• Journal of Applied Biological Chemistry
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• v.44 no.3
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• pp.113-118
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• 2001

Xylose (glucose) isomerase was purified to homogeneity from cell-extracts of Streptomyces chibaensis J-59 via ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, and gel filtration on Sephacryl S-300. The purified enzyme is a homotetramer with a native molecular mass of 180 kDa and a subunit molecular mass of 44 kDa. The amino acid N-terminal sequence of glucose isomerase from S. chibaensis J-59 was determined to be Ser-Tyr-Gln-Pro-Thr-Pro-Glu-Asp-Arg-Phe-Thr-Phe-Gly-Leu. The first 14 amino acids of the N-terminal sequence of the enzyme showed strong analogies with N-terminal sequences of glucose isomerase produced by other Streptomyces spp. The optimum pH and temperature for activity were 7.5 and 85, respectively. The purified enzyme required $Mg^{2+}$, $Co^{2+}$, and $Mn^{2+}$ for the activity, $Mg^{2+}$ being the most effective. The enzyme was not inhibited by $Ca^{2+}$, but was inhibited by $Hg^{2+}$, $Ag^+$, and $Cu^{2+}$. The $K_m$, $V_{max}$, and $k_{cat}$ values of S. chibaensis J-59 isomerase for glucose were 83 mM, 40.9 U/mg, and $1,843min^{-1}$, respectively. In the presence of $Co^{2+}$, cell-free enzymes retained 100% without loss of activities by the heat-treatment at $70^{\circ}C$ for 7 days. The enzyme retained 50% residual activity after heating at $85^{\circ}C$ for 13.5 h, at $90^{\circ}C$ for 126 min. The enzyme is more thermostable than any other glucose isomerases of Streptomyces spp.

• Microbiology and Biotechnology Letters
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• v.24 no.4
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• pp.385-392
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• 1996

Bacillus stearotheymophilus, a potent xylanolytic bacterium isolated from soil, was tested for the strain's strategies of pentose utilization and the evidence of substrate preferences. The strain metabolized glucose, xylose, ribose, maltose, cellobiose, sucrose, arabinose and xylitol. The efficacy of the sugars as a carbon and energy source in this strain was of the order named above. The organism, however, could not grow on glycerol as a sole growth substrate. During cultivation on a mixture of glucose and xylose or arabinose, the major hydrolytic products of xylan, B. stearothermophilus displayed classical diauxic growth in which glucose was utilized during the first phase. On the other hand, the pentose utilization was prevented immediately upon addition of glucose. Cellobiose was preferred over xylose or arabinose. In contrast, maltose and pentose were co-utilized, and also no preference on between xylose and arabinose. Enzymatic studies indicated that B. stearothermophilus possessed constitutive hexokinase, a key enzyme of the glucose metabolic system. While, the production of $^{D}$-xylose isomerase, $^{D}$-xylulokinase and $^{D}$-arabinose isomerase essential for pentose phosphate pathway were induced by xylose, xylan, and xylitol but repressed by glucose. Taken together, the results suggested that the sequential utilization of B. stearothermophilus would be mediated by catabolite regulatory mechanisms such as catabolite inhibition or inducer exclusion.

• Korean Journal of Microbiology
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• v.21 no.2
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• pp.51-60
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• 1983

Cultural characteristics of Strptomyces griseolus isolated from the soil were investigated. This strain was disclosed to utilize D-xylose, and D-glactose in preference order as a carbon source with the formation of glucose isomerase. The addition of sweet potato starch also proved effective promoting the total enzyme activity measured at 29% higher than the control. Corn cob, one of waste agricultural resources, was hydrolyzed in 2~3% $H_2SO_4$ solution at $100^{\circ}C$, 3~5 hours to produce a xylose syrup which gave rise to the recovery of 19.9% in a batch system and 28.2% in a repeated system. By the addition of both 2% of xylose syrup(Be'28) prepared by and us 65% of corn steep liquor (total nitrogen 1.2%), enzyme induction was maximized. The enzyme activity was stimulated by the xylose and the cell growth by the C.S.L. Also, remarkable increase of enzyme activity was noticed by the addition of protein acid hydrolysate 86.2% higher than the control. $QO_2$ of the biomass cultured in 30L capacity jarfermentor recorded low oxygen requirement of 251.2 1/hr. Maximum activity of glucose isomerase was observed noted at the 9th hour after inoculation which is 2 hours faster than the stationery was observed noted at the 9th hour after inoculation which is 2 hours faster than the stationery phase of the biomass growth. Glucose isomerase from the strain was activated by adding the $Co^{++}\;and\;Mg^{++}$ with optimum temperature of $73^{\circ}C$ and pH of 7.2. Conversion ratio of 60% glucose to frutose was 42.5% after 70 hours reaction.

