Cell lysate of Escherichia coli strain BL21 showed significant D-glucose isomerase activity. The rate of glucose conversion was increased up to 40% when cells were induced with 1% D-xylose. E. coli BL21 xylose isomerase (ECXI-BL21) was purified to homogeneity, up to 1.9-fold with overall 10.88% enzyme yield by heat shock, salting out and electro-elution. The molecular mass of ECXI-BL21 was estimated as 43.9 kDa on SDS-PAGE. pHopt. and Topt. of the enzyme were calculated as 7.0 and 50 °C, respectively. Activation energy (Ea) of ECXI-BL21 was 45 kJ/mol. Enzyme was stable from 30 to 55 °C and at pH range 6.0–8.0. ECXI-BL21(holo) was activated by 10 mM magnesium (35%), 0.5 mM cobalt (20%) and manganese (25%), and 0.5/10 mM Mn2+/Mg2+ (50%) and Co2+/Mg2+ (30%) as compared to ECXI-BL21(apo). Catalytic affinity (Km) of ECXI-BL21 for D-glucose was calculated as 0.82 mM, while maximum velocity (Vmax) of the reaction D-glucose(aldo) ⇌ D-fructose(keto) was 108 μmol/mg/min. D-fructose formed was identified on silica gel plate. This thermophilic enzyme, Tm = 75 °C, has great potential for high fructose syrup production used in food and soft drink industries.
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Ahlem C, Huisman W, Neslund G, Dahms AS (1982) Purification and properties of a periplasmic D-xylose-binding protein from Escherichia coli K-12. J Biol Chem 257:2926–2931
Ammar EM, Wang X, Rao CV (2018) Regulation of metabolism in Escherichia coli during growth on mixtures of the non-glucose sugars: arabinose, lactose, and xylose. Sci Rep 8:1–11. https://doi.org/10.1038/s41598-017-18704-0
Bhosale SH, Rao MB, Deshpande VV (1996) Molecular and industrial aspects of glucose isomerase. Microbiol Rev 60:280–300
Boyer RF (1993) Modern experimental biochemistry, second. The Benjamin/Cummings Publishing Company, Inc.
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. https://doi.org/10.1016/0003-2697(76)90527-3
Branco AT, dos Ferreira BS, de Souza Filho GA (2008) A paper-based electroelution system for protein recovery from stained sodium dodecyl sulfate-polyacrylamide gels. Anal Biochem 381:267–269. https://doi.org/10.1016/j.ab.2008.03.025
Brown SH, Sjoholm C, Kelly RM (1993) Purification and characterization of a highly thermostable glucose isomerase produced by the extremely thermophilic eubacterium, Thermotoga maritima. Biotechnol Bioeng 41:878–886. https://doi.org/10.1002/bit.260410907
Chan E-C, Ueng PP, Chen LF (1989) Metabolism of D-xylose in Schizosaccharomyces pombe cloned with a xylose isomerase gene. Appl Microbiol Biotechnol 31–31:524–528. https://doi.org/10.1007/BF00270788
Chanitnun K, Pinphanichakarn P (2012) Glucose (xylose) isomerase production by Streptomyces sp. CH7 grown on agricultural residues. Brazil J Microbiol 43:1084–1093. https://doi.org/10.1590/S1517-83822012000300035
Chiang LC, Gong CS, Chen LF, Tsao GT (1981) D-xylulose fermentation to ethanol by Saccharomyces cerevisiae. Appl Environ Microbiol 42:284–289
Chiang LC, Hsiao HY, P.P., et al (1981b) Ethanol production from xylose by enzymic isomerization and yeast fermentation. Biotechnol Bioeng Symp 11:Web
Collyer CA, Henrick K, Blow DM (1990) Mechanism for aldose-ketose interconversion by D-xylose isomerase involving ring opening followed by a 1,2-hydride shift. J Mol Biol 212:211–235. https://doi.org/10.1016/0022-2836(90)90316-E
David JD, Weismeyer H (1970) Control of xylose metabolism in Escherichia coli X289. Biochem Biophys Acta 201:497–499. https://doi.org/10.1016/0304-4165(70)90171-6
Dawson RM, Elliott DC, Elliott WH, Jones KM (1969) Data for biochemical research. Oxford University Press, London
Driska SP (1998) Using light scattering to locate less than a microgram of protein per band in polyacrylamide tube gels after isoelectric focusing. Anal Biochem 260:44–49. https://doi.org/10.1006/abio.1998.2696
