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.     

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.     

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     

Application of a compatible xylose isomerase in simultaneous bioconversion of glucose and xylose to ethanol

Simultaneous isomerisation and fermentation (SIF) of xylose and simultaneous isomerisation and cofermentation (SICF) of glucose-xylose mixture was carried out by the yeastSaccharomyces cerevisiae in the presence of a compatible xylose isomerase. The enzyme converted xylose to xylulose andS. cerevisiae fermented xylulose, along with glucose, to ethanol at pH 5.0 and 30°C. This compatible xylose isomerase fromCandida boidinii, having an optimum pH and temperature range of 4.5–5.0 and 30–50°C respectively, was partially purified and immobilized on an inexpensive, inert and easily available support, hen egg shell. An immobilized xylose isomerase loading of 4.5 IU/(g initial xylose) was optimum for SIF of xylose as well as SICF of glucose-xylose mixture to ethanol byS. cerevisiae. The SICF of 30 g/L glucose and 70 g xylose/L gave an ethanol concentration of 22.3 g/L with yield of 0.36 g/(g sugar consumed) and xylose conversion efficiency of 42.8%.     

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.     

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.     

High level expression of a thermostable Bacillus xylose (glucose) isomerase in Escherichia coli

Summary A thermophilic Bacillus sp. producing xylose (glucose) isomerase has been isolated. Its xy/A gene when cloned in Escherichia coli and expressed gave 37.5 and 12.8 units/ mg protein respectively for xylose and glucose isomerase activities at 85°C. A single heat treatment of the crude extract purified the enzyme further yielding the highest ever recorded activities of 150 and 49.02 units /mg protein.     

Catalytic properties of the immobilized Talaromyces thermophilus β-xylosidase and its use for xylose and xylooligosaccharides production

The present study explores the efficiency of Talaromyces thermophilus β-xylosidase, in the production of xylose and xylooligosaccharides. The β-xylosidase was immobilized by different methods namely ionic binding, entrapment and covalent coupling and using various carriers. Chitosan, pre-treated with glutaraldehyde, was selected as the best support material for β-xylosidase immobilization; it gave the highest immobilization and activity yields (94%, 87%, respectively) of initial activity, and also provided the highest stability, retaining 94% of its initial activity even after being recycled 25 times. Shifts in the optimal temperature and pH were observed for the immobilized β-xylosidase when compared to the free enzyme. The maximal activity obtained for the immobilized enzyme was achieved at pH 8.0 and 53 °C, whereas that for the free enzyme was obtained at pH 7.0 and 50 °C. The immobilized enzyme was more thermostable than the free β-xylosidase. We observed an increase of the K_m values of the free enzyme from 2.37 to 3.42 mM at the immobilized state. Native and immobilized β-xylosidase were found to be stimulated by Ca²⁺, Mn²⁺ and Co²⁺ and to be inhibited by Zn²⁺, Cu²⁺, Hg²⁺, Fe²⁺, EDTA and SDS. Immobilized enzyme was found to catalyze the reverse hydrolysis reaction, forming xylooligosaccharides in the presence of a high concentration of xylose. In order to examine the synergistic action of xylanase and β-xylosidase of T. thermophilus, these two enzymes were co-immobilized on chitosan. A continuous hydrolysis of 3% Oat spelt xylan at 50 °C was performed and better hydrolysis yields and higher amount of xylose was obtained.     

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.     

Production of Thermostable Clostridium thermohydrosulfuricum Xylose Isomerase in Bacillus brevis

The xylose isomerase gene from the thermophile Clostridium thermohydrosulfuricum has been cloned into Bacillus brevis. Under control of the strong cell wall protein promoter, the gene was efficiently expressed during the early stationary phase of growth, when cell densities were high. The expressed gene product was a soluble cytoplasmic protein and made up more than 20% of the total cellular protein. A simple heat treatment at 85°C for 10 min gave a virtually pure enzyme. Final isomerase yields were about 0.5 g isomerase per liter culture. The purified isomerase has an optimum temperature at 85°C, and an optimum pH around 7. The isomerase is stable at 85°C for several hours, opening possibilities for new uses.     

Cloning,expression and characterization of xylose isomerase from the marine bacterium Fulvimarina pelagi in Escherichia coli

Production of a xylose isomerase (XI) with high tolerance to the inhibitors xylitol and calcium, and high activity at the low pH and temperature conditions characteristic of yeast fermentations, is desirable for a simultaneous isomerization/fermentation process for cellulosic ethanol production. A putative XI gene (xylA) from the marine bacterium Fulvimarina pelagi was identified by sequence analysis of the F. pelagi genome, and was PCR amplified, cloned, and expressed in Escherichia coli. The rXI was produced in shake flask and fed‐batch fermentations using glucose as the growth substrate. The optimum pH for rXI was approximately 7, although activity was evident at pH as low as 5.5. The purified rXI had a molecular weight in 160 kDA, a V_max of 0.142 U/mg purified rXI, and a K_M for xylose in the range of 1.75–4.17 mM/L at pH 6.5 and a temperature of 35°C. The estimated calcium and xylitol K_I values for rXI in cell‐free extracts were 2,500 mg/L and >50 mM, respectively. The low K_M of the F. pelagi xylose isomerase is consistent with the low nutrient conditions of the pelagic environment. These results indicate that Ca²⁺ and xylitol are not likely to be inhibitory in applications employing the rXI from F. pelagi to convert xylose to xylulose in fermentations of complex biomass hydrolysates. A higher V_max at low pH (<6) and temperature (30°C) would be preferable for use in biofuels production. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1230–1237, 2016     

