Characterization of Gene Encoding Amylopullulanase from Plant-Originated Lactic Acid Bacterium, Lactobacillus plantarum L137(MICROBIAL PHYSIOLOGY AND BIOTECHNOLOGY)
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概要
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A starch-hydrolyzing lactic acid bacterium, Lactobacillus plantarum L137, was isolated from traditional fermented food made from fish and rice in the Philippines. A gene (apuA) encoding an amylolytic enzyme from Lactobacillus plantarum L137 was cloned, and its nucleotide sequence was determined. The apuA gene consisted of an open reading frame of 6171 bp encoding a protein of 2056 amino acids, the molecular mass of which was calculated to be 215,625Da. The catalytic domains of amylase and pullulanase were located in the same region within the middle of the N-terminal region. The deduced amino acid sequence revealed four highly conserved regions that are common among amylolytic enzymes. In the N-terminal region, a six-amino-acid sequence (Asp-Ala/Thr-Ala-Asn-Ser-Thr) is repeated 39 times, and a three-amino-acid sequence (Gln-Pro-Thr) is repeated 50 times in the C-terminal region. The apuA gene was subcloned in L. plantarum NCL21, which is a plasmid-cured derivative of the wild-type L137 strain and has no amylopullulanase activity, and the gene was overexpressed under the control of its own promoter. The ApuA enzyme from this recombinant L. plantarum NCL21 harboring apuA gene was purified. The enzyme has both α-amylase and pullulanase activities. The N-terminal sequence of the purified enzyme showed that the signal peptide was cleaved at Ala^<36> and the molecular mass of the mature extracellular enzyme is 211,537Da. The major reaction products from soluble starch were maltotriose (G3) and maltotetraose (G4). Only maltotriose (G3) was produced from pullulan. From these results, we concluded that ApuA is an amylolytic enzyme belonging to the amylopullulanase family.
- 社団法人日本生物工学会の論文
- 2008-11-25
著者
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Fukusaki Eiichiro
Department of Biotechnology, Graduate School of Engineering, Osaka University
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KIM Jong-Hyun
Department of Surgery, College of Medicine, Hallym University
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YAMASHITA MITSUO
Department of Agricultural Chemistry, Faculty of Bioresources, Mie University, Department of Biotech
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MUROOKA YOSHIKATSU
Department of Agricultural Chemistry, Faculty of Bioresources, Mie University, Department of Biotech
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Yamashita Mitsuo
Department Of Biotechnology Graduate School Of Engineering Osaka University:department Of Applied Ch
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Yamashita M
Department Of Biotechnology Graduate School Of Engineering Osaka University
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Murooka Yoshikatsu
Department Of Biotechnology Graduate School Of Engineering Osaka University:hiroshima Institute Of T
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Murooka Y
Department Of Biotechnology Graduate School Of Engineering Osaka University
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Sunako Michihiro
Sanwa Cornstarch Co., Ltd.
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Ono Hisayo
Department of Biotechnology, Graduate School of Engineering, Osaka University
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Kim Jong-hyun
Department Of Biotechnology Graduate School Of Engineering Osaka University
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Ono Hisayo
Department Of Biotechnology Graduate School Of Engineering Osaka University
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Ono H
Department Of Biotechnology Graduate School Of Engineering Osaka University
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Ono Hisayo
Department Of Biotechnology Faculty Of Engineering Osaka University
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Sunako Michihiro
Sanwa Cornstarch Co. Ltd.
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Fukusaki Eiichiro
Department Of Biotechnology Faculty Of Engineering Osaka University
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Murooka Yoshikatsu
Depart. Of Bioteclnology Graduate School Of Eng. Osaka Univ.
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Matsumoto Yukimasa
Department Of Biotechnology Graduate School Of Engineering Osaka University
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Yamashita Mitsuo
Department Of Agricultural Chemistry Faculty Of Bioresources Mie University Department Of Biotechnol
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Kim Jong-hyun
Department Of Anatomy College Of Oriental Medicine Institute Of Oriental Medicine Kyung Hee Universi
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