Studies on glucose isomerase from a Streptomyces species.

Production and properties of glucose isomerase from a Co2+-sensitive Streptomyces species were studied. After 4 days of shaking cultivation at 30 degrees C and 200 rpm, a maximum of 1.1 enzyme units per ml of broth was obtained. Cell-free glucose isomerase, obtained from mycelia heat-treated in the presence of 0.5 mM Co2+, showed a 3.5-fold increase in specific activity over enzyme obtained from untreated mycelia. The optimum pH and temperature for the glucose isomerase were 7 to 8 and 80 degrees C, respectively. The Michaelis constant for fructose was 0.40 M. Mg2+ was found to enhance the glucose isomerase activity, whereas the effect of Co2+ on enzyme activity depended on the manner in which the enzyme was prepared. This glucose isomerase was quite heat stable, with a half-life of 120 h at 70 degrees C.     

海栖热袍菌葡萄糖异构酶基因xylA的克隆、表达及酶学性质

海栖热袍菌(Thermotoga maritima)是嗜极端高温的厌氧细菌,其产生的葡萄糖异构酶由于其出色的耐热性有着潜在的工业应用价值.由于海栖热袍菌苛刻的培养条件导致其葡萄糖异构酶产量较低.通过PCR方法克隆编码T. maritima MSB8葡萄糖异构酶基因xylA,构建重组质粒pHsh-xylA,转入Escherichia coli JM109,通过热激诱导表达.通过热处理和离子交换层析纯化两步得到电泳纯的酶制品,纯化倍数和回收率分别为8.02和49.02.对酶学性质研究表明,该重组酶为金属离子激活性酶,Mg2 ,Co2 对相对酶活有很强的激活作用,其最适pH为7.0,最适反应温度为95℃,且在pH 6～8之间有着较好的稳定性,在95℃下半衰期长达5 h以上.以葡萄糖为底物时的表观Km和Vmax分别为105 mmol/L和45.2 mol/min·mg.     

Purification and characterization of a highly thermostable glucose isomerase produced by the extremely thermophilic eubacterium, Thermotoga maritima

Thermotoga maritima, among the most thermophilic eubacteria currently known, produces glucose isomerase when grow in the presence of xylose. The purified enzyme is a homotetramer with submit molecular Wight of about 45,000. It has a number of features in common with previously described glucose isomerases-pH optimum of 6.5 to 7.5, presence of activesite histidine, requirement for metal cations such as Co(2+) and Mg(2+), and preference for xylose as substrate. In addition, it has significant sequence/structural homology with other glucose isomerases, as shown by both N-terminal sequencing and immunological crossreactivity. The T. maritima enzyme is distinguished by its extreme thermostability-a temperature optimum of 105 to 110 degrees C, and an estimated half-life of 10 minutes at 120 degrees C, pH 7.0. The high degree of thermostability, coupled with a neutral to slightly acid pH optimum, reveal this enzyme to be a promising candidate for improvement of the industrial glucose isomerization process (c) 1993 Wiley & Sons, Inc.     

Preparation and properties of glucose isomerase of Streptomyces kanamyceticus cells entrapped in calcium alginate

Summary The properties of glucose isomerase in native, heat-treated and immobilized cells of Streptomyces kanamyceticus after heat and mineral treatment have been compared. The optimum pH for glucose isomerase in native cells was shifted from 8.2 to 8.6 by heat treatment and immobilization. There is no change in the optimum temperature (90°C) for activity of the enzyme by the above treatment. Heat-treated cells and immobilized cells show greater pH and thermal stability of the enzyme. The Km values of the enzyme of native cells, heat-treated cells and immobilized heat-mineral-treated cells are 208 mM, 212 mM and 166 mM respectively; Mg⁺⁺ and Co⁺⁺ enhance the activity of isomerase in all cases.     