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     

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 glucoamylase on DEAE-cellulose activated with chloride compounds

Partially purified glucoamylase (1,4-α-d-glucan glucohydrolase, EC 3.2.1.3) from Aspergillus niger NRRL 330 has been immobilized on DEAE-cellulose activated with cyanuric chloride in 0.2 m acetate buffer, pH 4.2. In the matrix-bound glucoamylase, enzyme yield was 20 mg g^?1 of support, corresponding to 40 200 units g^?1 of DEAE support. Binding of the enzyme narrows the pH optimum from 3.8–5.2 to 3.6. Thermal stability of the bound glucoamylase enzyme was decreased although it showed a higher temperature optimum (70°C) than the free form (55°C). The rate of reaction of glucoamylase was also changed after immobilization. V_max values for free and bound enzyme were 36.6 and 22.6 μmol d-glucose ml^?1 min^?1 and corresponding K_m values were 3.73 and 4.8 g l^?1 respectively. Free and immobilized enzyme when used in the saccharification process gave 84 and 56% conversion of starch to d-glucose, respectively. The bound enzyme was quite stable and in the batch process it was able to operate for about five cycles without any loss of activity.     

Xylose isomerase from polycentric fungus Orpinomyces: gene sequencing, cloning, and expression in Saccharomyces cerevisiae for bioconversion of xylose to ethanol

The cDNA sequence of the gene for xylose isomerase from the rumen fungus Orpinomyces was elucidated by rapid amplification of cDNA ends. The 1,314-nucleotide gene was cloned and expressed constitutively in Saccharomyces cerevisiae. The deduced polypeptide sequence encoded a protein of 437 amino acids which showed the highest similarity to the family II xylose isomerases. Further, characterization revealed that the recombinant enzyme was a homodimer with a subunit of molecular mass 49 kDa. Cell extract of the recombinant strain exhibited high specific xylose isomerase activity. The pH optimum of the enzyme was 7.5, while the low temperature optimum at 37°C was the property that differed significantly from the majority of the reported thermophilic xylose isomerases. In addition to the xylose isomerase gene, the overexpression of the S. cerevisiae endogenous xylulokinase gene and the Pichia stipitis SUT1 gene for sugar transporter in the recombinant yeast facilitated the efficient production of ethanol from xylose.     

Xylose metabolism in the anaerobic fungus<Emphasis Type="Italic"> Piromyces</Emphasis> sp. strain E2 follows the bacterial pathway

The anaerobic fungus Piromyces sp. strain E2 metabolizes xylose via xylose isomerase and d-xylulokinase as was shown by enzymatic and molecular analyses. This resembles the situation in bacteria. The clones encoding the two enzymes were obtained from a cDNA library. The xylose isomerase gene sequence is the first gene of this type reported for a fungus. Northern blot analysis revealed a correlation between mRNA and enzyme activity levels on different growth substrates. Furthermore, the molecular mass calculated from the gene sequence was confirmed by gel permeation chromatography of crude extracts followed by activity measurements. Deduced amino acid sequences of both genes were used for phylogenetic analysis. The xylose isomerases can be divided into two distinct clusters. The Piromyces sp. strain E2 enzyme falls into the cluster comprising plant enzymes and enzymes from bacteria with a low G+C content in their DNA. The d-xylulokinase of Piromyces sp. strain E2 clusters with the bacterial d-xylulokinases. The xylose isomerase gene was expressed in the yeast Saccharomyces cerevisiae, resulting in a low activity (25±13 nmol min^–1mg protein^-1). These two fungal genes may be applicable to metabolic engineering of Saccharomyces cerevisiae for the alcoholic fermentation of hemicellulosic materials.     

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.     

The Expression of the Bacterial Gene for Xylose(Glucose) Isomerase in Transgenic Tobacco Plants Affects Plant Morphology and Phytohormonal Balance

The gene encoding xylose(glucose) isomerase (P00944, EC 5.3.1.5) in Escherichia coli was put under the control of the 35S CaMV promoter and transferred to Nicotiana tabacum L. plants using an Agrobacterium tumefaciens vector. Transgenic plants, which synthesized an active bacterial enzyme, were characterized by the accelerated development of the root system, more rapid accumulation of total plant weight, and larger leaves. These changes were correlated with a changed hormonal balance and a changed activity of the chloroplast-gene expression.     

Characterization of extracellular substrate specific glucose and xylose isomerases of Chainia

Summary Specific glucose and xylose isomerases have been identified in cell-free culture filtrates of a Chainia species. Treatment with DEAE-cellulose selectively adsorbed xylose isomerase activity while only the glucose isomerase was adsorbed on CM-cellulose. Glucose isomerase was completely inhibited by xylose at 1.3 × 10^-4 M concentration. The differential identity of the extracellular glucose and xylose isomerases, unique to Chainia, is discussed.(NCL Communication 3562)     

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.