Dataset Information

Purification, Identification, and Characterization of an Endo-1,4-?-Xylanase from Wheat Malt.

ABSTRACT: In this study, an endo-1,4-?-xylanase was purified from wheat malt following the procedures of ammonium sulfate precipitation, cation-exchange chromatography, and two-step anion-exchange chromatography. The purified endo-1,4-?-xylanase had a specific activity of 3.94 u/mg, demonstrating a weight average molecular weight (Mw) of approximately 58,000 Da. After LC-MS/MS (Liquid chromatography-tandem mass spectrometry) identification, the purified enzyme had the highest matching degree with a GH10 (Glycoside Hydrolase 10) domain-containing protein from wheat, there were 23 match peptides with a score above the threshold and the prot-cover was 45.5%. The resulting purified enzyme was used to investigate its degradation ability on high viscosity wheat-derived water-extractable arabinoxylan (WEAX). Degradation experiments confirmed that the purified enzyme was a true endo-acting enzyme, which could degrade large WEAX into smaller WEAX. The average degree of polymerization (avDP) and the viscosity of WEAX decreased with the increasing reaction time. The enzyme could degrade a small amount of WEAX into arabinoxylan-oligosaccharides (AXOS) with a degree of polymerization of 2-6, but no monosaccharide was produced. The degradation occurred rapidly in the first 3.5 h and decreased with the further prolongation of reaction time.

SUBMITTER: Peng Z

PROVIDER: S-EPMC7180899 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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Publications

Purification, Identification, and Characterization of an Endo-1,4-β-Xylanase from Wheat Malt.

Peng Zhaojun Z Jin Yuhong Y

Molecules (Basel, Switzerland) 20200329 7

In this study, an endo-1,4-β-xylanase was purified from wheat malt following the procedures of ammonium sulfate precipitation, cation-exchange chromatography, and two-step anion-exchange chromatography. The purified endo-1,4-β-xylanase had a specific activity of 3.94 u/mg, demonstrating a weight average molecular weight (Mw) of approximately 58,000 Da. After LC-MS/MS (Liquid chromatography-tandem mass spectrometry) identification, the purified enzyme had the highest matching degree with a GH10 ( ...[more]

PMID: 32235408

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Project description:BACKGROUND: The coffee berry borer, Hypothenemus hampei, reproduces and feeds exclusively on the mature endosperm of the coffee seed, which has a cell wall composed mainly of a heterogeneous mixture of hemicellulose polysaccharides, including arabinoxylans. Xylanases are digestive enzymes responsible for the degradation of xylan based polymers, hydrolyzing them into smaller molecules that are easier to assimilate by insects. We report the cloning, expression and enzymatic characterization of a xylanase gene that was identified in the digestive tract of the coffee berry borer. METHODS: The complete DNA sequence encoding a H. hampei xylanase (HhXyl) was obtained using a genome walking technique in a cDNA library derived from the borer digestive tract. The XIP-I gene was amplified from wheat (Triticum aestivum variety Soisson). A Pichia pastoris expression system was used to express the recombinant form of these enzymes. The xylanase activity and XIP-I inhibitory activity was quantified by the 3,5-dinitrosalicylic (DNS). The biological effects of XIP-I on borer individuals were evaluated by providing an artificial diet enriched with the recombinant XIP-I protein to the insects. RESULTS: The borer xylanase sequence contains a 951 bp open reading frame that is predicted to encode a 317-amino acid protein, with an estimated molecular weight of 34.92 kDa and a pI of 4.84. Bioinformatic analysis revealed that HhXyl exhibits high sequence homology with endo-?-D-xylanases of Streptomyces bingchenggensis from glycosyl hydrolase 10 (GH10). The recombinant xylanase showed maximal activity at pH 5.5 and 37°C. XIP-I expressed as a recombinant protein inhibited HhXyl activity in vitro and caused individual H. hampei mortality in bioassays when included as a supplement in artificial diets. CONCLUSION: A xylanase from the digestive tract of the coffee berry borer was identified and functionally characterized. A xylanase inhibitor protein, XIP-I, from wheat was shown to be a potent inhibitor of this xylanase, suggesting that its deployment has potential as a strategy to control coffee berry borer colonization of coffee plants.

Dataset Information

Purification, Identification, and Characterization of an Endo-1,4-?-Xylanase from Wheat Malt.

Publications

Purification, Identification, and Characterization of an Endo-1,4-β-Xylanase from Wheat Malt.

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