Production and Purification of Extracellular D-Xylose Isomerase from an Alkaliphilic, Thermophilic Bacillus sp.

An alkaliphilic, thermophilic Bacillus sp. (NCIM 59) produced extracellular xylose isomerase at pH 10 and 50°C by using xylose or wheat bran as the carbon source. The distribution of xylose isomerase as a function of growth in comparison with distributions of extra- and intracellular marker enzymes such as xylanase and β-galactosidase revealed that xylose isomerase was truly secreted as an extracellular enzyme and was not released because of sporulation or lysis. The enzyme was purified to homogeneity by ammonium sulfate precipitation followed by gel filtration, preparative polyacrylamide gel electrophoresis, and ion-exchange chromatography. The molecular weight of xylose isomerase was estimated to be 160,000 by gel filtration and 50,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating the presence of three subunits. The enzyme is most active at pH 8.0 and with incubation at 85°C for 20 min. Divalent metal ions Mg²⁺, Co²⁺, and Mn²⁺ were required for maximum activity of the enzyme. The K_m values for D-xylose and D-glucose at 80°C and pH 7.5 were 6.66 and 142 mM, respectively, while K_cat values were 2.3 × 10² s^-1 and 0.5 × 10² s^-1, respectively.     

Purification and characterization of thermostable xylose(glucose) isomerase from Bacillus thermoantarcticus

Xylose isomerase produced by Bacillus thermoantarcticus was purified 73-fold to homogeneity and its biochemical properties were determined. It was a homotetramer with a native molecular mass of 200 kDa and a subunit molecular mass of 47 kDa, with an isoelectric point at 4.8. The enzyme had a K _m of 33 mM for xylose and also accepted D-glucose as substrate. Arrhenius plots of the enzyme activity of xylose isomerase were linear up to a temperature of 85°C. Its optimum pH was around 7.0, and it had 80% of its maximum activity at pH 6.0. This enzyme required divalent cations for its activity and thermal stability. Mn²⁺, Co²⁺ or Mg²⁺ were of comparable efficiency for xylose isomerase reaction, while Mg²⁺ was necessary for glucose isomerase reaction. Journal of Industrial Microbiology & Biotechnology (2001) 27, 234–240. Received 18 March 2001/ Accepted in revised form 03 July 2001     

Production of high-fructose syrup by a heat-fixed Lactobacillus sp.

A Lactobacillus sp. isolated from soil and capable of growing on xylose-containing medium exhibited high glucose isomerase activity. The enzyme was thermostable, stable toward dialysis, and activated by heat treatment. It did not show the presence of xylose or ribose isomerase activities; the K_m for glucose and xylose substrates were 0.48M and 0.513M, respectively. The heat treatment of ultrasonic crude extract gave insoluble fixed active glucose isomerase enzyme. The properties of free and immobilized enzyme in heat-fixed whole cells differed in many respects. The optimum temperature for enzyme activity changed from 70 to 85°C, the optimum substrate concentration changed from 1.0M to 2.4M, and the optimum pH from 7.4 to 6.0. Co²⁺ and Mg²⁺ ions activated the enzyme when used singly, but in combination they inhibited the enzyme and Mn²⁺ had no effect on the enzyme. Free and immobilized enzymes, when used in the used in the conversions of corn and bagasse hydrolysates to fructose, gave 58, 25.6%, and 50, 27.6% conversions, respectively. Immobilized enzyme retained a significant activity for more than 30 hr and was able to operate at higher glucose concentrations showing less products inhibition effect as compared to free enzyme. In the batch process it was able to operate for about eight cycles.     

