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Biochemical characterization of a novel dual-function arabinofuranosidase/xylosidase isolated from a compost starter mixture

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Abstract

The gene encoding a glycoside hydrolase family 43 enzyme termed deAX was isolated and subcloned from a culture seeded with a compost starter mixed bacterium population, expressed with a C-terminal His6-tag, and purified to apparent homogeneity. deAX was monomeric in solution and had a broad pH maximum between pH 5.5 and pH 7. A twofold greater k cat/K m for the p-nitrophenyl derivative of α-l-arabinofuranose versus that for the isomeric substrate β-d-xylopyranose was due to an appreciably lower K m for the arabinofuranosyl substrate. Substrate inhibition was observed for both 4-methylumbelliferryl arabinofuranoside and the xylopyranoside cogener. While no loss of activity was observed over 4 h at 40°C, the observed t 1/2 value rapidly decreased from 630 min at 49°C to 47 min at 53°C. The enzyme exhibited end-product inhibition, with a K i for xylose of 145 mM, 18.5 mM for arabinose, and 750 mM for glucose. Regarding natural substrate specificity, deAX had arabinofuranosidase activity on sugar beet arabinan, 1,5-α-l-arabinobiose, and 1,5-α-l-arabinotriose, and wheat and rye arabinoxylan, while xylosidase activity was detected for the substrates xylobiose, xylotriose, xylotetraose, and arabinoxylan from beech and birch. Thus, deAX can be classified as a dual-function xylosidase/arabinofuranosidase with respect to both artificial and natural substrate specificity.

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References

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    Article  CAS  Google Scholar 

  • Bajpai P (1997) Microbial xylanolytic enzyme system: properties and applications. Adv Appl Microbiol 43:141–195

    Article  CAS  Google Scholar 

  • Biely P (2003) Xylanolytic enzymes. In: Whitaker JR, Voragen AGJ, Wong DWS (eds) Handbook of food enzymology. Marcel Dekker, New York, NY, pp 879–915

    Google Scholar 

  • Biely P, Mislovièová D, Toman R (1985) Soluble chromogenic substrates for the assay of endo-1,4-β-xylanases and endo-1,4-β-glucanases. Anal Biochem 144:142–146

    Article  CAS  Google Scholar 

  • Brüx C, Ben-David A, Shallom-Shezifi D, Leon M, Niefind K, Shoham G, Shoham Y, Schomburg D (2006) The structure of an inverting GH43 β-Xylosidase from Geobacillus stearothermophilus with its substrate reveals the role of the three catalytic residues. J Mol Biol 359:97–109

    Article  Google Scholar 

  • Chow V, Nong G, Preston JF (2007) Structure, function, and regulation of the aldouronate utilization gene cluster from Paenibacillus sp. strain JDR-2. J Bacteriol 189:8863–8870

    Article  CAS  Google Scholar 

  • Coutinho PM, Henrissat B (1999) Carbohydrate-active enzymes: an integrated database approach. Cambridge, The Royal Society of Chemistry, Cambridge

    Google Scholar 

  • Jordan DB, Braker JD (2007) Inhibition of the two-subsite β-d-xylosidase from Selenomonas ruminantium by sugars: competitive, noncompetitive, double binding, and slow binding modes. Arch Biochem Biophys 465:231–246

    Article  CAS  Google Scholar 

  • Jordan DB, Li X-L (2007) Variation in relative substrate specificity of bifunctional β-d-xylosidase/α-l-arabinofuranosidase by single-site mutations: roles of substrate distortion and recognition. Biochim Biophys Acta 1774:1192–1198

    Article  CAS  Google Scholar 

  • Jordan DB, Li X-L, Dunlap CA, Whitehead TR, Cotta MA (2007a) β-d-Xylosidase from Selenomonas ruminantium of glycoside hydrolase family 43. Appl Biochem Biotechnol 136–140:93–104

    Google Scholar 

  • Jordan DB, Li X-L, Dunlap CA, Whitehead TR, Cotta MA (2007b) Structure–function relationships of a catalytically efficient β-d-xylosidase. Appl Biochem Biotechnol 141:51–76

    Article  CAS  Google Scholar 

  • Lee RC, Hrmova M, Burton RA, Lahnstein J, Fincher GB (2003) Bifunctional family 3 glycoside hydrolase from barley with α-l-arabinofuranosidase and β-d-xylosidase activity. J Biol Chem 278:5377–5387

