Comparison of primary structures and substrate specificities of two pullulan-hydrolyzing α-amylases, TVA I and TVA II, from Thermoactinomyces vulgaris R-47

Tonozuka, Takashi; Mogi, Shin-ichi; Shimura, Yoichiro; Shimizu‐Ibuka, Akiko; Sakai, Hiroshi; Matsuzawa, Hiroshi; Sakano, Yuji; Ohta, Takao

doi:10.1016/0167-4838(95)00101-y

Cited by 59 publications

(41 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The pH range of AmyC activity (pH 7.5 to 9.0) is unusually basic for an intracellular enzyme. Other ␣-amylases known to be local- ized in the cytoplasm are reported to have a pH optimum at neutral pH or below, e.g., AmyA from E. coli has a pH optimum of pH 7.2, PFIA from Pyrococcus furiosus has a pH optimum of pH 7.0, and AmyC from the thermoalkaliphilic Anaerobranca gottschalkii and TVAII from Thermoactinomyces vulgaris display maximum activity at pH 6.0 (2,13,21,26). The activity of AmyC is reduced by nucleotide triphosphates.…”

Section: Discussionmentioning

confidence: 99%

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

Ballschmiter

Fütterer

Liebl

2006

Appl Environ Microbiol

View full text Add to dashboard Cite

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...

show abstract

Section: Discussionmentioning

confidence: 99%

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

Ballschmiter

Fütterer

Liebl

2006

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…This specificity of CDs is similar to that of -amylase I from Thermoactinomyces vulgaris R-47, which is a neopullulanase-like -amylase, that hydrolyzes starch, pullulan, and CDs. 27) But AmyL did not hydrolyze pullulan, which is a good substrate for T. vulgaris R-47 -amylase I.…”

Section: 9)mentioning

confidence: 96%

Purification and Characterization of a Liquefying α-Amylase from Alkalophilic ThermophilicBacillussp. AAH-31

Kim

Morimoto

Saburi

et al. 2012

Bioscience, Biotechnology, and Biochemistry

View full text Add to dashboard Cite

“…Kinetic parameters (including parameters for pNPG) were determined as follows: A reaction mixture (1 ml) containing the substrate, 50 mM McIlvaine buffer (pH 6.5), and bsPNPGH or bsO16G was incubated at 60 C, and the portions (125 ml) were taken at 5-min intervals to confirm the linearity of the reaction. The reaction was stopped by adding equal amounts of 40 mM sodium hydroxide, and the amount of liberated glucose was measured using glucose-oxidase and peroxidase as described 1,14) for all the substrates listed in Table 2. For evaluation of the ratio of the enzymatic activities for trehalose 6-phosphate and pNPG (5 mM each), 10 mM Tris-HCl buffer (pH 7.0) was used instead of McIlvaine buffer to eliminate the effect of phosphate on the hydrolysis.…”

Section: )mentioning

confidence: 99%

Molecular Cloning and Characterization of an Enzyme Hydrolyzingp-Nitrophenyl α-D-Glucoside fromBacillus stearothermophilusSA0301

Kobayashi

Tonozuka

Sato

et al. 2006

Bioscience, Biotechnology, and Biochemistry

Self Cite

View full text Add to dashboard Cite

Comparison of primary structures and substrate specificities of two pullulan-hydrolyzing α-amylases, TVA I and TVA II, from Thermoactinomyces vulgaris R-47

Cited by 59 publications

References 26 publications

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

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

Purification and Characterization of a Liquefying α-Amylase from Alkalophilic ThermophilicBacillussp. AAH-31

Molecular Cloning and Characterization of an Enzyme Hydrolyzingp-Nitrophenyl α-D-Glucoside fromBacillus stearothermophilusSA0301

Contact Info

Product

Resources

About