Immobilization of glucose isomerase to ion-exchange materials

Glucose isomerase (D -xylose ketol-isomerase EC 5.3.1.5) from Bacillus Coagulans was partially purified and immobilized by adsorption to anion exchangers. The highest activities were obtained when the enzyme was adsorbed to DEAE-cellulose. On immobilization to DEAE-cellulose the measured optimum pH value for enzyme activity shifted from 7.2 to 6.8. There was no appreciable difference between the heat stabilities of soluble and immobilized enzyme. The K_{m app} values for the immobilized enzyme were found to be 0.25M in the presence of 0.01M Mg²⁺ and 0.19M with 0.005M Mg²⁺, while those enzyme were 0.11 and 0.17M, re spectively. Under conditions of contimuous of D -glucose, a decrease of activity with time was observed, but this decrease was less at a low Mg²⁺ concentration and was affected by column geometry. There were no appreciable diffusional limitation effects in packed-bed columns.     

Production of Glucose Isomerase by Streptomyces flavogriseus

A microorganism that produces glucose isomerase was isolated from soil and identified as a strain of Streptomyces flavogriseus. The organism produced a large quantity of glucose isomerase when grown on straw hemicellulose, xylan, xylose, and H₂SO₄ hydrolysate of ryegrass straw. The organism produced glucose isomerase both intra- and extra-cellularly. The highest level of intracellular glucose isomerase (3.5 U/ml) was obtained in about 36 h by a culture grown on straw hemicellulose; the extracellular enzyme (1.5 U/ml) appeared in cultures grown for about 72 h. About equal levels of enzyme were produced in cultures grown on straw hemicellulose, xylan, xylose, and H₂SO₄ hydrolysate of straw, but production of the enzyme was drastically reduced when the organism was grown on other carbon sources. As a nitrogen source, corn steep liquor produced the best results. Soy flour extract, yeast extract, and various peptones also were adequate substrates for glucose isomerase production. Addition of Mg²⁺, Mn²⁺, or Fe²⁺ to the growth medium significantly enhanced enzyme production. The organism, however, did not require Co²⁺, which is commonly required by microorganisms used in the production of glucose isomerase.     

Cloning of <Emphasis Type="Italic">Escherichia coli</Emphasis> K12 xylose isomerase (glucose isomerase) gene and studying the enzymatic properties of its expression product

The coding region of Escherichia coli K12 xylose (glucose) isomerase gene was inserted into the pRAC expression vector and cloned in E. coli BL21(DE3) cells. After induction of expression of the cloned gene, the proportion of recombinant xylose isomerase accounted for 40% of the total protein content. As a result of one-stage purification by affinity chromatography, a protein preparation of 90% purity was obtained. The recombinant enzyme catalyzed the isomerization of glucose to fructose and exhibited maximum activity (0.8 U/mg) at 45°C and pH 6.8. The enzyme required Mg²⁺ ions as a cofactor. When Mg²⁺ and Co²⁺ ions were simultaneously present in the reaction medium, the enzyme activity increased by 15–20%. Complete replacement of Mg²⁺ with Co²⁺ decreased the enzyme activity. In the presence of Ca²⁺ at concentrations comparable to the concentration of Mg²⁺, the enzyme was not inhibited, although published data reported inhibition of similar enzymes by Ca²⁺. The recombinant enzyme exhibited a very low thermostability: it underwent a slow inactivation when incubated at 45°C and was completely inactivated after incubation at 65°C for 1 h.     

