Close evolutionary relatedness among functionally distantly related members of the (α/β)8‐barrel glycosyl hydrolases suggested by the similarity of their fifth conserved sequence region

Janeček, Štefan

doi:10.1016/0014-5793(95)01309-1

Cited by 41 publications

(30 citation statements)

References 16 publications

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“…2). A motif search showed a short stretch ( 209 QPDLN) between the S␤3 and N␣3 regions of PalI which is a proposed selection marker of the ␣-amylase family (9). The sequence alignment with the two OGLs showed that PalI also contains a catalytic triad consisting of three carboxylic amino acids and the two histidine residues essential for enzyme activity.…”

Section: Resultsmentioning

confidence: 99%

Isomaltulose Synthase from Klebsiella sp. Strain LX3: Gene Cloning and Characterization and Engineering of Thermostability

Zhang¹,

Li²,

Zhang³

2002

Appl Environ Microbiol

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The gene (palI) encoding isomaltulose synthase (PalI) from a soil bacterial isolate, Klebsiella sp. strain LX3, was cloned and characterized. PalI converts sucrose into isomaltulose, trehalulose, and trace amounts of glucose and fructose. Sequence domain analysis showed that PalI contains an ␣-amylase domain and (␤/␣) 8 -barrel structures, suggesting that it belongs to the ␣-amylase family. Sequence alignment indicated that the five amino acid residues of catalytic importance in ␣ Isomaltulose (6-O-␣-D-glucopyranosyl-D-fructose, commonly referred to as palatinose) is a sucrose isomer with physical and organoleptic properties similar to those of sucrose. It has been suggested as a noncariogenic alternative to sucrose (1) and has now been widely used as a sugar substitute in food. Unlike ingestion of sucrose, ingestion of isomaltulose has only a minor effect on the concentration of glucose in blood, indicating its potential as a parenteral nutrient acceptable to diabetics and nondiabetics (6). Several microorganisms have been found to form isomaltulose and trehalulose from sucrose, for example Protaminobacter rubrum (39), Serratia plymuthica (5), Erwinia rhapontici (4), Klebsiella planticola CCRC 19112 (7), Pseudomonas mesoacidophila MX-45 (18), and Agrobacterium radiobacter .Isomaltulose synthase, also known as sucrose isomerase, has been purified from S. plymuthica (14, 33), E. rhapontici (4), a Klebsiella sp. (24), and Pseudomonas mesoacidophila . In addition to isomerizing sucrose to produce isomaltulose and trehalulose, the enzyme reaction also releases small amounts of glucose and fructose as by-products (7,14,31,33). The isomaltulose synthase from S. plymuthica even converted sucrose to isomaltose and isomelezitose (33). The product composition varies depending on the bacterial strain used. Protaminobacter rubrum (15), S. plymuthica NCIB 8285 (5), E. rhapontici NCPPB 1579 (4), and K. planticola CCRC 19112 (7) produce mainly isomaltulose (75 to 85%), whereas Pseudomonas mesoacidophila MX-45 (17) and A. radiobacter MX-232 (19) produce more trehalulose (90%) than isomaltulose. The reaction of these enzymes is strongly influenced by temperature. The optimum temperature for isomaltulose production by the enzyme from S. plymuthica ATCC 15928 is around 30°C, and no product is produced at 55°C (33), indicating the thermolability of isomaltulose synthase. Isomaltulose synthase sequence information is required to clarify the molecular mechanism of isomerization and assess the feasibility of engineering thermostability.We recently identified a new isomaltulose-producing bacterial isolate, Klebsiella sp. strain LX3 (L.-H. Zhang, unpublished data). Biochemical analyses showed that this isolate converts sucrose mainly into isomaltulose, with small amounts of trehalulose and trace amounts of glucose and fructose. In the present study, we report the cloning and characterization of the gene (palI) encoding isomaltulose synthase (PalI) from Klebsiella sp. strain LX3. The recombinant enzyme was overexpressed in Escherichia co...

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Section: Resultsmentioning

confidence: 99%

Isomaltulose Synthase from Klebsiella sp. Strain LX3: Gene Cloning and Characterization and Engineering of Thermostability

Zhang¹,

Li²,

Zhang³

2002

Appl Environ Microbiol

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“…A typical -amylase from A. oryzae (Taka-amylase), like most -amylases, possesses Ca 2þ -binding sites. 26,27) On the other hand, several amino acid residues composing the Ca 2þ -binding site were replaced with other amino acids in the primary structure of MalA. Amino acid substitution can probably alter the Ca 2þ -binding property of MalA.…”

Section: Characterization Of Recombinant Malamentioning

confidence: 99%

Gene Analysis, Expression, and Characterization of an Intracellular α-Amylase from the Extremely Halophilic Archaeon Haloarcula japonica

Onodera

Yatsunami

Tsukimura

et al. 2013

Bioscience, Biotechnology, and Biochemistry

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“…3). Furthermore, the amino acid sequence of the neopullulanase exhibited the ¢fth conserved sequence region (189-MPDLN) described for the sequences of some K-amylases and related enzymes [20] (Fig. 3).…”

Section: Sequence Of Nplamentioning

confidence: 99%

Expression and secretion of Bacillus polymyxa neopullulanase in Saccharomyces cerevisiae

Yebra¹

1999

FEMS Microbiology Letters

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We have isolated the gene encoding the neopullulanase enzyme from Bacillus polymyxa CECT 155. It consists of an open reading frame of 1545 bp that could code for a protein of 515 amino acids. This open reading frame was expressed in Bacillus subtilis and the corresponding transformants produced extracellular neopullulanase. The neopullulanase gene was also expressed in Saccharomyces cerevisiae placing it under the control of the yeast actin gene (ACT1) promoter. Clones containing the intact neopullulanase gene, including its own bacterial signal sequence, gave rise to the synthesis of active, but intracellular, enzyme by S. cerevisiae transformants. When sequences specifying the signal sequence and leader region of the yeast mating pheromone K-factor (MFK1) were fused upstream of the gene encoding the neopullulanase enzyme, the enzyme was secreted by S. cerevisiae. The secreted protein presented the same biochemical properties and the same apparent molecular mass as the Bacillus polymyxa original enzyme. The predicted amino acid sequence of the neopullulanase protein contained sequence motifs conserved among amylolytic enzymes. Northern blot analysis indicated that the transcription of the neopullulanase gene in B. polymyxa was induced by the presence of the substrate, pullulan, in the culture, and was repressed by glucose. z

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Close evolutionary relatedness among functionally distantly related members of the (α/β)₈‐barrel glycosyl hydrolases suggested by the similarity of their fifth conserved sequence region

Cited by 41 publications

References 16 publications

Isomaltulose Synthase from Klebsiella sp. Strain LX3: Gene Cloning and Characterization and Engineering of Thermostability

Isomaltulose Synthase from Klebsiella sp. Strain LX3: Gene Cloning and Characterization and Engineering of Thermostability

Gene Analysis, Expression, and Characterization of an Intracellular α-Amylase from the Extremely Halophilic Archaeon <i>Haloarcula japonica</i>

Expression and secretion of Bacillus polymyxa neopullulanase in Saccharomyces cerevisiae

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