Preparation and Some Properties of Chitosan Bound Enzymes

An immobilization method using chitosan prepared from chitin as an insoluble carrier was investigated. Glucose isomerase, urease, glucamylase, trypsin and glucose oxidase were attached to chitosan by the aid of water soluble carbodiimide. Their activity yields were as follows; glucose isomerase 32%, urease 44%, glucamylase 8%, trypsin 10%, glucose oxidase 37%.

Immobilized glucose isomerase showed no significant changes in optimal temperature and heat stability. But pH optimum of reaction and pH stability range were somewhat lowered. The inhibitory effects of bivalent metal ions were considerably reduced by immobilization and similar tendency was observed for buffer reagents such as Tris or veronal. Immobilized glucose isomerase was inhibited by 8 m urea or 6 m guanidine hydrochloride in nearly the same way as free enzyme. With SDS, cysteine or mercaptoethanol free glucose isomerase was scarcely affected by these reagents, while immobilized enzyme considerably suffered to a loss of its activity.     

Expression, enzymatic characterization, and high-level production of glucose isomerase from Actinoplanes missouriensis CICIM B0118(A) in Escherichia coli

High-level production of recombinant glucose isomerase (rGI) is desirable for lactulose synthesis. In this study, the xylA gene encoding glucose isomerase from Actinoplanes missouriensis CICIM B0118(A) was cloned and expressed in E. coli BL21(DE3), and high-level production was performed by optimization of the medium composition. rGI was purified from a recombinant E. coli BL21(DE3) and characterized. The optimum pH value of the purified enzyme was 8.0 and it was relatively stable within the pH range of 7.0-9.0. Its optimum temperature was around 85 degrees C, and it exhibited good thermostability when the temperature was lower than 90 degrees C. The maximum enzyme activity required the presence of both Co2+ and Mg2+, at the concentrations of 200 microM and 8 mM, respectively. With high-level expression and the simple one-step chromatographic purification of the His-tagged recombinant enzyme, this GI could be used in industrial production of lactulose as a potential economic tool.     

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.     

Fructokinase (Fraction IV) of Pea Seeds

A fructokinase (EC 2.7.1.4) was obtained from pea (Pisum sativum L.) seeds. This enzyme, termed fructokinase (fraction IV), was specific for fructose as substrate and had little activity with glucose or mannose. Excess fructose inhibited the enzyme at the optimum pH (8.2) but not at pH 6.6. MgATP was inhibitory at pH 6.6. The apparent Michaelis-Menten constants at pH 8.2 were 0.057 mm for fructose and 0.10 mm for MgATP. Mg(2+) ions were essential for activity; Mn(2+) could partially replace Mg(2+). Fructokinase (fraction IV) had a requirement for K(+) ions which could be substantially replaced by Rb(+) or NH(4) (+) but not by Na(+). The enzyme was inhibited by MgADP. The possible significance of fructokinase (fraction IV) in plant carbohydrate metabolism is discussed.     

Characterization of a mutant glucose isomerase from <Emphasis Type="Italic">Thermoanaerobacterium saccharolyticum</Emphasis>

A series of site-directed mutant glucose isomerase at tryptophan 139 from Thermoanaerobacterium saccharolyticum strain B6A were purified to gel electrophoretic homogeneity, and the biochemical properties were determined. W139F mutation is the most efficient mutant derivative with a tenfold increase in its catalytic efficiency toward glucose compared with the native GI. With a maximal activity at 80 °C of 59.58 U/mg on glucose, this mutant derivative is the most active type ever reported. The enzyme activity was maximal at 90 °C and like other glucose isomerase, this mutant enzyme required Co²⁺ or Mg²⁺ for enzyme activity and thermal stability (stable for 20 h at 80 °C in the absence of substrate). Its optimum pH was around 7.0, and it had 86 % of its maximum activity at pH 6.0 incubated for 12 h at 60 °C. This enzyme was determined as thermostable and weak-acid stable. These findings indicated that the mutant GI W139F from T. saccharolyticum strain B6A is appropriate for use as a potential candidate for high-fructose corn syrup producing enzyme.     