El-shora HM, El-shobaky AM, Ghoneim JE (2016) Activity of glucose isomerase from Bacillus thuringiensis under different treatments. Int J Curr Microbiol Appl Sci 5:579–589. 10.20546/ijcmas.2016.502.065
Epting KL, Vieille C, Zeikus JG, Kelly RM (2005) Influence of divalent cations on the structural thermostability and thermal inactivation kinetics of class II xylose isomerases. FEBS J 272:1454–1464. https://doi.org/10.1111/j.1742-4658.2005.04577.x
Fatima B, Aftab MN, Haq IU (2016) Cloning, purification, and characterization of xylose isomerase from Thermotoga naphthophila RKU-10. J Basic Microbiol 56:949–962. https://doi.org/10.1002/jobm.201500589
Gong CS, Chen LF, Flickinger MC et al (1981) Production of ethanol from D-xylose by using D-xylose isomerase and yeasts. Appl Environ Microbiol 41:430–436
Jenkins J, Janin J, Rey F et al (1992) Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 1. Crystallography and site-directed mutagenesis of metal binding sites. Biochemistry 31:5449–5458. https://doi.org/10.1021/bi00139a005
Jensen VJ, Rugh S (1987) Industrial-scale production and application of immobilized glucose isomerase. Methods Enzymol 136:356–370. https://doi.org/10.1016/S0076-6879(87)36035-5
Kasumi T, Hayashi K, Tsumura N (1982) Roles of magnesium and cobalt in the reaction of glucose isomerase from Streptomyces griseofuscus S-41. Agric Biol Chem 46:31–39. https://doi.org/10.1271/bbb1961.46.21
Kasumi T, Mori S, Kaneko S, Koyama Y (2012) Molecular cloning and characterization of D-xylose isomerase from a novel actinobacteria, Thermobifida fusca MBL10003. J Appl Glycosci 59:43–46. https://doi.org/10.5458/jag.jag.JAG-2011
Kim BC, Yu SN, Kim KY et al (2010) Cloning, expression and characterization of xylose isomerase, xyl-A, from Caldanaerobacter subterraneus subsp. yonseiensis. Biotechnol Lett 32:929–933. https://doi.org/10.1007/s10529-010-0255-3
Kluskens LD, Zeilstra J, Geerling ACM et al (2010) Molecular characterization of the glucose isomerase from the thermophilic bacterium Fervidobacterium gondwanense. Environ Technol 31:1083–1090. https://doi.org/10.1080/09593330903486673
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. https://doi.org/10.1038/227680a0
Lam VM, Daruwalla KR, Henderson PJ, Jones-Mortimer MC (1980) Proton-linked D-xylose transport in Escherichia coli. J Bacteriol 143:396–402
Li GQ, Shao J, Guo CG et al (2012) A simple monolithic column electroelution for protein recovery from gel electrophoresis. Anal Biochem 430:24–31. https://doi.org/10.1016/j.ab.2012.07.008
Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. J Am Chem Soc 56:658–666
Natake M, Yoshimura S (1964) Studies of glucose isomerase of bacteria: Part II, The glucose isomerizing activity of Escherichia intermedia strain HN-500. Agri Biol Chem 28:505–509
Nguyen HT, Tran G-B (2018) Optimization of fermentation conditions and media for production of glucose isomerase from Bacillus megaterium using response surface methodology. scientifica (Cairo) 1:1–11. https://doi.org/10.1155/2018/6842843
Rozanov AS, Zagrebelny SN, Beklemishev AB (2009) Cloning of Escherichia coli K12 xylose isomerase (glucose isomerase) gene and studying the enzymatic properties of its expression product. Appl Biochem Microbiol 45:38–44. https://doi.org/10.1134/s0003683809010062
Schellenberg GD, Sarthy A, Larson AE et al (1984) Xylose isomerase from Escherichia coli. Characterization of the protein and the structural gene. J Biol Chem 259:6826–6832
Schenk M, Bisswanger H (1998) A microplate assay for D-xylose/D-glucose isomerase. Enzyme Microb Technol 22:721–723
Schneider H, Wang PY, Johnson BF (1981) Current developments in yeast research. Pergamon Press, pp 81–85
Shamannat DK, Sanderson KE (1979) Uptake and catabolism of D-xylose in Salmonella typhimurium LT2. J Bacteriol 139:64–70
