Purification and sequence analysis of the atypical maltohexaose-forming α-amylase of the B. stearothermophilus US100

Ali, Mamdouh Ben; Mhiri, Sonda; Mezghani, Monia; Béjar, Samír

doi:10.1016/s0141-0229(01)00294-0

Cited by 65 publications

(29 citation statements)

References 17 publications

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“…Temperature optima for the catalytic activity of Bacillus spp. including B. stearothermophilus a-amylases are generally in the 50-80°C range (Vihinen and Mäntsälä 1990;Wind et al 1994;Ben Ali et al 2001). Thermostability experiments revealed that 50% of the original activity was retained after 45-min incubation at 80°C (Fig.…”

Section: Purification and Biochemical Characterizationmentioning

confidence: 97%

Atypical Ca2+-independent, raw-starch hydrolysing α-amylase from Bacillus sp. GRE1: characterization and gene isolation

Haki

Anceno

Rakshit

2008

World J Microbiol Biotechnol

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Bacillus sp. GRE1 isolated from an Ethiopian hyperthermal spring produced raw-starch digesting, Ca 2+ -independent thermostable a-amylase. Enzyme production in shake flask experiments using optimum nutrient supplements and environmental conditions was 2,360 U l -1 . Gel filtration chromatography yielded a purification factor of 33.6-fold and a recovery of 46.5%. The apparent molecular weight of the enzyme was 55 kDa as determined by SDS-PAGE. Presence or absence of Ca 2+ produced similar temperature optima of 65-70°C. The optimum pH was in the range of 5.5-6.0. The enzyme maintained 50% of its original activity after 45 min of incubation at 80°C and was stable at a pH range of 5.0-9.0. The V max and K m values for soluble starch were 42 mg reducing sugar min -1 and 4.98 mg starch ml -1 , respectively. Strong inhibitors of enzyme activity included Cu 2+ , Zn 2+ and Fe 2+ . The enzyme coding gene and the deduced protein translation revealed a characteristic but markedly atypical homology to Bacillus species a-amylase sequences. The enzyme hydrolyzed wheat, corn and tapioca starch granules efficiently below their gelatinization temperatures. Rather than the higher oligosaccharides normally produced by Bacillus a-amylases operating at high temperatures, maltose was the major hydrolysis product with the present enzyme.

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Section: Purification and Biochemical Characterizationmentioning

confidence: 97%

Atypical Ca2+-independent, raw-starch hydrolysing α-amylase from Bacillus sp. GRE1: characterization and gene isolation

Haki

Anceno

Rakshit

2008

World J Microbiol Biotechnol

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“…The optimum temperature was obtained measuring its activity, for 10 min at temperatures between 10 and 95°C ± 2°C, using some soluble starch solution in 0.1M acetate buffer at pH 4.8 (Aguilar et al, 2000;Ben Ali et al, 2001;Duedal-Olsen et al, 2000;Mohamed 2004;Wanderley et al, 2004). Initial rates of starch hydrolysis were determined at various substrate concentrations (0.01-20 mg/mL).…”

Section: Enzyme Characterizationmentioning

confidence: 99%

“…The kinetic constants K m and V max were estimated by Lineweaver-Burk method. SDS-PAGE was performed on mini-PROTEAN II cell (Bio-Rad, USA) with 12 % acrylamide gel, using buffer protein molecular weight marker (Aguilar et al, 2000;Ben Ali et al, 2001;Duedal-Olsen et al, 2000;Mohamed 2004). …”

Section: Enzyme Characterizationmentioning

confidence: 99%

“…It was probable that the molecular weights could be of α and β-amylases, as several authors have cited that molecular weight of the first enzyme, commonly, was between 50-230 kDa and of the second enzyme between 20-50 kDa (Dixon and Webb, 1971;Wiseman, 1987). According to literature, the molecular weight is dependent of α-amylase source; α-amylase from Bacillus clausii (Duedal-Olsen et al, 2000) and H. bacteriophora 47 kDa, from A. suum 83 kDa, from H. dromedarii 106 kDa (Mohamed, 2004), from B. stearothermophilus 59 kDa (Ben Ali et al, 2001) and from L. manihotivoras 135 kDa (Aguilar et al, 2000).…”

Section: +mentioning

confidence: 99%

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Production and characterization of amylases from Zea mays malt

Biazus

Souza

Marquez

et al. 2009

Braz. arch. biol. technol.

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“…The specific binding of an enzyme to its substrate has been exploited in several cases to constitute a useful purification step (Wilchek et al 1984). aamylases from various species have been purified to homogeneity by either binding to starch (Shaw et al 1995;Ben Ali et al 2001) or by using cross-linked starch Li & Geng 1992;Stredansky et al 1993). Not only the binding of an enzyme to the affinity column but also its subsequent elution constitutes an important step in purification.…”

Section: Introductionmentioning

confidence: 99%

Rapid method for the affinity purification of thermostable α-amylase from Bacillus licheniformis

Rao

Ratnam

VenkataRamesh

et al. 2005

World J Microbiol Biotechnol

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A rapid and efficient method the exploiting affinity of a-amylase for its substrate starch is described. a-amylase from Bacillus licheniformis was purified to homogeneity by ammonium sulphate precipitation and affinity chromatography with 230-fold purification. The a-amylase adsorption to various starches was examined in order to screen its ability for highest binding to starch. The a-amylase was bound to starch more tenaciously, hence various eluants like maltose, soluble starch and high salts could not elute the bound a-amylase. However, the bound a-amylase was instantly eluted using 2% (w/v) dextrin. The purified enzyme showed a single polypeptide on SDS-PAGE, with a molecular weight of 58 kD. Western blot analysis confirmed the specificity of antibody raised against purified a-amylase.

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Purification and sequence analysis of the atypical maltohexaose-forming α-amylase of the B. stearothermophilus US100

Cited by 65 publications

References 17 publications

Atypical Ca2+-independent, raw-starch hydrolysing α-amylase from Bacillus sp. GRE1: characterization and gene isolation

Atypical Ca2+-independent, raw-starch hydrolysing α-amylase from Bacillus sp. GRE1: characterization and gene isolation

Production and characterization of amylases from Zea mays malt

Rapid method for the affinity purification of thermostable α-amylase from Bacillus licheniformis

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