Cloning, Expression, and Characterization of a Thermostable GH51 α-L-Arabinofuranosidase from Paenibacillus sp DG-22

被引:15
作者
Lee, Sun Hwa [1 ]
Lee, Yong-Eok [1 ]
机构
[1] Dongguk Univ, Dept Biotechnol, Gyeongju 780714, South Korea
关键词
alpha-L-Arabinofuranosidase; Paenibacillus sp DG-22; gene cloning and expression; XYLANOLYTIC ENZYMES; BACILLUS-PUMILUS; L-ARABINOSIDASE; PURIFICATION; XYLAN; SEQUENCE; DEGRADATION; GENERATION; ACID; GENE;
D O I
10.4014/jmb.1308.08078
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
The gene encoding alpha-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 degrees C, respectively. The enzyme activity was completely inhibited by 1 mM HgCl2. rAbfA was active only towards p-nitrophephenyl alpha-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.
引用
收藏
页码:236 / 244
页数:9
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