Smith CA, Rangarajan M, Hartley BS (1991) D-Xylose (D-glucose) isomerase from Arthrobacter strain N.R.R.L. B3728: Purification and properties. Biochem J 277:255–261. https://doi.org/10.1042/bj2770255
Sriprapundh D, Vieille C, Zeikus JG (2000) Molecular determinants of xylose isomerase thermal stability and activity: analysis of thermozymes by site-directed mutagenesis. Protein Eng 13:259–265. https://doi.org/10.1093/protein/13.4.259
Sriprapundh D, Vieille C, Zeikus JG (2003) Directed evolution of Thermotoga neapolitana xylose isomerase: high activity on glucose at low temperature and low pH. Protein Eng Des Sel 16:683–690. https://doi.org/10.1093/protein/gzg082
Staudigl P, Haltrich D, Peterbauer CK (2014) L-Arabinose isomerase and D-xylose isomerase from Lactobacillus reuteri: Characterization, coexpression in the food grade host Lactobacillus plantarum, and application in the conversion of D-galactose and D-glucose. J Agric Food Chem 62:1617–1624. https://doi.org/10.1021/jf404785m
Suekane M, Tamura M, Tomimura C (1978) Physico-chemical and enzymatic properties of purified glucose isomerases from Streptomyces olivochromogenes and Bacillus stearothermophilus. Agric Biol Chem 42:909–917. https://doi.org/10.1271/bbb1961.42.909
Terpe K (2006) Overview of bacterial expression systems for heterologous protein production: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol Biotechnol 72:211–222. https://doi.org/10.1007/s00253-006-0465-8
Van Bastelaere PB, Callens M, Vangrysperre WA, Kersters-Hilderson HL (1992) Binding characteristics of Mn2+, Co2+ and Mg2+ ions with several D-xylose isomerases. Biochem J 286:729–735
Van Bastelaere PBM, Kersters-Hilderson HLM, Lambeir AM (1995) Wild-type and mutant D-xylose isomerase from Actinoplanes missouriensis: Metal-ion dissociation constants, kinetic parameters of deuterated and non-deuterated substrates and solvent-isotope effects. Biochem J 307:135–142. https://doi.org/10.1042/bj3070135
Vangrysperre W, Van Damme J, Vandekerckhove J et al (1990) Localization of the essential histidine and carboxylate group in D-xylose isomerases. Biochem J 265:699–705. https://doi.org/10.1042/bj2650699
Vieille C, Zeikus GJ (2001) Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability. Microbiol Mol Biol Rev 65:1–43. https://doi.org/10.1128/MMBR.65.1.1-43.2001
Vieille C, Hess JM, Kelly RM, Zeikus JG (1995) xyl-A cloning and sequencing and biochemical characterization of xylose isomerase from Thermotoga neapolitana. Appl Environ Microbiol 61:1867–1875
Walker G, Ricci BA, Goodwin JC (1965) Thin layer chromatography of sugar beet carbohydrates. J A S S B T 13:503–508
Wang PY, Johnson BF, Schneider H (1980a) Fermentation of D-xylose by yeasts using glucose isomerase in the medium to convert D-xylose to D-xylulose. Biotechnol Lett 2:273–278. https://doi.org/10.1007/BF00239856
Wang PY, Shopsis C, Schneider H (1980b) Fermentation of a pentose by yeasts. Biochem Biophys Res Commun 94:248–254. https://doi.org/10.1016/S0006-291X(80)80213-0
Yanmis D, Karaoglu H, Colak DN et al (2014) Characterization of a novel xylose isomerase from Anoxybacillus gonensis G2T. Turkish J Biol 38:586–592. https://doi.org/10.3906/biy-1403-76
Yassien MAM, Jiman-fatani AAM, Hani AZ (2013) Purification, characterization and immobilization of glucose isomerase from Streptomyces albaduncus. Afr J Microbiol Res 7:2682–2688. https://doi.org/10.5897/AJMR2012.2482
Provision of laboratory facilities by University of Veterinary and Animal Sciences (UVAS) Lahore and Institute of Public Health (IPH) Lahore to undertake this research is gratefully acknowledged.
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Fatima, B., Javed, M.M. Production, purification and physicochemical characterization of D-xylose/glucose isomerase from Escherichia coli strain BL21. 3 Biotech 10, 39 (2020). https://doi.org/10.1007/s13205-019-2036-6
- Escherichia coli
- Thin-layer chromatography
- D-glucose isomerase