Derya YANMIŞ, Hakan KARAOĞLU, Dilşat Nigar ÇOLAK, Fulya AY ŞAL, Sabriye ÇANAKÇI, Ali Osman BELDÜZ

Characterization of a novel xylose isomerase from Anoxybacillus gonensis G2T

The xylA gene encoding xylose isomerase from Anoxybacillus gonensis G2T has been cloned and successfully expressed in E. coli. Xylose isomerase was purified 10.98-fold by heat-shock and sequential column chromatography techniques to homogeneity, and the biochemical properties of the enzyme were characterized. The optimum temperature of the enzyme was 85 °C and maximum activity was observed at a pH of 6.5. Its Km and Vmax values were calculated as 25 ± 2 mM and 0.12958 ± 0.002 mumol/min/mg protein, respectively. The effects of various metal ions on the xylose isomerase were examined. Divalent cations Co2+, Mg2+, and Mn2+ were essential for xylose isomerase activity; however, bivalent metal ions (Ca2+, Hg2+, Ni2+, Zn2+, Fe2+, and Cu2+) showed inhibitory effects. This is the first report of characterization of the xylose isomerase of Anoxybacillus spp. According to results obtained from this study, xylose isomerase is a promising candidate for industrial applications in production of xylulose and ribose.

Anahtar Kelimeler:

Xylose isomerase, Anoxybacillus, characterization, thermophilic

Characterization of a novel xylose isomerase from Anoxybacillus gonensis G2T

Keywords:

