Cloning and sequencing of kojibiose phosphorylase gene from Thermoanaerobacter brockii ATCC35047

Yamamoto, Tameyoshi; Maruta, Kazuhiko; Mukai, Kazuhisa; Yamashita, Hiroshi; Nishimoto, Tetsuya; Kubota, Michio; Fukuda, Shigeharu; Kurimoto, Masashi; Tsujisaka, Yoshio

doi:10.1016/s1389-1723(04)70249-2

Cited by 24 publications

(20 citation statements)

References 18 publications

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“…4) The Km values of this mutant for kojibiose, Pi, β G1P, and glucose were 1.06, 1.15, 0.47 and 3.72 mM (the wild type; 0.80, 0.75, 0.79 and 3.84 mM), respectively. D513N had specific activities and Km values that were almost the same as those of the wild type.…”

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confidence: 87%

“…We have already purified KP and TP, 2,3) and cloned these phosphorylase genes from T. brockii. 4,5) Furthermore, various oligosaccharides were synthesizing by using KP or TP. 6 9) Protein engineering techniques have often been employed to improve the characteristics of targeted enzymes.…”

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confidence: 99%

“…15) The residue Asp513 of KP corresponds to Asn499 of MP, which is included in an helix (residues 496 513). Since the regions around these residues are highly homologous to each other, 4) Asp513 of KP might be a component of an α helix. An aspartic acid residue was reported to be unfavorable for forming an α helix.…”

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confidence: 99%

“…The regions around Ser676 and Asn687 of KP are homologous to those of MP. 4) Based on the tertiary structure of MP, these residues of KP would be adjacent to the phosphate binding sites, S627 and S628, of MP and located at the inside of the active site pocket. These mutations may affect the binding of phosphate to the site of KP and the active site structure of KP, and may change the affinity for oligosaccharides, probably in favor of larger oligosaccharides as glucosyl acceptors.…”

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Improvement of the Enzymatic Properties of Kojibiose Phosphorylase from Thermoanaerobacter brockii by Random Mutagenesis and Chimerization

拓生¹,

友之²,

博人³

et al. 2006

J. Appl. Glycosci.

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Phosphorylases that have been reported to date are classified into fourteen kinds by the substrates phosphorolyzed.1) All of them catalyze an exowise phosphorolysis at the nonreducing end of the glycosyl linkage and, more specifically, most phosphorolyze glucosyl linkages. Kojibiose phosphorylase (KP; EC 2.4.1.230) from Thermoanaerobacter brockii ATCC35047 catalyzes the reversible phosphorolysis of the α 1,2 glucosyl bond of kojibiose (α D glucopyranosyl (1 2) D glucopyranose). T. brockii also produces trehalose phosphorylase (TP; EC 2.4.1.64) that reversibly phosphorolyzes the α,α 1,1 glucosyl bond of trehalose (α D glucopyranosyl (1 1) α D glucopyranoside). KP and TP produce β D glucose 1 phosphate (β G1P) and D glucose by the phosphorolysis of each substrate. Functionally, KP and TP catalyze the phosphorolysis of the α glucosyl bond with an inversion of the anomeric configuration. Structurally, they are classified into glycoside hydrolase family 65 (GH65) based on the amino acid sequence (http: afmb.cnrs mrs.fr CAZY GH.html). We have already purified KP and TP,2,3) and cloned these phosphorylase genes from T. brockii. 4,5) Furthermore, various oligosaccharides were synthesizing by using KP or TP. Protein engineering techniques have often been employed to improve the characteristics of targeted enzymes. For industrial purposes, the enhancement of thermostability of targeted enzymes is generally favorable, and the change in specificities of enzymes could increase the yield of the main products, thereby reducing unfavorable by products, or would facilitate the synthesis of novel products. Random mutagenesis has been employed to improve the characteristics of various enzymes.10 12) Chimerization is also used to improvement on the enzymatic properties. Since it is possible to exchange or insert regions in the target enzymes, chimerization could result in remarkable improvements in the characteristics of enzymes.13)The regiospecificities of phosphorylases are very strict and they phosphorolyze only their specific type of glycosyl linkage. These strict specificities are important in the exploitation of these enzymes in the synthesis of oligosaccharides by their synthetic reaction. Namely, oligosaccharides with specific glycosyl linkages can be synthesized using phosphorylases. Therefore, improvement of enzymatic properties of phosphorylases would open an opportunity for production of useful oligosaccharides.In this review, we introduce our recent studies about the random mutagenesis of KP, and the construction and characterization of chimeric enzymes from KP and TP. J. Appl. Glycosci., 53, 123 129 (2006) ! C 2006 The Japanese Society of Applied Glycoscience Proceedings of the Symposium on Amylases and Related Enzymes, 2005 Improvement of the Enzymatic Properties of Kojibiose Phosphorylase fromThermoanaerobacter brockii by Random Mutagenesis and Chimerization (Received December 20, 2005) Takuo Yamamoto, Abstract: Random mutagenesis by error-prone PCR was introduced to kojibiose phosphorylase (KP; EC 2.4.1.230) fro...