IMMOBILIZATION OF CARBONIC ANHYDRASE ENZYME PURIFIED FROM Bacillus subtilis VSG-4 AND ITS APPLICATION AS CO2 SEQUESTERER

The purification, immobilization, and characterization of carbonic anhydrase (CA) secreted by Bacillus subtilis VSG-4 isolated from tropical soil have been investigated in this work. Carbonic anhydrase was purified using ammonium sulfate precipitation, Sephadex-G-75 column chromatography, and DEAE-cellulose chromatography, achieving a 24.6-fold purification. The apparent molecular mass of purified CA obtained by SDS-PAGE was found to be 37 kD. The purified CA was entrapped within a chitosan–alginate polyelectrolyte complex (C-A PEC) hydrogel for potential use as an immobilized enzyme. The optimum pH and temperature for both free and immobilized enzymes were 8.2 and 37°C, respectively. The immobilized enzyme had a much higher storage stability than the free enzyme. Certain metal ions, namely, Co²⁺, Cu²⁺, and Fe³⁺, increased the enzyme activity, whereas CA activity was inhibited by Pb²⁺, Hg²⁺, ethylenediamine tetraacetic acid (EDTA), 5,5′-dithiobis-(2-nitrobenzoic acid (DTNB), and acetazolamide. Free and immobilized CAs were tested further for the targeted application of the carbonation reaction to convert CO₂ to CaCO₃. The maximum CO₂ sequestration potential was achieved with immobilized CA (480 mg CaCO₃/mg protein). These properties suggest that immobilized VSG-4 carbonic anhydrase has the potential to be used for biomimetic CO₂ sequestration.     

The addition of Co2+ enhances the catalytic efficiency and thermostability of recombinant glucose isomerase from Thermobifida fusca

Glucose isomerase is an important industrial enzyme that catalyzes the reversible isomerization of glucose to fructose. In this study, the effect of cobalt ions (Co²⁺) on the catalytic efficiency and thermostability of recombinant glucose isomerase from Thermobifida fusca was analyzed. The activity of glucose isomerase from engineered Escherichia coli supplemented with 1 mM Co²⁺ (C-GI) reached 41 U/ml, 2.1-fold higher than enzyme prepared from E. coli without additive (GI). The purified C-GI also exhibited an increased specific activity (23.8 U/mg compared to 12.1 U/mg for GI) and a greater thermostability (half-life of 17 h at 75 °C, 11.3-fold higher than GI (1.5 h)). The optimal temperature for C-GI shifted from 80 °C to 85 °C and demonstrated higher activity over pH 7.0–9.0. The k_cat/K_m value of C-GI (89.3 M^?1 s^?1) for the isomerization of glucose to fructose was nearly 1.75-fold higher than that of GI. In addition, the engineered cells were immobilized with the method of flocculation-cross linking. The immobilized cells supplemented with 1 mM Co²⁺ (C-IGI) had a better operational performance than cells without additives (IGI); at the end of 6 cycles, the conversion rate of C-IGI was still 43.1%, meeting the conversion rate requirement.     

ISOLATION,PURIFICATION, AND CHARACTERIZATION OF THERMOPHILIC T80 ISOENZYME OF XYLOSE ISOMERASE FROM THE XEROPHYTE Cereus pterogonus

A thermostable isoenzyme (T₈₀) of xylose isomerase from the eukaryote xerophyte Cereus pterogonus was purified to homogeneity by precipitation with ammonium sulfate and column chromatography on Dowex-1 ion exchange, with Sephadex G-100 gel filtration, resulting in an approximately 25.55-fold increase in specific activity and a final yield of approximately 17.9%. Certain physiochemical and kinetic properties (K_m and V_max) of the T₈₀ xylose isomerase isoenzyme were investigated. The molecular mass of the purified T₈₀ isoenzyme was 68 kD determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Polyclonal antibodies against the purified T₈₀ isoenzyme recognized a single polypeptide band on Western blots. The activation energy required for the thermal denaturation of the isoenzyme was determined to be 61.84 KJ mol^?1. The use of differential scanning calorimetry established the melting temperature of the CPXI isoenzyme to be 80°C, but when studied with added metal ions, melting temperature increases to more than the normal. Fluorescence spectroscopy of T₈₀ isoenzymes yielded an emission peak with λ_em at 320 nm and 340 nm, respectively, confirming the presence of Trp residue in these proteins. Electron paramagnetic resonance (EPR) analysis at liquid nitrogen temperature established the presence of Mn²⁺ and Co²⁺ associated with each isoenzyme. These enzyme species exhibited different thermal and pH stabilities compared to their mesophilic counterparts and offered greater efficiency in functioning as a potential alternate catalytic converter of glucose in the production of high-fructose corn syrup (HFCS) for the sweetener industry and for ethanol production.     