Xylose isomerase, Anoxybacillus, characterization, thermophilic,

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Amore R, Hollenberg C (1989). Xylose isomerase from Adinoplanes missouriensis: primary structure of the gene and the protein. Nucleic Acids Res 17: 7515–7515.
Belduz AO, Dulger S, Demirbag Z (2003). Anoxybacillus gonensis sp. nov., a moderately thermophilic, xylose-utilizing, endospore- forming bacterium. Int J Syst Evol Microbiol 53: 1315–1320.
Belfaquih N, Penninckx MJ (2000). A bifunctional beta-xylosidase- xylose isomerase from Streptomyces sp EC 10. Enzyme Microb Technol 27: 114–121.
Bhosale SH, Rao MB, Deshpande VV (1996). Molecular and indus- trial aspects of glucose isomerase. Microbiol Rev 60: 280–300.
Bradford MM (1976). A rapid and sensitive method for the quantita- tion of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.
Brown SH, Sjİholm C, Kelly RM (1993). Purification and character- ization of a highly thermostable glucose isomerase produced by the extremely thermophilic eubacterium, Thermotoga mari- tima. Biotechnol Bioeng 41: 878–886.
Cha JH, Cho YJ, Whitaker RD, Carrell HL, Glusker JP, Karplus PA, Batt CA (1994). Perturbing the metal site in D-xylose isomer- ase. Effect of mutations of His-220 on enzyme stability. J Biol Chem 269: 2687–2694.
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: 524–528.
Chen W-P (1980). Glucose isomerase (a review). Process Biochem 15: 30–35.
Chen X, Huang Z, Zhou B, Wang H, Jia G, Qiao J (2014). Expression and purification of porcine Akirin2 in Escherichia coli. Turk J Biol 38: 339–345.
Chiang L-C, Gong C-S, Chen L-F, Tsao GT (1981). D-Xylulose fer- mentation to ethanol by Saccharomyces cerevisiae. Appl Envi- ron Microbiol 42: 284–289.
De Raadt A, Ebner M, Ekhart C, Fechter M, Lechner A, Strobi M, Stütz A (1994). Glucose isomerase (EC 5.3. 1.5) as a reagent in carbohydrate synthesis: success and failures with the isomeri- sation of non-natural derivatives of d. Catal Today 22: 549–561.
Dekker K, Yamagata H, Sakaguchi K, Udaka S (1991). Xylose (glu- cose) isomerase gene from the thermophile Clostridium ther- mohydrosulfuricum; cloning, sequencing, and expression in Escherichia coli. Agric Biol Chem 55: 221–227.
Ertunga NS, Colak A, Belduz AO, Canakci S, Karaoglu H, Sandalli C (2007). Cloning, expression, purification and characteriza- tion of fructose-1, 6-bisphosphate aldolase from Anoxybacillus gonensis G2. J Biochem 141: 817–825.
Gong C-S, Chen L-F, Flickinger MC, Chiang L-C, Tsao GT (1981). Production of ethanol from D-xylose by using D-xylose isom- erase and yeasts. Appl Environ Microbiol 41: 430–436.
Gregory Zeikus J (1996). Molecular determinants of thermozyme activity and stability: Analysis of xylose isomerase and amylo- pullulanase. Prog Biotechnol 12: 145–161.
Haki G, Rakshit S (2003). Developments in industrially important thermostable enzymes: a review. Bioresour Technol 89: 17–34.
Jensen VJ, Rugh S (1987). Industrial-scale production and applica- tion of immobilized glucose isomerase. Methods Enzymol 136: 356–370.
Joo G-J, Shin J-H, Heo G-Y, Kim Y-M, Rhee I-K (2005). Molecu- lar cloning and expression of a thermostable xylose (glucose) isomerase gene, xylA, from Streptomyces chibaensis J-59. The Journal of Microbiology 43: 34–37.
Karaoglu H, Yanmis D, Sal FA, Celik A, Canakci S, Belduz AO (2013). Biochemical characterization of a novel glucose isom- erase from Anoxybacillus gonensis G2 T that displays a high level of activity and thermal stability. J Mol Catal B: Enzym 97: 215–224.
Kikuchi T, Itoh Y, Kasumi T, Fukazawa C (1990). Molecular cloning of the xylA gene encoding xylose isomerase from Streptomyces griseofuscus S-41: primary structure of the gene and its prod- uct. Agric Biol Chem 54: 2469–2472.
Kim B-C, Grote R, Lee D-W, Antranikian G, Pyun Y-R (2001). Ther- moanaerobacter yonseiensis sp. nov., a novel extremely ther- mophilic, xylose-utilizing bacterium that grows at up to 85 degrees C. Int J Syst Evol Microbiol 51: 1539–1548.
Lama L, Nicolaus B, Calandrelli V, Romano I, Basile R, Gambacorta A (2001). Purification and characterization of thermostable xy- lose (glucose) isomerase from Bacillus thermoantarcticus. J Ind Microbiol Biotechnol 27: 234–240.
Liu S-Y, Wiegel J, Gherardini FC (1996). Purification and cloning of a thermostable xylose (glucose) isomerase with an acidic pH optimum from Thermoanaerobacterium strain JW/SL-YS 489. J Bacteriol 178: 5938–5945.
Lönn A, Träff-Bjerre K, Cordero Otero R, Van Zyl W, Hahn-Häg- erdal B (2003). Xylose isomerase activity influences xylose fer- mentation with recombinant Saccharomyces cerevisiae strains expressing mutated xylA from Thermus thermophilus. Enzyme Microb Technol 32: 567–573.
Saari G, Kumar A, Kawasaki G, Insley M, O’Hara P (1987). Sequence of the Ampullariella sp. strain 3876 gene coding for xylose isomerase. J Bacteriol 169: 612–618.
Sandallı C, Saral A, Ülker S, Karaoğlu H, Beldüz AO, Çopur-Çiçek A (2014). Cloning, expression, and characterization of a novel CTP synthase gene from Anoxybacillus gonensis G2. Turk J Biol 38: 111–117.
Schellenberg G, Sarthy A, Larson A, Backer M, Crabb J, Lidstrom M, Hall B, Furlong C (1984). Xylose isomerase from Escherichia coli. Characterization of the protein and the structural gene. J Biol Chem 259: 6826–6832.
Schenck F (2000). High fructose syrups - a review. Indian Sugar 50: 281–287.
Vieille C, Hess JM, Kelly RM, Zeikus JG (1995). xylA cloning and sequencing and biochemical characterization of xylose isom- erase from Thermotoga neapolitana. Appl Environ Microbiol 61: 1867–1875.
Wang P, Johnson B, Schneider H (1980). Fermentation of D-xylose by yeasts using glucose isomerase in the medium to convert D- xylose to D-xylulose. Biotechnol Lett 2: 273–278.
Wilhelm M, Hollenberg CP (1985). Nucleotide sequence of the Bacil- lus subtilis xylose isomerase gene: extensive homology between the Bacillus and Escherichia coli enzyme. Nucleic Acids Res 13: 5717–5722.
Wovcha MG, Steuerwald DL, Brooks KE (1983). Amplification of D- xylose and D-glucose isomerase activities in Escherichia coli by gene cloning. Appl Environ Microbiol 45: 1402–1404.