Purification, Properties and Structural Aspects of a Thermoacidophilic α‐Amylase from <i>Alicyclobacillus Acidocaldarius</i> Atcc 27009

Schwermann, Birgit; Pfau, Karsten; Liliensiek, Birgit; Schleyer, Manfred; Fischer, Thomas; Bakker, Evert P.

doi:10.1111/j.1432-1033.1994.00981.x

Cited by 72 publications

(51 citation statements)

References 61 publications

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“…Selective agar plates (pH 4), containing basal salts, 0.5% konjac glucomannan, and 1.5% agar (w/v), were prepared as described elsewhere (26). Mud and water samples from an acidic hot spring in Tengchong (Yunnan province, China) were spread on the selective agar plates at 100 l/plate; this was followed by incubation for 48 h at 60°C.…”

Section: Methodsmentioning

confidence: 99%

“…The colonies thus obtained were replicated on selective agar plates and cultured. Mannanase-producing colonies were detected by flooding the plates with 0.1% (w/v) Congo Red solution (26,27). Strain Tc-12-31, which gave the largest clear lysis zone, was selected.…”

Section: Methodsmentioning

confidence: 99%

“…Then the strain was designated A. acidocaldarius Tc-12-31. To prepare genomic DNA, A. acidocaldarius Tc-12-31 was grown under the conditions described elsewhere (26), but the medium was devoid of glycerol and soluble starch. Escherichia coli DH5␣ (Invitrogen), E. coli BL21(DE3) (Novagen), and pUC18 (TaKaRa) and pET28a (Novagen) plasmids were used.…”

Section: Methodsmentioning

confidence: 99%

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Biochemical and Structural Characterization of the Intracellular Mannanase AaManA of Alicyclobacillus acidocaldarius Reveals a Novel Glycoside Hydrolase Family Belonging to Clan GH-A

Zhang

Peng

et al. 2008

Journal of Biological Chemistry

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An intracellular mannanase was identified from the thermoacidophile Alicyclobacillus acidocaldarius Tc-12-31. This enzyme is particularly interesting, because it shows no significant sequence similarity to any known glycoside hydrolase. Gene cloning, biochemical characterization, and structural studies of this novel mannanase are reported in this paper. The gene consists of 963 bp and encodes a 320-amino acid protein, AaManA. Based on its substrate specificity and product profile, AaManA is classified as an endo-␤-1,4-mannanase that is capable of transglycosylation. Kinetic analysis studies revealed that the enzyme required at least five subsites for efficient hydrolysis. The crystal structure at 1.9 Å resolution showed that AaManA adopted a (␤/␣) 8 -barrel fold. Two catalytic residues were identified: Glu 151 at the C terminus of ␤-stand ␤4 and Glu 231 at the C terminus of ␤7. Based on the structure of the enzyme and evidence of its transglycosylation activity, AaManA is placed in clan GH-A. Superpositioning of its structure with that of other clan GH-A enzymes revealed that six of the eight GH-A key residues were functionally conserved in AaManA, with the exceptions being residues Thr 95 and Cys 150 . We propose a model of substrate binding in AaManA in which Glu 282 interacts with the axial OH-C(2) in ؊2 subsites. Based on sequence comparisons, the enzyme was assigned to a new glycoside hydrolase family (GH113) that belongs to clan GH-A.Mannans are polysaccharides found in plants and consist of a backbone of ␤-1,4-linked mannose and glucose units. Mannose residues often carry an ␣-galactosyl substitute at O-6, and the degree of substitution depends on the plant of origin (Fig. 1A) (1). Mannans are widely distributed in nature in parts of the hemicellulose fraction in hardwoods and softwoods (1), legume seeds (2), and beans of carob trees (3).Endo-␤-1,4-mannanases (mannanases; EC 3.2.1.78) are glycoside hydrolases that randomly cleave the ␤-1,4-linkage in mannans (Fig. 1, A and B) (4); these enzymes have been isolated from bacteria, fungi, plants, and some mollusks (5-8). Interest in these enzymes has been increasing due to their importance in hemicellulose hydrolysis and various other applications (5, 9, 10). During the last 2 decades, more than 80 sequences of the catalytic domains of mannanase have been reported (see the CAZy site on the World Wide Web) and classified into glycoside hydrolase (GH) 2 families 5 and 26, based on their sequence similarities (11). In recent years, crystallographic resolution of the structures of these enzymes has yielded information on their structure. At present, the tertiary structures of seven mannanases have been determined, of which five are from GH family 5 (12-16) and two are from family 26 (17, 18). All of these mannanases share a common (␤/␣) 8 barrel fold, a retaining reaction mechanism (Fig. 1C), and two conserved catalytic residues (two glutamate residues at the C termini of ␤4 and ␤7, respectively). Therefore, all of these enzymes have been assigned to the same GH ...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Biochemical and Structural Characterization of the Intracellular Mannanase AaManA of Alicyclobacillus acidocaldarius Reveals a Novel Glycoside Hydrolase Family Belonging to Clan GH-A

Zhang

Peng

et al. 2008

Journal of Biological Chemistry

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“…Cleavage of the protein during the preparation is believed to be due to the action of an extracellular protease. 8 Dynamic light scattering experiments showed this sample represented a monomer of molecular weight 42 kDa in the temperature range þ 5 8C to þ 50 8C.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…This Gram-positive bacterium has a pH optimum of 3.6 and a temperature optimum of 57 8C. 5,6 Comparison of the core (ba) 8 -barrel domain of the secreted a-amylase (amylopullulanase) 7 from A. acidocaldarius with the available structures of homologous proteins from mesophilic organisms indicated that charged amino acid residues were often replaced with polar but uncharged ones in the thermoacidophile's protein, especially on the molecular surface. 8 This trend had not been identified in thermostable proteins, and so was believed to reflect the enzymes's acidostability.…”