Purification, Immobilization, and Some Properties of Glucose Isomerase from Streptomyces flavogriseus

Glucose isomerase (EC 5.3.1.5) produced from Streptomyces flavogriseus was purified by fractionation with (NH₄)₂SO₄ and chromatography on diethylaminoethyl (DEAE)-cellulose and DEAE-Sephadex A-50 columns. The purified enzyme was homogeneous as shown by ultracentrifugation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Benzyl DEAE-cellulose, triethylaminoethyl-cellulose, and DEAE-cellulose were effective in the immobilization of partially purified glucose isomerase. Several differences in properties were found between purified soluble enzyme, immobilized enzyme (DEAE-cellulose-glucose isomerase), and heat-treated whole cells. Glucose and xylose served as substrate for the enzyme. Whole cells had the highest K_m values for glucose and xylose; the soluble enzyme had the lowest values. The optimum temperature for activity of the soluble and immobilized enzymes was 70°C; that for whole cells was 75°C. The pH optimum for the three enzyme preparations was 7.5. Magnesium ion or Co²⁺ was required for enzyme activity; an addition effect resulted from the presence of both Mg²⁺ and Co²⁺. The enzyme activity was inhibited by Hg²⁺, Ag⁺, or Cu²⁺. The conversion ratio of the enzyme for isomerization was about 50%. The soluble and immobilized enzymes showed a greater heat stability than whole cells. The soluble enzyme was stable over a slightly wider pH (5.0 to 9.0) range than the immobilized enzyme and whole cells (pH 5.5 to 9.0). The molecular weight of the enzyme determined by the sedimentation equilibrium method was 171,000. A tetrameric structure for the enzyme was also indicated. After operating at 70°C for 5 days, the remaining enzyme activity of the immobilized enzyme and whole cells, which were used for the continuous isomerization of glucose in a plug-flow type of column in the presence of Mg²⁺ and Co²⁺, was 75 and 55%, respectively. Elimination of Co²⁺ decreased operational stability.     

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     

Purification and cloning of a thermostable xylose (glucose) isomerase with an acidic pH optimum from Thermoanaerobacterium strain JW/SL-YS 489.

An unusual xylose isomerase produced by Thermoanaerobacterium strain JW/SL-YS 489 was purified 28-fold to gel electrophoretic homogeneity, and the biochemical properties were determined. Its pH optimum distinguishes this enzyme from all other previously described xylose isomerases. The purified enzyme had maximal activity at pH 6.4 (60 degrees C) or pH 6.8 (80 degrees C) in a 30-min assay, an isoelectric point at 4.7, and an estimated native molecular mass of 200 kDa, with four identical subunits of 50 kDa. Like other xylose isomerases, this enzyme required Mn2+, Co2+, or Mg2+ for thermal stability (stable for 1 h at 82 degrees C in the absence of substrate) and isomerase activity, and it preferred xylose as a substrate. The gene encoding the xylose isomerase was cloned and expressed in Escherichia coli, and the complete nucleotide sequence was determined. Analysis of the sequence revealed an open reading frame of 1,317 bp that encoded a protein of 439 amino acid residues with a calculated molecular mass of 50 kDa. The biochemical properties of the cloned enzyme were the same as those of the native enzyme. Comparison of the deduced amino acid sequence with sequences of other xylose isomerases in the database showed that the enzyme had 98% homology with a xylose isomerase from a closely related bacterium, Thermoanaerobacterium saccharolyticum B6A-RI. In fact, only seven amino acid differences were detected between the two sequences, and the biochemical properties of the two enzymes, except for the pH optimum, are quite similar. Both enzymes had a temperature optimum at 80 degrees C, very similar isoelectric points (pH 4.7 for strain JW/SL-YS 489 and pH 4.8 for T. saccharolyticum B6A-RI), and slightly different thermostabilities (stable for 1 h at 80 and 85 degrees C, respectively). The obvious difference was the pH optimum (6.4 to 6.8 and 7.0 to 7.5, respectively). The fact that the pH optimum of the enzyme from strain JW/SL-YS 489 was the property that differed significantly from the T. saccharolyticum B6A-RI xylose isomerase suggested that one or more of the observed amino acid changes was responsible for this observed difference.     