• Microbiology and Biotechnology Letters
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• v.4 no.4
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• pp.138-144
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• 1976

five strains of Streptomyces spp. with high Productivity of glucose isomerase (15-30 units/ml) were obtained among 280 microbial strains isolated from 150 soil samples. These strains produced glucose isomerase with xylose as an inducer. These 5 strains were also identified to be different strains of Streptomyces spp.：streptomyces sp. K-14, K-53, K-71, K-77 and K-733. It was found that Streptomyces sp. K-14 produced the highest enzyme activity. The spore chains of these strains were rectiflexible and spore surface was smooth except Steptomyces sp. K-77 and K-733, with spiny surface.

• Applied Biological Chemistry
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• v.39 no.6
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• pp.430-436
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• 1996

The induction by xylose and repression by glucose of xylose isomerase(XI) were investigated to elucidate the regulation for production of XI in Escherichia coli. Regulation for expression of xyIA gene which codes XI is under control of xylR which is a regulatory gene for xylose catabolism. When xyIR gene was resided in chromosome, the inductions of XI by the addition of 0.4% xylose were increased to 1.9 and 1.7-fold in case of locating on multicopy(pEX202/DH77) and low copy Plasmid(pEX102/DH77), respectively, as compared with that of xylA gene which was resided in chromosome(JM109). xyIR gene product derived from xyIR gene on chromosome might react to xylA gene on the plasmid as same as xylA gene on chromosome. In JM109 and xylA transformant; pEX202/DH77 and pEX102/DH77, the inductions of XI were completely repressed by the addition of 0.2% glucose and these catabolite repressions were derepressed by the addition of 1 mM cAMP In comparison with the addition of 0.4% xylose only for the induction XI was inductively produced 1.7 to 2-fold with the addition of xylose plus 1 mM cAMP in DM minimal media. pEX13/TP2010, xylA transformant of the deficient mutant($xyl^-,\;cya^-$; TP2010) of XI and cAMP production, did not induce XI by the addition of xylose only but induced in case of simultaneous addition of xylose and cAMP. These results show that cAMP and xylose are the indispensable effectors for the induction and derepression of Xl in E. coli.

• Applied Biological Chemistry
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• v.26 no.4
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• pp.222-230
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• 1983

This study was designed to develop a process for the immobilization of xylose isomerase(D-xylose ketol isomerase, EC 5.3.1.5) from Streptomyces griseolus previously isolated by the authors and its application on a pilot plant scale for the production of high fructose corn syrup. The biomass which has endo-excreted xylose isomerase was homogenized under a pressure of $500kg/cm^2$ and 90.8% of the enzyme recovery of the native activity was obtained as compared to 54.7% recovery by the lysozyme treatment. Ionic bonding method was adopted for the enzyme immobilization due to its many reported merits. It was found that the porous resins such as Diaion HP 20, Duolite A-7, Amberlite IRA 93 and 94 were effective in immobilizing the enzyme. In addition, it was disclosed that the regeneration form of $BO_4--$ is effective for Amberlite IRA 93 and $HCO_3-$ for Diaion HP 20. Optimal immobilization condition for Amberlite IRA 93 was pH 8.0 and $55^{\circ}C$ yielding 80.6% of immobilization. Activity decay test showed half life of the immobilized enzyme with Amberlite IRA 93 was more than 24 days at $65^{\circ}C$. The carrier was evaluated to be resuable and its result showed the relative immobilization yields were 98.2, 93.3, 90.7 and 87.5%, respectively at second, third, forth and fifth rebinding test of the enzyme on Amberlite IRA 93. Optimal temperature of the immobilized enzyme was slightly lowered and the range widened to $60\sim70^{\circ}C$, while optimal pH moved toward $8.0\sim8.3$ in its isomerization reaction. The trial production result of high fructose corn syrup in pilot scale immobilization showed that one liter of immobilized xylose isomerase (350 IXIU/ml-R) is capable producing about 293l high fructose corn syrup(75% dry substance) in 30 days.

• Journal of Microbiology and Biotechnology
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• v.29 no.2
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• pp.244-255
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• 2019

Xylose isomerase (XI; E.C. 5.3.1.5) catalyzes the isomerization of xylose to xylulose, which can be used to produce bioethanol through fermentation. Therefore, XI has recently gained attention as a key catalyst in the bioenergy industry. Here, we identified, purified, and characterized a XI (PbXI) from the psychrophilic soil microorganism, Paenibacillus sp. R4. Surprisingly, activity assay results showed that PbXI is not a cold-active enzyme, but displays optimal activity at $60^{\circ}C$. We solved the crystal structure of PbXI at $1.94-{\AA}$ resolution to investigate the origin of its thermostability. The PbXI structure shows a $({\beta}/{\alpha})_8$-barrel fold with tight tetrameric interactions and it has three divalent metal ions (CaI, CaII, and CaIII). Two metal ions (CaI and CaII) located in the active site are known to be involved in the enzymatic reaction. The third metal ion (CaIII), located near the ${\beta}4-{\alpha}6$ loop region, was newly identified and is thought to be important for the stability of PbXI. Compared with previously determined thermostable and mesophilic XI structures, the ${\beta}1-{\alpha}2$ loop structures near the substrate binding pocket of PbXI were remarkably different. Site-directed mutagenesis studies suggested that the flexible ${\beta}1-{\alpha}2$ loop region is essential for PbXI activity. Our findings provide valuable insights that can be applied in protein engineering to generate low-temperature purpose-specific XI enzymes.

