Characterization of Gene Encoding Amylopullulanase from Plant-Originated Lactic Acid Bacterium, Lactobacillus plantarum L137

Jong Hyun Kim, Michihiro Sunako, Hisayo Ono, Yoshikatsu Murooka, Eiichiro Fukusaki, Mitsuo Yamashita

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

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 215,625 Da. 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 Ala36 and the molecular mass of the mature extracellular enzyme is 211,537 Da. 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.

Original languageEnglish
Pages (from-to)449-459
Number of pages11
JournalJournal of Bioscience and Bioengineering
Volume106
Issue number5
DOIs
Publication statusPublished - 2008 Nov 1
Externally publishedYes

Keywords

  • Lactobacillus plantarum L137
  • amylopullulanase
  • repeating amino acid sequence

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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