Glucose isomerase activity of streptomyces kanamyceticus

Summary Streptomyces kanamyceticus produces a significant level of intracellular glucose isomerase when grown in submerged culture. The optimum temperature for enzyme activity is 90°C, but the optimum pH is changed by the kinds of buffer solution used. The activity is higher at pH 7.0–9.5. Treatment of cells with cetyl trimethyl ammonium bromide extracts almost the same amount of the enzyme as ultrasonic treatment. The selection of the method of treatment for enzyme extraction depends, however, on the nature of cell growth in synthetic or complex medium.     

树状黄杆菌变株U-616葡萄糖异构酶的研究

以树状黄杆菌（Ｆｌａｖｏｂａｃｔｅｒｉｕｍａｒａｂｏｒｅｓｃｅｎｓ）ＮＲＲＬ１１０２２为出发菌株,用紫外线对其进行诱变,经筛选得到一株葡萄糖异构酶的高产菌株Ｕ－６１６,其酶活力提高３１％。经保存三年和多次传代复测,其产酶能力保持稳定。其生长和产酶需较高的溶氧水平,最适产酶温度为３０℃,最适产酶ｐＨ为７．０－７．５,铁离子对其生长和产酶无明显的影响。所产葡萄糖异构酶的最适温度为６０－８０℃,最适ｐＨ为７．５－８．５,Ｃｏ２＋和Ｍｇ２＋对酶有激活作用,对金属离子耐受性较强,对Ｃａ２＋不敏感,热稳定性较好。树状黄杆菌变株Ｕ－６１６是一株产胞内葡萄糖异构酶的优良菌株。     

Properties of glucose isomerase from Streptomyces robeus S-606

Optimal conditions of the glucose isomerase fixation in a cell are determined by thermal treatment of Str. robeus S-606 biomass. Under these conditions the maximal enzyme activation (by 50-55 percent) is simultaneously observed. Basic properties of glucose isomerase fixed inside the cell are studied in comparison with the enzymic cell-free extract of this enzyme. The pH-optimum for preparations coincides and is observed at pH 7.5; the temperature optimum for the soluble enzyme is 70 degrees C, and for the intracellular enzyme it is higher by 5 degrees C. Thermostability of the intracellular enzyme is also higher than that of the soluble one. The Michaelis constants are calculated for the glucose isomerase preparations in a form of producer cells and enzymic extract: they equal to 0.375 M and 0.285 M, respectively. A comparison of properties permits considering intracellular glucose isomerase as an immobilized enzymic preparation.     

Purification and general properties of δ-aminolaevulate dehydratase from Nicotiana tabacum L

1. delta-Aminolaevulate dehydratase (EC 4.2.1.24) was purified 80-fold from tobacco leaves and its properties were studied. 2. The enzyme had optimum pH7.4 in potassium phosphate buffer, K(m)6.25x10(-4)m at 37 degrees and pH7.4, optimum temperature 45 degrees and an activation energy of 11100 cal./mole. 3. The enzyme lost activity when prepared in the absence of cysteine, and this activity was only partly restored by the later addition of thiols. Reagents for thiol groups inactivated the enzyme. 4. Mg(2+) was essential for activity, and EDTA and Fe(2+) were inhibitory; Mn(2+) was an activator or an inhibitor depending on the concentration.     

Glucose phosphate isomerase deficiency with hereditary hemolytic anemia in a Spanish family: clinical and familial studies.

