An isoamylase with neutral pH optimum from a Flavobacterium species: cloning, characterization and expression of the iam gene

Krohn, Bradley M.; Barry, Gerard F.; Kishore, Ganesh M.

doi:10.1007/s004380050441

Cited by 20 publications

(10 citation statements)

References 36 publications

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“…pH 3.5, 5.5, 5.0 and 5.6±6.4 for enzymes from P. amyloderamosa (Yokobayashi et al 1970), Cytophaga (Gunja- Smith et al 1970), Bacillus amyloliquefaciens (Urlaub and Wober 1975) and E. coli (Jeanningros et al 1976), respectively. Recently, however, a neutral pH optimum (6±7) for Flavobacterium isoamylase (Krohn et al 1997) and an alkaline pH optimum (9.0) for isoamylase from an alkalophilic strain of Bacillus sp. KSM-3309 (Ara et al 1993) have been reported.…”

Section: Discussionmentioning

confidence: 98%

Purification, characterization, and cDNA structure of isoamylase from developing endosperm of rice

Fujita

Kubo

Francisco

et al. 1999

Planta

114

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Isoamylase (EC 3.2.1.68) in rice (Oryza sativa L.) was efficiently purified within a day to homogeneity, as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), from developing endosperm by sequential use of Q Sepharose HP anion-exchange chromatography, ammonium sulfate fractionation, and TSKgel G4000SWXL and G3000SWXL gel filtration chromatography. Although the protein exhibited a molecular size of ca. 83 kDa on SDS-PAGE, the apparent size of the native enzyme was approximately 340 and 490 kDa on TSKgel G3000SWXL and G4000SWXL gel filtration chromatograms, respectively, suggesting that rice isoamylase exists in a homo-tetramer to homo-hexamer form in developing endosperm. The purified rice isoamylase was able to debranch glycogen, phytoglycogen and amylopectin but could not attack pullulan. The optimum pH and temperature for isoamylase activity were found to be pH 6.5 to 7.0 and 30 degrees C, respectively. The enzyme activity was completely inhibited by HgCl2 and p-chloromercuribenzoate at 1 mM. These results indicate that rice isoamylase possesses properties which are distinct from those reported for bacterial isoamylase. Complementary-DNA clones for rice endosperm isoamylase were isolated with a polymerase-chain-reaction product as probe which was generated by primers designed from nucleotides conserved in cDNA for maize Sugary-1 isoamylase (M.G. James et al., 1995. Plant Cell 7: 417-429) and a Pseudomonas amyloderamosa gene encoding isoamylase (A. Amemura et al. 1988, J Biol Chem 263: 9271-9275). The nucleotide sequence and deduced amino acid sequence of the longest clone showed a high similarity to those of maize Surgary-1 isoamylase, but a lesser similarity to those of Pseudomonas amyloderamosa isoamylase. Southern blot analysis and gene mapping analysis indicated that the isoamylase gene exists as a single copy in the rice genome and is located on chromosome 8 of cv. Nipponbare which belongs to the Japonica rice group. Phylogenetic analysis indicated that isoamylases from maize and rice are more closely related to a number of glgX gene products of the blue green alga Synechocystis and various bacteria than to isoamylases from Pseudomonas and Flavobacterium. Hence, it is proposed that glgX proteins are classified as isoamylase-type debranching enzymes. Our tree also showed that all starch- and glycogen-debranching enzymes from plants and bacteria tested can be classified into two distinct types, an isoamylase-type and a pullulanase-type.

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Section: Discussionmentioning

confidence: 98%

Purification, characterization, and cDNA structure of isoamylase from developing endosperm of rice

Fujita

Kubo

Francisco

et al. 1999

Planta

114

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“…R. tropici GlgX also shows 56% similarity with the Flavobacterium sp. isoamylase enzyme, which degrades ␣-1,6 glycosidic bonds (23). This similarity is primarily around the four domains (indicated in Fig.…”

Section: Isolation and Characterization Of An R Tropici Nadimentioning

confidence: 99%

Enhanced Symbiotic Performance by Rhizobium tropici Glycogen Synthase Mutants

et al. 2001

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We isolated a Tn5-induced Rhizobium tropici mutant that has enhanced capacity to oxidize N,N-dimethylp-phenylendiamine (DMPD) and therefore has enhanced respiration via cytochrome oxidase. The mutant had increased levels of the cytochromes c 1 and CycM and a small increase in the amount of cytochrome aa 3 . In plant tests, the mutant increased the dry weight of Phaseolus vulgaris plants by 20 to 38% compared with the control strain, thus showing significantly enhanced symbiotic performance. The predicted product of the mutated gene is homologous to glycogen synthases from several bacteria, and the mutant lacked glycogen. The DNA sequence of the adjacent gene region revealed six genes predicted to encode products homologous to the following gene products from Escherichia coli: glycogen phosphorylase (glgP), glycogen branching enzyme (glgB), ADP glucose pyrophosphorylase (glgC), glycogen synthase (glgA), phosphoglucomutase (pgm), and glycogen debranching enzyme (glgX). All six genes are transcribed in the same direction, and analysis with lacZ gene fusions suggests that the first five genes are organized in one operon, although pgm appears to have an additional promoter; glgX is transcribed independently. Surprisingly, the glgA mutant had decreased levels of high-molecular-weight exopolysaccharide after growth on glucose, but levels were normal after growth on galactose. A deletion mutant was constructed in order to generate a nonpolar mutation in glgA. This mutant had a phenotype similar to that of the Tn5 mutant, indicating that the enhanced respiration and symbiotic nitrogen fixation and decreased exopolysaccharide were due to mutation of glgA and not to a polar effect on a downstream gene.

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“…In addition to intracellular glg operon-encoded isoamylases homologous to E. coli GlgX, some bacteria produce an extracellular isoamylase that is able to cleave substrates such as glycogen and amylopectin but not pullulan. Examples are the extracellular isoamylases produced by Pseudomonas amyloderamosa (2) and Flavobacterium odoratum (17). In higher plants, three classes of isoamylase genes and one class of pullulanase genes are found.…”

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confidence: 99%

Role of the Escherichia coli glgX Gene in Glycogen Metabolism

et al. 2005

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A role for the Escherichia coli glgX gene in bacterial glycogen synthesis and/or degradation has been inferred from the sequence homology between the glgX gene and the genes encoding isoamylase-type debranching enzymes; however, experimental evidence or definition of the role of the gene has been lacking. Construction of E. coli strains with defined deletions in the glgX gene is reported here. The results show that the GlgX gene encodes an isoamylase-type debranching enzyme with high specificity for hydrolysis of chains consisting of three or four glucose residues. This specificity ensures that GlgX does not generate an extensive futile cycle during glycogen synthesis in which chains with more than four glucose residues are transferred by the branching enzyme. Disruption of glgX leads to overproduction of glycogen containing short external chains. These results suggest that the GlgX protein is predominantly involved in glycogen catabolism by selectively debranching the polysaccharide outer chains that were previously recessed by glycogen phosphorylase.

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An isoamylase with neutral pH optimum from a Flavobacterium species: cloning, characterization and expression of the iam gene

Cited by 20 publications

References 36 publications

Purification, characterization, and cDNA structure of isoamylase from developing endosperm of rice

Purification, characterization, and cDNA structure of isoamylase from developing endosperm of rice

Enhanced Symbiotic Performance by Rhizobium tropici Glycogen Synthase Mutants

Role of the Escherichia coli glgX Gene in Glycogen Metabolism

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