[논문]Cloning, Expression, and Characterization of a Thermostable GH51 <tex>${\\alpha}-\\small{L}$</tex>-Arabinofuranosidase from Paenibacillus sp. DG-22

Lee, Sun Hwa; Lee, Yong-Eok

doi:10.4014/jmb.1308.08078

[국내논문] Cloning, Expression, and Characterization of a Thermostable GH51 ${\\alpha}-\\small{L}$-Arabinofuranosidase from Paenibacillus sp. DG-22 원문보기

Journal of microbiology and biotechnology, v.24 no.2, 2014년, pp.236 - 244

Lee, Sun Hwa (Department of Biotechnology, Dongguk University) , Lee, Yong-Eok (Department of Biotechnology, Dongguk University)

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The gene encoding ${\alpha}-\small{L}$-arabinofuranosidase (AFase) from Paenibacillus sp. DG-22 was cloned, sequenced, and expressed in Escherichia coli. The AFase gene (abfA) comprises a 1,509 bp open reading frame encoding 502 amino acids with a molecular mass of 56,520 daltons. The deduced amino acid sequence of the gene shows that AbfA is an enzyme consisting of only a catalytic domain, and that the enzyme has significant similarity to AFases classified into the family 51 of the glycosyl hydrolases. abfA was subcloned into the pQE60 expression vector to fuse it with a six-histidine tag and the recombinant AFase (rAbfA) was purified to homogeneity. The specific activity of the recombinant enzyme was 96.7 U/mg protein. Determination of the apparent molecular mass by gel-filtration chromatography indicated that AbfA has a tetrameric structure. The optimal pH and temperature of the enzyme were 6.0 and $60^{\circ}C$, respectively. The enzyme activity was completely inhibited by 1 mM $HgCl_2$. rAbfA was active only towards p-nitrophephenyl ${\alpha}-\small{L}$-arabinofuranoside and exhibited $K_m$ and $V_{max}$ values of 3.5 mM and 306.1 U/mg, respectively. rAbfA showed a synergistic effect in combination with endoxylanase on the degradation of oat spelt xylan and wheat arabinoxylan.

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DNA sequencing was conducted at the Genotech DNA sequencing facility (Daejeon, Korea) by automated sequencing using the dideoxynucleotide chain termination method. The nucleotide sequence was analyzed using the National Center for Biotechnology Information (NCBI) Open Reading Frame (ORF) Finder tool.
DNA sequencing was conducted at the Genotech DNA sequencing facility (Daejeon, Korea) by automated sequencing using the dideoxynucleotide chain termination method. The nucleotide sequence was analyzed using the National Center for Biotechnology Information (NCBI) Open Reading Frame (ORF) Finder tool. The signal peptide in the deduced amino acid sequence was predicted by the SignalP 4.
The protein content was determined by the Bradford method [6] with Protein Assay reagent (Bio-Rad) using bovine serum albumin as the standard. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on a 15% running gel [17] and resolved proteins were visualized by staining with Coomassie Brilliant Blue R-250 (Sigma).
aReducing sugar was measured using the DNS method [23].

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[국내논문] Cloning, Expression, and Characterization of a Thermostable GH51 ${\\alpha}-\\small{L}$-Arabinofuranosidase from Paenibacillus sp. DG-22 원문보기

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[국내논문] Cloning, Expression, and Characterization of a Thermostable GH51 ${\\alpha}-\\small{L}$-Arabinofuranosidase from Paenibacillus sp. DG-22 원문보기

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