A new case of glucose phosphate isomerase deficiency associated with cogenital nonspherocytic hemolytic anemia is described in a 12-year-old girl of Spanish origin. The parents exhibited erythrocyte glucose phosphate isomerase activity between 50 and 60% of normal. The enzyme of the propositus had normal Michaelis-Menten constants both for F-6-P and G-6-P, but abnormal pH optimum and decreased heat stability at 48 degrees C. On starch-gel electrophoresis the father's enzyme was normal but the mother's showed a cathodic migrating band in addition to the normal one. The enzyme from the propositus exhibited only one band with cathodal mobility of 116% of the main band found in normal subjects. It is postulated that the propositus is double heterozygous for two abnormal alleles, and the mother contributes a mutant allele with abnormal electrophoretic mobility and thermolability at 48 degrees C whereas the father contributes an allele without enzymatic activity.     

Production of saccharogenic and dextrinogenic amylases by Rhizomucor pusillus A 13.36

A newly-isolated thermophilic strain of the zygomycete fungus Rhizomucor pusillus 13.36 produced highly active dextrinogenic and saccharogenic enzymes. Cassava pulp was a good alternative substrate for amylase production. Dextrinogenic and saccharogenic amylases exhibited optimum activities at a pH of 4.0-4.5 and 5.0 respectively and at a temperature of 75 degrees C. The enzymes were highly thermostable, with no detectable loss of saccharogenic or dextrinogenic activity after 1 h and 6 h at 60 degrees C, respectively. The saccharogenic activity was inhibited by Ca(2+) while the dextrinogenic was indifferent to this ion. Both activities were inhibited by Fe(2+) and Cu(2+) Hydrolysis of soluble starch by the crude enzyme yielded 66% glucose, 19.5% maltose, 7.7% maltotriose and 6.6% oligosaccharides.     

Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose

AIMS: Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase used to increase the production rate of D-tagatose. METHODS AND RESULTS: A mutated gene was obtained by an error-prone polymerase chain reaction using L-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322-->Val, Ser393-->Thr, and Val408-->Ala), compared with the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8.0, 65 degrees C, and 1.0 mM Co2+, while the wild-type enzyme had a maximum activity at pH 8.0, 60 degrees C, and 1.0-mM Mn2+. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerization activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose. CONCLUSIONS: The mutated L-arabinose isomerase from G. stearothermophilus is valuable for the commercial production of D-tagatose. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes knowledge on the characterization of a mutated L-arabinose isomerase, and allows an increased production rate for D-tagatose from D-galactose using the mutated enzyme.     

Cloning and expression of the Clostridium thermosulfurogenes glucose isomerase gene in Escherichia coli and Bacillus subtilis.

The gene that encodes thermostable glucose isomerase in Clostridium thermosulfurogenes was cloned by complementation of glucose isomerase activity in a xylA mutant of Escherichia coli. A new assay method for thermostable glucose isomerase activity on agar plates, using a top agar mixture containing fructose, glucose oxidase, peroxidase, and benzidine, was developed. One positive clone, carrying plasmid pCGI38, was isolated from a cosmid library of C. thermosulfurogenes DNA. The plasmid was further subcloned into a Bacillus cloning vector, pTB523, to generate shuttle plasmid pMLG1, which is able to replicate in both E. coli and Bacillus subtilis. Expression of the thermostable glucose isomerase gene in both species was constitutive, whereas synthesis of the enzyme in C. thermosulfurogenes was inducible by D-xylose. B. subtilis and E. coli produced higher levels of thermostable glucose isomerase (1.54 and 0.46 U/mg of protein, respectively) than did C. thermosulfurogenes (0.29 U/mg of protein). The glucose isomerases synthesized in E. coli and B. subtilis were purified to homogeneity and displayed properties (subunit Mr, 50,000; tetrameric molecular structure; thermostability; metal ion requirement; and apparent temperature and pH optima) identical to those of the native enzyme purified from C. thermosulfurogenes. Simple heat treatment of crude extracts from E. coli and B. subtilis cells carrying the recombinant plasmid at 85 degrees C for 15 min generated 80% pure glucose isomerase. The maximum conversion yield of glucose (35%, wt/wt) to fructose with the thermostable glucose isomerase (10.8 U/g of dry substrate) was 52% at pH 7.0 and 70 degrees C.