Section: Introductionmentioning

confidence: 99%

X-ray Structures of the Maltose–Maltodextrin-binding Protein of the Thermoacidophilic Bacterium Alicyclobacillus acidocaldarius Provide Insight into Acid Stability of Proteins

Schäfer

Magnusson

Scheffel

et al. 2004

Journal of Molecular Biology

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Maltose-binding proteins act as primary receptors in bacterial transport and chemotaxis systems. We report here crystal structures of the thermoacidostable maltose-binding protein from Alicyclobacillus acidocaldarius, and explore its modes of binding to maltose and maltotriose. Further, comparison with the structures of related proteins from Escherichia coli (a mesophile), and two hyperthermophiles (Pyrococcus furiosus and Thermococcus litoralis) allows an investigation of the basis of thermo-and acidostability in this family of proteins.The thermoacidophilic protein has fewer charged residues than the other three structures, which is compensated by an increase in the number of polar residues. Although the content of acidic and basic residues is approximately equal, more basic residues are exposed on its surface whereas most acidic residues are buried in the interior. As a consequence, this protein has a highly positive surface charge. Fewer salt bridges are buried than in the other MBP structures, but the number exposed on its surface does not appear to be unusual. These features appear to be correlated with the acidostability of the A. acidocaldarius protein rather than its thermostability.An analysis of cavities within the proteins shows that the extremophile proteins are more closely packed than the mesophilic one. Proline content is slightly higher in the hyperthermophiles and thermoacidophiles than in mesophiles, and this amino acid is more common at the second position of b-turns, properties that are also probably related to thermostability. Secondary structural content does not vary greatly in the different structures, and so is not a contributing factor.

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Acidophiles

Oren¹

2010

Encyclopedia of Life Sciences

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Microorganisms growing in highly acidic environments (pH values below 3) are found in all three domains of life: Archaea, Bacteria and Eucarya. Such organisms thrive in acidic sulfur springs and in association with mining activities where microbial oxidation of pyrite and other reduced sulfur compounds lead to the formation of sulfuric acid. Acidophilic prokaryotes are also involved in the industrial leaching of copper and other metals from ores. Some acidophiles grow at high temperatures. Physiologically, the acidophiles are very diverse: there are aerobic and facultative anaerobic chemolithotrophs and different types of heterotrophic prokaryotes, photoautotrophic eukaryotes, predatory protozoa and others. Acidophilic microorganisms maintain their intracellular pH close to neutrality and their cytoplasmic membrane may support proton gradients up to five orders of magnitude; their membrane potential is often reversed in comparison with neutrophiles and alkaliphiles, with an intracellular positive charge. Key concepts: Microbial life is possible in strongly acidic environments and even at pH values below zero. The low pH of highly acidic environments in volcanic areas and environments associated with metal ore mining is caused by the activities of acidophilic microorganisms that oxidize reduced sulfur compounds to sulfuric acid. Acidophilic microorganisms are found in all three domains of life: Archaea, Bacteria and Eucarya. Unicellular eukaryotic green algae such as Dunaliella acidophila and Chlamydomonas acidophila perform photosynthesis at pH values down to 0–1 and the red alga Cyanidium caldarium grows as a photoautotroph in acidic hot springs up to 57°C and at pH<2–4. Some thermoacidophilic Archaea thrive at low pH up to very high temperatures. Sulfolobus and Acidanus spp. grow up to 96°C at pH 1–5; Picrophilus oshimae tolerates pH<0 and grows up to 65°C. Acidophilic microorganisms keep their intracellular pH at values close to neutrality and maintain a proton gradient over their cytoplasmic membranes of up to five orders of magnitude. In contrast to neutrophilic and alkaliphilic microorganisms that maintain an outside‐positive membrane potential, the membrane potential over the cytoplasmic membrane can be reversed, positive‐inside, in acidophilic microorganisms. Extracellular enzymes of acidophilic microorganisms are optimally active at low pH. Acid mine drainage is caused by the chemolithotrophic oxidation of pyrite and other reduced sulfur compounds in the ores by acidophilic prokaryotes such as Acidithiobacillus , Leptospirillum and Ferroplasma . The activities of the same types of organisms that cause acid mine drainage are exploited industrially for the bioleaching of copper and other metals from ores.

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Purification, Properties and Structural Aspects of a Thermoacidophilic α‐Amylase from Alicyclobacillus Acidocaldarius Atcc 27009

Cited by 72 publications

References 61 publications

Biochemical and Structural Characterization of the Intracellular Mannanase AaManA of Alicyclobacillus acidocaldarius Reveals a Novel Glycoside Hydrolase Family Belonging to Clan GH-A

Biochemical and Structural Characterization of the Intracellular Mannanase AaManA of Alicyclobacillus acidocaldarius Reveals a Novel Glycoside Hydrolase Family Belonging to Clan GH-A

X-ray Structures of the Maltose–Maltodextrin-binding Protein of the Thermoacidophilic Bacterium Alicyclobacillus acidocaldarius Provide Insight into Acid Stability of Proteins

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