cu²⁺ and Na⁺ activated laccase while Ca²⁺ treatment increased laccase activity up to 3 mM, beyond which it inhibited laccase. Co²⁺, Hg²⁺, DTT, SDS and EDTA showed an inhibition of laccase activity. The Leucaena laccase was found to be fairly tolerant to organic solvents; upon exposure for 1 h individually to 50% (v/v) each of ethanol, DMF, DMSO and benzene, more than 50% of the activity was retained, while in the presence of 50% (v/v) each of methanol, isopropanol and chloroform, a 40% residual activity was observed. The purified laccase efficiently decolorized synthetic dyes such as indigocarmine and congo red in the absence of any redox mediator.     

Application of factorial and Doehlert designs for optimization of protopectinase production by a Geotrichum klebahnii strain

Two successive factorial designs followed by two response surface methodology were applied to optimize protopectinase production by Geotrichum klebahnii. Factorial designs were used to study the effect of 11 variables (mineral pool and pH) on enzyme production. Only pH and Fe²⁺ had significant effect on the protopectinase production in the conditions of the assay, the interaction between them not being significant. According to this result pH and Fe²⁺ were multivariate according to a Doelhert design. The central points were pH 3.5 and Fe²⁺ 1.8 μmol L⁻¹. The results show a negative effect of pH and a positive (quadratic) effect of Fe²⁺ on enzyme production. The second Doelhert design was centered at pH 3.3. A relative maximum was obtained at pH 2.8 and Fe²⁺ 0.540 μmol L⁻¹, where the enzyme activity obtained was 236 U/mL. This value is two times higher than the value reported elsewhere.     

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.