    Article  CAS  Google Scholar 

  • Mai V, Wiegel J, Lorenz WW (2000) Cloning, sequencing, and characterization of the bifunctional xylosidase–arabinosidase from the thermophile Thermoanaerobacter ethanolicus. Gene 247:137–143

    Article  CAS  Google Scholar 

  • Malet C, Planas A (1997) Mechanism of Bacillus 1,3-1,4-β-d-glucan 4-glucanohydrolase: kinetics and pH studies with 4-methylumbelliferyl β-d-glucan oligosaccharides. Biochemistry 36:13838–13848

    Article  CAS  Google Scholar 

  • Rahman AKMS, Sugitani N, Hatsu M, Takamizawa K (2003) A role of xylanase, α-l-arabinofuranosidase, and xylosidase in xylan degradation. Can J Microbiol 49:58–64

    Article  CAS  Google Scholar 

  • Schwimmer S (1944) Regeneration of heat-inactivated peroxidase. J Biol Chem 154:487–495

    CAS  Google Scholar 

  • Shallom D, Shoham Y (2003) Microbial hemicellulases. Curr Opin Microbiology 6:219–228

    Article  CAS  Google Scholar 

  • Shallom D, Leon M, Bravman T, Ben-David A, Zaide G, Belakhov V, Shoham G, Schomburg D, Baasov T, Shoham Y (2005) Biochemical characterization and identification of the catalytic residues of a family 43 β-d-Xylosidase from Geobacillus stearothermophilus T-6. Biochemistry 44:387–397

    Article  CAS  Google Scholar 

  • Shulami S, Gat O, Sonenshein AL, Shoham Y (1999) The glucuronic acid utilization gene cluster from Bacillus stearothermophilus T-6. J Bacteriol 181(12):3695–3704

    Article  CAS  Google Scholar 

  • Sørensen HR, Pedersen S, Viksø-Nielsen A, Meyer AS (2005) Efficiencies of designed enzyme combinations in releasing arabinose and xylose from wheat arabinoxylan in an industrial ethanol fermentation residue. Enzyme Microb Technol 36:773–784

    Article  Google Scholar 

  • Spagnuolo M, Crecchio C, Pizzigallo MDR, Ruggiero P (1999) Fractionation of sugar beet pulp into pectin, cellulose, and arabinose by arabinases combined with ultrafiltration. Biotechnol Bioeng 64:685–691

    Article  CAS  Google Scholar 

  • Timell T (1964) Wood hemicelluloses: part I. Adv Carbohydr Chem Biochem 19:247–299

    CAS  Google Scholar 

  • Tsujibo H, Kosaka M, Ikenishi S, Sato T, Katsushiro M, Inamori Y (2004) Molecular characterization of a high-affinity xylobiose transporter of Streptomyces thermoviolaceus OPC-520 and its transcriptional regulation. J Bacteriol 186(4):1029–1037

    Article  CAS  Google Scholar 

  • Van Doorslaer E, Kersters-Hilderson H, De Bruyne CK (1985) Hydrolysis of β-d-xylo-oligosaccharides by β-d-xylosidase from Bacillus pumilus. Carbohydr Res 140:342–346

    Article  Google Scholar 

  • Wagschal K, Franqui-Espiet D, Lee CC, Robertson GH, Wong DWS (2005) Enzyme-coupled assay for β-xylosidase hydrolysis of natural substrates. Appl Environ Microbiol 71(9):5318–5323

    Article  CAS  Google Scholar 

  • Wagschal K, Franqui-Espiet D, Lee CC, Kibblewhite-Accinelli RE, Robertson GH, Wong DWS (2007) Genetic and biochemical characterization of an α-l-arabinofuranosidase isolated from a compost starter mixture. Enzyme Microb Technol 40:747–753

    Article  CAS  Google Scholar 

  • Zykwinska A, Thibault J-F, Ralet M-C (2007) Organization of pectic arabinan and galactan side chains in association with cellulose microfibrils in primary cell walls and related models envisaged. J Exp Bot 58:1795–1802

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors would like to thank Gregory Gray for GLC analysis. Reference to a company and/or products is for purposes of information and does not imply approval or recommendation of the product to the exclusion of others which may also be suitable. All programs and services of the US Department of Agriculture are offered on a non-discriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap.

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Wagschal, K., Heng, C., Lee, C.C. et al. Biochemical characterization of a novel dual-function arabinofuranosidase/xylosidase isolated from a compost starter mixture. Appl Microbiol Biotechnol 81, 855–863 (2009). https://doi.org/10.1007/s00253-008-1662-4

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  • DOI: https://doi.org/10.1007/s00253-008-1662-4

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