Isolation and analysis of genes for amylolytic enzymes of the hyperthermophilic bacterium Thermotoga maritima

Bibel, M

doi:10.1016/s0378-1097(97)00420-5

Cited by 43 publications

(81 citation statements)

References 13 publications

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“…(3) The C-terminal domain of MTase is not significantly related to other proteins. In family 13 glycosyl hydrolases/transferases, the (β/A) 8 -barrel is typically succeeded by a C-domain which may or may not bear sequence similarity to the C-domains of other members of the enzyme family. In those cases where crystal structures are known, i.e.…”

Section: Discussionmentioning

confidence: 99%

“…These facts, and the observation that addition of EDTA has no effect on transferase activity, leads to the conclusion that MTase is Ca 2ϩ -independent. In Ca 2ϩ -dependent A-amylases, the metal ion links domain B to the (β/A) 8 -barrel domain A (both A and B are involved in substrate binding), making the structure more rigid; the absence of this structural Ca 2ϩ ion in MTase could increase the flexibility of domain B. (3) Interestingly, MTase also lacks two highly conserved histidines at positions corresponding to His122 and His296 in the active site region of A. oryzae A-amylase (Thr206 and Pro467 in MTase).…”

Section: Discussionmentioning

confidence: 99%

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Thermotoga maritima maltosyltransferase, a novel type of maltodextrin glycosyltransferase acting on starch and malto‐oligosaccharides

Meissner¹,

Liebl²

1998

European Journal of Biochemistry

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A novel enzyme acting on starch and malto-oligosaccharides was identified and characterised. The non-hydrolytic enzyme, designated maltosyltransferase (MTase), of the hyperthermophilic bacterium Thermotoga maritima MSB8 disproportionates malto-oligosaccharides via glycosyl transfer reactions. The enzyme has a unique transfer specificity strictly confined to the transfer of maltosyl units. Incubation of MTase with starch or its constituents, i.e. amylose and amylopectin, led to the formation of a set of multiples of maltose (i.e. maltose, maltotetraose, maltohexaose etc.). Malto-oligosaccharides with a degree of polymerization (DP) X were disproportionated to products with a DP of X Ϯ2n (with Xу 3 and n ϭ 0,1, 2, ...). Maximum activity in a 10-min assay was recorded at pH 6.5 and 85Ϫ90°C. The enzyme displayed extraordinary resistance to thermal inactivation. For example, at 90, 85, and 70°C (pH 6.5, 0.34 mg ml Ϫ1 protein), MTase half-lives of about 2.5 h, 17 h, and 21 days, respectively, were recorded.The gene for MTase, designated mmtA, was isolated from a gene library of T. maritima strain MSB8. Analysis of the MTase primary structure as deduced from the nucleotide sequence of mmtA revealed that the enzyme is not closely related to known protein sequences. However, low-level local similarity between MTase and the A-amylase enzyme family (glycosyl hydrolase family 13) was detected, including conserved acidic residues essential for catalysis. Therefore, MTase should be assigned to this family. Based on detailed sequence analyses and comparison with amylolytic enzymes of known crystal structure we propose that MTase contains a (β/A) 8 -fold as the core supersecondary structure which is typical for the Aamylase family. On the other hand, MTase is unique in that it lacks several residues highly conserved throughout this family. Also, MTase possesses an extraordinarily large domain B (a domain typical for the A-amylase family, inserted between β-strand 3 and A-helix 3 of the (β/A) 8 -barrel fold).Keywords : glycosyl transfer; mmtA gene; nucleotide sequence; hyperthermophilic bacterium; thermostability.Enzymes from organisms inhabiting extremely hot environments represent fascinating objects for basic as well as applied research. On the one hand, the industry is interested in thermostable biocatalysts for biotechnological processes demanding elevated temperatures. On the other hand, considerable efforts have been made over the last years to understand how the proteins of extreme thermophiles maintain their structure and function at high temperatures (for a review, see [1]). We have focused our interests on the enzymes involved in carbohydrate breakdown and utilisation of the hyperthermophilic bacterium, Thermotoga maritima. This organism, which represents one of the deepest branches in the phylogenetic tree of the bacterial domain [2], is characterised by a growth range between 55°C and 90°C with an optimum at 80°C [3], making it one of the most thermophilic bacteria known to date. T. maritima MSB8 (DSM 3109), the typ...

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Thermotoga maritima maltosyltransferase, a novel type of maltodextrin glycosyltransferase acting on starch and malto‐oligosaccharides

Meissner¹,

Liebl²

1998

European Journal of Biochemistry

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“…T. neapolitana shares with other Thermotogales, specifically Thermotoga maritima, both the capacity to catabolize a wide variety of ␣-and ␤-linked glucans and a fermentative metabolism. While Thermotogales elaborate hydrolases such as amylases, cellulases, glucosidases, galactosidases, mannanases, and xylanases (6,7,10,11,23,37), neither T. neapolitana (D. Y. Yernool, G. Swiatek, and J.-D. Bok, unpublished data) nor T. maritima (27) has been shown to have a gene for a cellobiohydrolase, a key enzyme in classical cellulolytic systems.In this paper, we report the characterization of a catabolic gene cluster in T. neapolitana encoding two enzymes: a cellobiose phosphorylase (CbpA) and a ␤-glucan glucohydrolase (GghA). Cellobiose phosphorylases (EC 2.4.1.20) cleave cellobiose by phosphorolysis, yielding glucose-1-phosphate as one of the products, while ␤-glucan glucohydrolases or exoglucohydrolases (1,4-␤-D-glucan glucohydrolase [EC 3.2.1.74]) preferentially act on cellooligomers, releasing glucose (17,31,37,43).…”