Purification and Characterization of Xylose Isomerase of a Methanol Yeast,Candida boidinii,Which is Involved in Sorbitol Production from Glucose

d-Xylose (xylose) isomerase was extracted from xylose-grown cells of a methanol yeast, Candida boidinii (Kloeckera sp.) No. 2201. The enzyme was purified 70-fold, over the original cell- free extract, with a yield of 2.4% in a homogeneous state, as judged on sodium dodecyl sulfate- polyacrylamide gel electrophoresis and high performance liquid chromatography. The molecular weight of the enzyme was determined to be 130,000, the enzyme being composed of two subunits of 65,000. The optimum pH and temperature for activity were 4.5 and 37~45°C, respectively. The enzyme activity was markedly enhanced by Mn²⁺, Mg²⁺ and Co²⁺, and the enzyme isomerized aldopentoses and aldohexoses. The Km values for xylose and d-glucose were 5.6 × 10?¹m and 4.1 × 10?¹m, and the V_max values were 5.8 × 10² and 3.3 × 10² µmol/min/mg, respectively. NaHAsO₄ 7H₂O and NaCN strongly inhibited the activity, but HgCl₂, NaN₃, dithiothreitol, monoiodoacetate and polyols such as d-sorbitol, xylitol and d-mannitol did not inhibit the activity.     

A novel xylose isomerase from Neurospora crassa

Summary Highest production of xylose Isomerase by Neurospora crassa grown with different carbon sources was at 0.014 U mg^-1 with D-xylose. The enzyme exhibited maximum activity at pH 8.0 and 70°C and retained 100% activity at 45°C for 30 min at pH 8.0. It was activated by 8 mM Mg²⁺ whereas 2 mM Co²⁺ afforded protection against inactivation by heat. The K _m for xylose was 10 mM and 22 mM for xylose Isomerase and xylose reductase respectively at 28°C and pH 7.0. This is the first report on the presence of xylose isomerase in N. crassa and the existence of two different pathways for the utilization of D-xylose.     

Purification and characterization fo a phosvitin kinase from the thyroid gland

1. Two cyclic AMP independent protein kinases phosphorylating preferentially acidic substrates have been identified in soluble extract from human, rat and pig thyroid glands/ Both enzymes were retained on DEAE-cellulose. The first enzyme activity eluted between 60 and 100 mM phosphate (depending on the species), phosphorylated both casein and phosvitin and was retained on phosphocellulose; this enzyme likely corresponds to a casein kinase already described in many tissues. The second enzyme activity eluted from DEAE-cellulose at phosphate concentrations higher than 3000 mM, phosphorylated only phosvitin and was not retained on phophocellulose. These enzymes were neither stimulated by cyclic AMP, cyclic GMP and calcium, nor inhbiited by the inhibitor of the cyclic AMP dependent protein kinases. 2. The second enzyme activity was purified from pig thyroid gland by the association of affinity chromatography on insolubilized phosvitin and DEAE-cellulose chromatography. Its specific activity was increased by 8400. 3. The purified enzyme (phosvitin kinase) was analyzed for biochemical and enzymatic properties. Phosvitin kinase phosphorylated phosvitin with an apparent K_m of 100 μg/ml; casein, histone, protamine and bovine serum albumin were not phosphorylated. The enzyme utilized ATP as well as GTP as phosphate donor with an apparent K_m of 25 and 28 μM, respectively. It had an absolute requirement for Mg²⁺ with a maximal activity at 4 mM and exhibited an optimal activity at pH 7.0. The molecular weight of the native enzyme was 110 000 as determined by Sephacryl S300 gel filtration. The analysis by SDS-polyacrylamide gel electrophoresis revealed a major band with a molecualr weight of 35 000 suggesting a polymeric structure of the enzyme.     