• Korean Journal of Microbiology
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• v.23 no.2
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• pp.138-146
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• 1985

A Bacillus licheniformis ATCC31667 gene coding for a glucose isomerase has been cloned and expressed in glucose isomerase negative mutant of Escherichia coli. A recombinant plasmid, constructed by ligation of a EcoRI fragment of B.licheniformis chromosomal DNA to vector plasmid pBR322, was expressed glucose isomerase positive in E.coli LE392-6 with growth on minimal medium containing xylose as a sole carbon source. This recombinant plasmid, designated pBGI6, had the insery of 4.1Kb of Bacillus gene in EcoRI site, and restriction map of the plasmid was established. The plasmid pBG16 was very stable after 10days of serial transfer to a fresh medium. The activity of glucose isomerase from the transformed cell containing pBGI6 was increased about 20 fold than its wild type of host.

• Korean Journal of Microbiology
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• v.16 no.2
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• pp.47-61
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• 1978

Strptomyces albus T-12 which ahd been isolated and identified in the laboratory, was selected for the studies on the cultural conditions on the production of D-xylose iosmerase and the enzymological characteristics using the partially purified enzyme. The best results in the enzyme production came from D-xylose medium than wheat bran. The divalent metla ions as $Co^{2+},\;Fe^{2+},\;Zn^{2+}\;and\;Cu^{2+}$ retard or inhibit the cell-growth at the early stages of mycelia propagations, and T-12 strain is especially sensitive to $Co^{2+}$. After 60 hours of shaking cultivation at $30^{\circ}C$ and 200 rpm, a maximum enzyme activitz, 0.49 enzyme units, was obtained. Cell-free enzyme obtained from mycelia heat-treated in the prescence of 0.5mM $Co^{2+}$, showed a 2.4-fold increase in specific than the enzyme from untreated mycelia. The specific activity of the purified enzyme through Sephadex G-150 columm showed 180 fold to the crude enzyme. The effective activators of the enzyme appeared to be $Mg^{2+}\;and\;Co^{2+}$ ions, and it exhibited the maximal enzyme activity showed at pH 7.0 and at tempersture around $80^{\circ}C$ when $Mg^{2+}\;and\;Co^{2+}$ ions were added. The enzyme isomerized D-glucose, D-xylose, D-ribose, L-arabinose, D-mannose, and L-rhamnose in the present of $Mg^{2+}\;and\;Co^{2+}$ ions as an activatiors. $Mg^{2+}\;and\;Co^{2+}$ ions were non-competitively bound at different allosterix sites of enzyme molecule. $Mg^{2+}(5mM)\;or\;Co^{2+}(1.0mM)$ protected against the thermal denaturations of the enzyme activities. The michelis constant(Km) and $V_{max}$ values of the emzyme for D-glucose and D-xylose were 0.52M, $2.12{\mu}moles/ml{\cdot}min.\;and\;0.28M,\;0.65moles/ml{\cdot}min.$, respectively.

• Microbiology and Biotechnology Letters
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• v.7 no.1
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• pp.9-14
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• 1979

The hemicellulose fraction of ryegrass straw was extracted with NaOH and used for production of glucose isomerase by Streptomyces flavogriseus. Up to 25％ crude hemicellulose (20％ as pentosan) could he obtained by treating straw with 1 to 24％ NaOH. The yield of hemicellulose was increased proportionately with increasing NaOH concentration up to 4％, but the rate of increase was slowed thereafter. The optimum condition for hemicellulose extraction from ryegrass straw was to treat straw with 4％ NaOH for 3hrs at 9$0^{\circ}C$ or 24hrs at 3$0^{\circ}C$. Highest level of glucose isomerase activity (3.04 units/ml culture) was obtained when the organism was grown for 2 days at 3$0^{\circ}C$ on 2％ straw hemicellulose. The organism also produced a good quantity of glucose isomerase on xylan, xylose or H$_2$SO$_4$-hydrolysate of straw. The hemicellulose-extracted straw residue could be used as animal feed, because the residue had 75％ higher digestibility and 20％ better feed efficiency for weanling meadow voles than the untreated straw.