Cloning and characterization of a thermostable intracellular α-amylase gene from the hyperthermophilic bacterium Thermotoga maritima MSB8

Lim, Woo Jin; Park, Sang Ryeol; An, Chang Long; Lee, Jong Yeoul; Hong, Su Young; Shin, Eun Chule; Kim, Sang Hyun; Kim, Jong Ok; Kim, Hoon; Yun, Han Dae

doi:10.1016/j.resmic.2003.09.005

Cited by 39 publications

(20 citation statements)

References 17 publications

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“…The active fractions were combined and purified by ion exchange chromatography on a Q-Sepharose column (Amersham Pharmacia Biotech, Pennsylvania, USA). 30) For the final purification step, the dialyzed sample was loaded onto an anion exchange Mono Q HR (Amersham Pharmacia Biotech, Pennsylvania, USA) column preequilibrated with 20 mM MOPS buffer, pH 6.5. The fractions with -glucosidase activity eluted as a single protein peak and the purity of the enzyme was assessed by SDS-PAGE.…”

Section: Methodsmentioning

confidence: 99%

Cloning and Comparison of Third β-Glucoside Utilization (bglEFIA) Operon with Two Operons ofPectobacterium carotovorumsubsp.carotovorumLY34

Hong¹,

An²,

Cho³

et al. 2006

Bioscience, Biotechnology, and Biochemistry

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Section: Methodsmentioning

confidence: 99%

Cloning and Comparison of Third β-Glucoside Utilization (bglEFIA) Operon with Two Operons ofPectobacterium carotovorumsubsp.carotovorumLY34

Hong¹,

An²,

Cho³

et al. 2006

Bioscience, Biotechnology, and Biochemistry

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“…Considerable genomic plasticity has been observed even within the Thermotoga genus, with respect to the gene content of carbohydrate active enzymes and transporter subunits, which may to some extent relate to lateral gene transfer events (48,49). Despite the range of sugar-active enzymes found within T. maritima MSB8 genome (Table S1 in the supplemental material) (6,9,10,23,27,34,35,37,38,39,42,45,46,54,70,71), a PTS (phosphoenolpyruvatedependent phosphotransferase system) similar to those used by other species for preferential uptake of selected sugars is apparently absent (48). No homologs of the PTS components EI and HPr (phosphocarrier proteins) and no sugar-specific EII sugar transporter subunits have been identified in the Thermotogales.…”

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An Expression-Driven Approach to the Prediction of Carbohydrate Transport and Utilization Regulons in theHyperthermophilic BacteriumThermotoga maritima

et al. 2005

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Comprehensive analysis of genome-wide expression patterns during growth of the hyperthermophilic bacterium Thermotoga maritima on 14 monosaccharide and polysaccharide substrates was undertaken with the goal of proposing carbohydrate specificities for transport systems and putative transcriptional regulators. Saccharide-induced regulons were predicted through the complementary use of comparative genomics, mixedmodel analysis of genome-wide microarray expression data, and examination of upstream sequence patterns. The results indicate that T. maritima relies extensively on ABC transporters for carbohydrate uptake, many of which are likely controlled by local regulators responsive to either the transport substrate or a key metabolic degradation product. Roles in uptake of specific carbohydrates were suggested for members of the expanded Opp/Dpp family of ABC transporters. In this family, phylogenetic relationships among transport systems revealed patterns of possible duplication and divergence as a strategy for the evolution of new uptake capabilities. The presence of GC-rich hairpin sequences between substrate-binding proteins and other components of Opp/Dpp family transporters offers a possible explanation for differential regulation of transporter subunit genes. Numerous improvements to T. maritima genome annotations were proposed, including the identification of ABC transport systems originally annotated as oligopeptide transporters as candidate transporters for rhamnose, xylose, ␤-xylan, and ␤-glucans and identification of genes likely to encode proteins missing from current annotations of the pentose phosphate pathway. Beyond the information obtained for T. maritima, the present study illustrates how expression-based strategies can be used for improving genome annotation in other microorganisms, especially those for which genetic systems are unavailable.Thermotoga maritima, a hyperthermophilic anaerobe with an optimal growth temperature of 80°C, has been found in diverse high-temperature locations and is capable of using a wide variety of simple and complex carbohydrate substrates for growth. The complexity of its carbohydrate utilization strategies, revealed by genome sequencing (48) and through previous work (11,12,47,51), is surprising, given the primitive features of this microorganism. Considerable genomic plasticity has been observed even within the Thermotoga genus, with respect to the gene content of carbohydrate active enzymes and transporter subunits, which may to some extent relate to lateral gene transfer events (48,49). Despite the range of sugar-active enzymes found within T. maritima MSB8 genome (Table S1 in the supplemental material) (6,9,10,23,27,34,35,37,38,39,42,45,46,54,70,71), a PTS (phosphoenolpyruvatedependent phosphotransferase system) similar to those used by other species for preferential uptake of selected sugars is apparently absent (48). No homologs of the PTS components EI and HPr (phosphocarrier proteins) and no sugar-specific EII sugar transporter subunits have been identified ...