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confidence: 87%

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confidence: 99%

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confidence: 99%

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confidence: 99%

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Improvement of the Enzymatic Properties of Kojibiose Phosphorylase from Thermoanaerobacter brockii by Random Mutagenesis and Chimerization

拓生¹,

友之²,

博人³

et al. 2006

J. Appl. Glycosci.

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“…ST04 kojibiose phosphorylase) in this study, it showed low sequence homology with other established DPases, such as kojibiose phosphorylases from Th. brockii (37%) (27) and C. saccharolyticus (37%) (28) and trehalose phosphorylases from Th. brockii (30%) (29), Ca.…”

Section: Disaccharide Hydrolysis Pattern Of Pyrococcus Sp Strain St04mentioning

confidence: 99%

Identification and Characterization of an Archaeal Kojibiose Catabolic Pathway in the Hyperthermophilic Pyrococcus sp. Strain ST04

Jung

Seo

Holden

et al. 2014

Journal of Bacteriology

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A unique gene cluster responsible for kojibiose utilization was identified in the genome of Pyrococcus sp. strain ST04. The proteins it encodes hydrolyze kojibiose, a disaccharide product of glucose caramelization, and form glucose-6-phosphate (G6P) in two steps. Heterologous expression of the kojibiose-related enzymes in Escherichia coli revealed that two genes, Py04_1502 and Py04_1503, encode kojibiose phosphorylase (designated PsKP, for Pyrococcus sp. strain ST04 kojibiose phosphorylase) and ␤-phosphoglucomutase (PsPGM), respectively. Enzymatic assays show that PsKP hydrolyzes kojibiose to glucose and ␤-glucose-1-phosphate (␤-G1P). The K m values for kojibiose and phosphate were determined to be 2.53 ؎ 0.21 mM and 1.34 ؎ 0.04 mM, respectively. PsPGM then converts ␤-G1P into G6P in the presence of 6 mM MgCl 2 . Conversion activity from ␤-G1P to G6P was 46.81 ؎ 3.66 U/mg, and reverse conversion activity from G6P to ␤-G1P was 3.51 ؎ 0.13 U/mg. The proteins are highly thermostable, with optimal temperatures of 90°C for PsKP and 95°C for PsPGM. These results indicate that Pyrococcus sp. strain ST04 converts kojibiose into G6P, a substrate of the glycolytic pathway. This is the first report of a disaccharide utilization pathway via phosphorolysis in hyperthermophilic archaea.

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The quest for a thermostable sucrose phosphorylase reveals sucrose 6′-phosphate phosphorylase as a novel specificity

Verhaeghe

Aerts

Diricks

et al. 2014

Appl Microbiol Biotechnol

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Sucrose phosphorylase is a promising biocatalyst for the glycosylation of a wide range of compounds, but its industrial application has been hampered by the low thermostability of known representatives. Hence, in this study, the putative sucrose phosphorylase from the thermophile Thermoanaerobacterium thermosaccharolyticum was recombinantly expressed and fully characterised. The enzyme showed significant activity on sucrose (optimum at 55 °C), and with a melting temperature of 79 °C and a half-life of 60 h at the industrially relevant temperature of 60 °C, it is far more stable than known sucrose phosphorylases. Substrate screening and detailed kinetic characterisation revealed however a preference for sucrose 6'-phosphate over sucrose. The enzyme can thus be considered as a sucrose 6'-phosphate phosphorylase, a specificity not yet reported to date. Homology modelling and mutagenesis pointed out particular residues (Arg134 and His344) accounting for the difference in specificity. Moreover, phylogenetic and sequence analysis suggest that glycoside hydrolase 13 subfamily 18 might harbour even more specificities. In addition, the second gene residing in the same operon as sucrose 6'-phosphate phosphorylase was identified as well, and found to be a phosphofructokinase. The concerted action of both these enzymes implies a new pathway for the breakdown of sucrose, in which the reaction products end up at different stages of the glycolysis.

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Cloning and sequencing of kojibiose phosphorylase gene from Thermoanaerobacter brockii ATCC35047

Cited by 24 publications

References 18 publications

Improvement of the Enzymatic Properties of Kojibiose Phosphorylase from Thermoanaerobacter brockii by Random Mutagenesis and Chimerization

Improvement of the Enzymatic Properties of Kojibiose Phosphorylase from Thermoanaerobacter brockii by Random Mutagenesis and Chimerization

Identification and Characterization of an Archaeal Kojibiose Catabolic Pathway in the Hyperthermophilic Pyrococcus sp. Strain ST04

The quest for a thermostable sucrose phosphorylase reveals sucrose 6′-phosphate phosphorylase as a novel specificity

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