Cloning, expression and characterization of xylose isomerase, XylA, from Caldanaerobacter subterraneus subsp. yonseiensis

The xylA gene, coding for xylose isomerase, from the extreme thermophile, Caldanaerobacter subterraneus subsp. yonseiensis was cloned, sequenced, and expressed in Escherichia coli. The nucleotide sequence of the xylA gene encoded a polypeptide of 438 residues with a calculated molecular weight of 50,170 Da. The purified XylA showed high sequence homology (92% identity) with that of Thermoanaerobacter thermohydrosulfuricus. The recombinant enzyme expressed in Escherichia coli was purified by heat treatment and gel chromatography. The purified enzyme was thermostable with optimal activity at 95°C. The enzyme required divalent cations including Zn²⁺ for its maximal activity and thermostability.     

Bacillus subtilis aminopeptidase: purification, characterization and some enzymatic properties.

The extracellular aminopeptidase from Bacillus subtilis was purified 300-fold by a simple procedure which gave a high recovery of enzyme. The native enzyme was shown to be a monomer of molecular weight 46,500 and to contain 1 g-atom of Zn²⁺ per mole of protein. Amino acid analyses demonstrated the protein to be rich in acidic residues and Lys, to possess about 3 residues of Met, and to be devoid of Cys. When activated with 5 mm Co(NO₃)₂ for 90 min the activity of the native enzyme was increased; the amount of activation depended on the identity of the substrate. Cobalt activation involved the reversible binding of 1 g-atom of Co²⁺ per mole of protein, without displacing the native Zn²⁺; K_Co was 1.25 mm. Zinc ions competed with Co²⁺ during activation, a process characterized by a _KZn of 28 μm. Ions other than Co²⁺ did not appreciably activate the enzyme.     

An inducible hydrolase from Mortierella isabellina (basidiomycetes). The deacylation of (+)-usnic acid

An inducible enzyme catalysing the hydrolysis of (+)-usnic acid to (+)-2-desacetylusnic acid and acetic acid has been purified 150-fold from the mycelium of Mortierella isabellina grown in the presence of (+)-usnic acid. Purification was achieved by treatment with protamine sulfate, (NH₄)₂SO₄ fractionation, negative adsorption on alumina Cγ gel and hydroxylapatite followed by chromatography on DEAE-cellulose and Sephadex G-200. The elution pattern from a Sephadex G-200 column indicated a MW of ca 7.6 × 10⁴ for the enzyme. The apparent K_m value for (+)-usnic acid at the pH optimum (pH 7) was 4.0 × 10^?5 M. The enzyme was specific for (+)-usnic acid and inactive towards (?)-usnic acid, (+)-isousnic acid or certain phloracetophenone derivatives. Its activity was enhanced in the presence of divalent metal ions such as Co²⁺, Ni²⁺, Mn²⁺, Mg²⁺ and Zn²⁺.     

Medium optimization for the production of glucose isomerase from thermophilic Streptomyces thermonitrificans

A thermophilic strain of Streptomyces thermonitrificans produced a high activity of intracellular glucose isomerase (12 U/ml) when grown in a medium containing 1% (w/v) xylose, supplemented with 2% (w/v) sorbitol as the second carbon source, at 50°C for 16 h. Addition of Mg²⁺ enhanced enzyme production but the organism could grow and produce the enzyme in the absence of Co²⁺.The authors are with the Division of Biochemical Sciences, National Chemical Laboratory, Pune 411 008, IndiaNCL Communication No. 5813     

Brain arylamidase. Purification and characterization of the soluble bovine enzyme

1. An enzyme acting on aminoacyl-β-naphthylamides has been isolated from the soluble fraction of bovine brain and purified 205-fold by means of ammonium sulphate fractionation, hydroxyapatite adsorption and DEAE-Sephadex column chromatography. 2. Arylamidase requires thiol groups for retention of its activity, is heat-labile and is susceptible to freezing. p-Chloromercuribenzoate and N-ethylmaleimide inactivate the enzyme rapidly. 3. Metal ions are not required for its activity, but stimulation by Mn²⁺ and Mg²⁺ and inactivation by Co²⁺ and Zn²⁺ are observed. 4. Optimum pH7·5 in phosphate buffer was exhibited for all substrates tested except l-leucyl-β-naphthylamide, for which optimum pH is 6·5. 5. K_m values for a number of substrates have been obtained and substrate inhibition at high concentrations was demonstrated. 6. The molecular weight is approx. 70000 as determined by Sephadex-gel filtration.     