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confidence: 99%

Cloning and Characterization of the Glucooligosaccharide Catabolic Pathway β-Glucan Glucohydrolase and Cellobiose Phosphorylase in the Marine HyperthermophileThermotoga neapolitana

et al. 2000

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Characterization in Thermotoga neapolitana of a catabolic gene cluster encoding two glycosyl hydrolases, 1,4-␤-D-glucan glucohydrolase (GghA) and cellobiose phosphorylase (CbpA), and the apparent absence of a cellobiohydrolase (Cbh) suggest a nonconventional pathway for glucan utilization in Thermotogales. GghA purified from T. neapolitana is a 52.5-kDa family 1 glycosyl hydrolase with optimal activity at pH 6.5 and 95°C. GghA releases glucose from soluble glucooligomers, with a preference for longer oligomers: k cat /K m values are 155.2, 76.0, and 9.9 mM ؊1 s ؊1 for cellotetraose, cellotriose, and cellobiose, respectively. GghA has broad substrate specificity, with specific activities of 236 U/mg towards cellobiose and 251 U/mg towards lactose. With p-nitrophenyl-␤-glucoside as the substrate, GghA exhibits biphasic kinetic behavior, involving both substrateand end product-directed activation. Its capacity for transglycosylation is a factor in this activation. Cloning of gghA revealed a contiguous upstream gene (cbpA) encoding a 93.5-kDa cellobiose phosphorylase. Recombinant CbpA has optimal activity at pH 5.0 and 85°C. It has specific activity of 11.8 U/mg and a K m of 1.42 mM for cellobiose, but shows no activity towards other disaccharides or cellotriose. With its single substrate specificity and low K m for cellobiose (compared to GghA's K m of 28.6 mM), CbpA may be the primary enzyme for attacking cellobiose in Thermotoga spp. By phosphorolysis of cellobiose, CbpA releases one activated glucosyl molecule while conserving one ATP molecule per disaccharide. CbpA is the first hyperthermophilic cellobiose phosphorylase to be characterized.To utilize polysaccharides such as glucans to meet carbon and energy requirements, heterotrophic organisms depend on a catabolic pathway involving the interaction of multiple hydrolytic enzymes, transporter complexes, and regulatory systems coordinating gene expression of pathway-specific proteins (41). During hydrolysis of the homopolymer cellulose, for example, the consortium of catalytic enzymes consists of endoglucanases (1,4-␤-D-glucan 4-glucohydrolase [EC 3.2.1.4]), which randomly hydrolyze internal ␤-1,4 glycosidic bonds; cellobiohydrolases (␤-D-glucan cellobiohydrolase [EC 3.2.1.91]), also referred to as exoglucanases, which remove cellobiose from either the nonreducing or reducing ends of cellooligomers; and ␤-glucosidases (␤-D-glucoside glucohydrolase [EC 3.2.1.21]), which convert cellobiose to glucose. These classes of enzymes have been documented in a number of fungal and bacterial systems (4, 5, 39), while other glucan-catabolyzing enzymes are less well understood.Thermotoga neapolitana, a marine hyperthermophile isolated from geothermally heated biotopes, belongs to the order Thermotogales. T. neapolitana shares with other Thermotogales, specifically Thermotoga maritima, both the capacity to catabolize a wide variety of ␣-and ␤-linked glucans and a fermentative metabolism. While Thermotogales elaborate hydrolases such as amylases, cellulases, glucosidases, galac...

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“…This domain is usually localized at the C-terminal end 393 of enzymes (Svensson et al 1989). A few exceptions are the Rhizopus oryzae glucoamylase (Ashikari et al 1986;Takahashi et al 1985), the Thermoactinomyces vulgaris "α-amylase" (Abe et al 2004) and the Thermotoga maritima pullulanase (Bibel et al 1998), which present their SBDs at the N-terminus. Production of α-amylase byB.…”

Section: Introductionmentioning

confidence: 99%

Molecular cloning, overexpression and characterization of the raw-starch-digesting α-amylase of Bacillus amyloliquefaciens

et al. 2010

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Raw starch is the most abundant source of glucose in the world. Therefore, finding enzymes capable of digesting raw starch would find high industrial demand. The α-amylase gene of Bacillus amyloliquefaciens ATCC 23842 was amplified, cloned and overexpressed in Escherichia coli BL21 (DE3) cells. The recombinant enzyme was purified to apparent homogeneity using ion exchange and gel filtration chromatography. The raw-starch digestibility of the purified enzyme was characterized by studying the hydrolysis and adsorption rate on a variety of raw starches (potato, cassava, corn, wheat and rice). The raw-starch digestion was further confirmed by scanning electron microscopy studies, which revealed an effective rate of hydrolysis. The kinetic studies revealed a relatively low Km of 2.76 mg/mL, exhibiting high affinity towards the soluble starch as the most preferred substrate and the inhibition kinetic studies revealed a high Ki value (350 mM).

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Isolation and analysis of genes for amylolytic enzymes of the hyperthermophilic bacterium Thermotoga maritima

Cited by 43 publications

References 13 publications

Thermotoga maritima maltosyltransferase, a novel type of maltodextrin glycosyltransferase acting on starch and malto‐oligosaccharides

Thermotoga maritima maltosyltransferase, a novel type of maltodextrin glycosyltransferase acting on starch and malto‐oligosaccharides

Cloning and Characterization of the Glucooligosaccharide Catabolic Pathway β-Glucan Glucohydrolase and Cellobiose Phosphorylase in the Marine HyperthermophileThermotoga neapolitana

Molecular cloning, overexpression and characterization of the raw-starch-digesting α-amylase of Bacillus amyloliquefaciens

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