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“…maritima has the highest number of genes for carbohydrate-degrading and -modifying enzymes in relation to its 1.8-Mbp genome known to date in free-living prokaryotes (19). Among the polysaccharide-degrading enzymes of T. maritima described so far are two ␣-amylases, one an extracellular putative lipoprotein (AmyA) (15) and one located in the cytoplasm (AmyB) (16).…”

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Identification and Characterization of a Novel Intracellular Alkaline α-Amylase from the Hyperthermophilic Bacterium Thermotoga maritima MSB8

Ballschmiter

Fütterer

Liebl

2006

Appl Environ Microbiol

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The gene for a novel ␣-amylase, designated AmyC, from the hyperthermophilic bacterium Thermotoga maritima was cloned and heterologously overexpressed in Escherichia coli. The putative intracellular enzyme had no amino acid sequence similarity to glycoside hydrolase family (GHF) 13 ␣-amylases, yet the range of substrate hydrolysis and the product profile clearly define the protein as an ␣-amylase. Based on sequence similarity AmyC belongs to a subgroup within GHF 57. On the basis of amino acid sequence similarity, Glu185 and Asp349 could be identified as the catalytic residues of AmyC. Using a 60-min assay, the maximum hydrolytic activity of the purified enzyme, which was dithiothreitol dependent, was found to be at 90°C. AmyC displayed a remarkably high pH optimum of pH 8.5 and an unusual sensitivity towards both ATP and EDTA.␣-Amylases (EC 3.2.1.1) are glycoside hydrolases which cleave the ␣-1,4-glycosidic linkages in starch and maltodextrins with an endo mechanism, i.e., in a random fashion within the polysaccharide molecule. Due to the retaining catalytic mechanism the released cleavage products have an ␣-anomeric configuration. Amylases are of commercial interest, e.g., for starch processing in the food industry. In the amino acid sequencebased classification system for carbohydrate active enzymes (CAZY) the vast majority of ␣-amylases are sorted into the glycoside hydrolase family (GHF) 13 (6). All enzymes of this family share a (␤/␣) 8 -barrel as the common supersecondary structure. Some ␣-amylases are also classified into GHF 57. This enzyme family is considerably smaller than GHF 13 and less well investigated.The hyperthermophilic bacterium Thermotoga maritima was first isolated from geothermally heated marine sediments (7). The bacterium grows anaerobically at temperatures up to 90°C with an optimum at 80°C. As an obligate heterotrophic organism it utilizes various polysaccharides and proteinaceous substrates. Sugars are mainly metabolized via the classical, unmodified Embden-Meyerhof pathway and to 15% via the EntnerDoudoroff pathway (7,23,24).T. maritima has the highest number of genes for carbohydrate-degrading and -modifying enzymes in relation to its 1.8-Mbp genome known to date in free-living prokaryotes (19). Among the polysaccharide-degrading enzymes of T. maritima described so far are two ␣-amylases, one an extracellular putative lipoprotein (AmyA) (15) and one located in the cytoplasm (AmyB) (16).Here we report the recombinant expression in Escherichia coli and the biochemical characterization of an additional intracellular ␣-amylase from T. maritima. The enzyme does not belong in the GHF 13 amylase group but has significant similarity to the less well characterized GHF 57. MATERIALS AND METHODSLibrary screening. A T. maritima MSB8 (type strain) chromosomal gene library, whose construction was described earlier (22), was screened for amylolytic activity. E. coli JM83 transformant colonies were grown overnight at 37°C on Luria-Bertani agar plates containing 0.5% soluble starch (Merck, Darmstadt...

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Cloning and characterization of a thermostable intracellular α-amylase gene from the hyperthermophilic bacterium Thermotoga maritima MSB8

Cited by 39 publications

References 17 publications

Cloning and Comparison of Third β-Glucoside Utilization (bglEFIA) Operon with Two Operons ofPectobacterium carotovorumsubsp.carotovorumLY34

Cloning and Comparison of Third β-Glucoside Utilization (bglEFIA) Operon with Two Operons ofPectobacterium carotovorumsubsp.carotovorumLY34

An Expression-Driven Approach to the Prediction of Carbohydrate Transport and Utilization Regulons in theHyperthermophilic BacteriumThermotoga maritima

Identification and Characterization of a Novel Intracellular Alkaline α-Amylase from the Hyperthermophilic Bacterium Thermotoga maritima MSB8

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