Enzymatic detection of heavy metal ions in aqueous solution from vegetable wastes by immobilizing pumpkin (<Emphasis Type="Italic">Cucumis melo</Emphasis>) urease in calcium alginate beads

Enzyme urease is extracted from the discarded seeds of pumpkin. Urease was purified to apparent homogeneity (5.2 fold) by heat treatment at 48 ± 1°C and gel filtration through Sephadex G-200. Effect of model metal ions on the activity of the homogeneous enzyme preparation (sp. activity 353 U/mg protein, A₂₈₀/A₂₆₀ = 1.12) of soluble as well as immobilized enzyme was investigated. The soluble and immobilized urease has been used for the quantitative estimation of general water pollution with heavy metal ions like Hg²⁺, Cu²⁺, Cd²⁺, and Co²⁺. The measurements of the urease residual activity have been carried out in tris-acetate buffer after pre-incubation of model metal salt. The inhibition was found to be biphasic with an initial rapid loss of activity and remainder in slow phase of 10∼15 min. The immobilization was done in 3.5% alginate beads leading to 86% of entrapment. There was no leaching of the enzyme over a period of 15 days at 4°C. The beads were fairly stable up to 50°C and exhibited activity even at −10°C. The inhibition by these ions was non-competitive and irreversible, hence could not be restored by dialysis. Based on the values of inhibition constant Ki the heavy-metal ions were found to inhibit urease in the following order Hg²⁺ > Cu²⁺ > Cd²⁺ > Co²⁺.     

Influence of temperature,pH and metal ions on guaiacol oxidation of purified laccase from Leptographium qinlingensis

The bark beetle Dendroctonus armandi is able to kill living Pinus armandi and has caused serious damage to pine forest in Northern China. As the most important symbiotic fungus of D. armandi, Leptographium qinlingensis plays an important role in the invasion process of the bark beetle. The laccase secreted by it are involved in lignin degradation to provide utilizable nutrition for D. armandi, and catalyze some biochemical reactions, causing the damages of tree tissue. In present study, the extracellular laccase of L. qinlingensis was purified by using the ammonium sulfate precipitation and DEAE-cellulose (DE-52) column chromatography. Furthermore, the effects of temperature, pH value and metal ions on it were investigated and characterized. The purified enzyme exerted its optimal activity with guaiacol. The catalytic efficiencies K_m and V_max determined for substrate guaiacol were 15.4 μM and 372.9 IU mg^?1, respectively. The optimum pH and temperature for the purified enzyme was 4.4 and 45 °C, respectively, with the highest enzyme specific activity of 7,000 IU mg^?1. Moreover, the metal ions, Co²⁺, Mn²⁺, Ca²⁺, Mg²⁺, Fe²⁺ and Cd²⁺, especially Hg²⁺, showed significantly inhibition effects on its activity. To understand the characteristics of this laccase might provide an opportunity and theoretical basis to promote integrated pest management of D. armandi.     

Studies on Sugar-isomerizing Enzyme Purification,Crystallization and Some Properties of Glucose Isomerase from Streptomyces sp.

Glucose isomerase was purified by means of acetone fractionation, DEAE-cellulose column chromatography, DEAE-Sephadex column chromatography and crystallization. The purified enzyme appeared to be homogeneous on ultracentrifugation and electrophoresis. The sedimentation coefficient, s_20,w, the diffusion coefficient, D_20,w, and partial specific volume of the enzyme were 8.0S, 4 × 10^?7cm²/sec and 0.69 ml/g, respectively. The molecular weight of the enzyme was estimated to be 157,000 from the sedimentation and diffusion measurements. The crystalline glucose isomerase contained cobalt and magnesium ions. The properties of the enzyme were also studied.