[Show abstract][Hide abstract]ABSTRACT: Anoxybacillus sp. DT3-1 is a newly isolated bacterium with amylolytic activity. The gene that encodes the α-amylase was recently cloned and expressed in E. coli system. However, the expression level was far too low to be used for further analysis. The main objective of this study is to enhance the recombinant α-amylase (ADTA) expression level extracellularly. In medium comparison, LB/amp medium was found to be the best medium to support the cell growth and extracellular ADTA production. Subsequently, three factors that affect the ADTA expression which are cells absorbance during induction, concentrations of IPTG and yeast extract were screened using 23 full factorial design. Cells absorbance during induction and IPTG concentration were found to be the significant variables that affected the ADTA production. In the consequently central composite rotatable design (CCRD), the optimized condition for maximum extracellular ADTA production was determined as OD600nm 1.52, 0.01 mM IPTG and 0.30% (w/v) yeast extract. The extracellular ADTA production was successfully increased from 30 U in the original medium to 82.29 U.
[Show abstract][Hide abstract]ABSTRACT: The Bacillaceae family members are a good source of bacteria for bioprocessing and biotransformation involving whole cells or enzymes. In contrast to Bacillus and Geobacillus, Anoxybacillus is a relatively new genus that was proposed in the year 2000. Because these bacteria are alkali-tolerant thermophiles, they are suitable for many industrial applications. More than a decade after the first report of Anoxybacillus, knowledge accumulated from fundamental and applied studies suggests that this genus can serve as a good alternative in many applications related to starch and lignocellulosic biomasses, environmental waste treatment, enzyme technology, and possibly bioenergy production. This current review provides the first summary of past and recent discoveries regarding the isolation of Anoxybacillus, its medium requirements, its proteins that have been characterized and cloned, bioremediation applications, metabolic studies, and genomic analysis. Comparisons to some other members of Bacillaceae and possible future applications of Anoxybacillus are also discussed.
Full-text · Article · Jan 2013 · Applied Microbiology and Biotechnology
[Show abstract][Hide abstract]ABSTRACT: Two thermophilic bacteria (SK3-4 and DT3-1) were isolated from the Sungai Klah (SK) and Dusun Tua (DT) hot springs in Malaysia. The cells from both strains were rod-shaped, stained Gram positive and formed endospores. The optimal growth of both strains was observed at 55 degrees C and pH 7. Strain DT3-1 exhibited a higher tolerance to chloramphenicol (100 microg ml(-1)) but showed a lower tolerance to sodium chloride (2%, w/v) compared to strain SK3-4. Phylogenetic analysis based on 16S rRNA gene sequences revealed that both strains belong to the genus Anoxybacillus. High concentrations of 15:0 iso in the fatty acid profiles support the conclusion that both strains belong to the genus Anoxybacillus and exhibit unique fatty acid compositions and percentages compared to other Anoxybacillus species. The DNA G + C contents were 42.0 mol% and 41.8 mol% for strains SK3-4 and DT3-1, respectively. Strains SK3-4 and DT3-1 were able to degrade pullulan and to produce maltotriose and glucose, respectively, as their main end products. Based on phenotypic and chemotaxonomic characteristics, 16S rRNA gene sequences, and the DNA G + C content, we propose that strains SK3-4 and DT3-1 are new pullulan-degrading Anoxybacillus strains.
Full-text · Article · Jun 2012 · Environmental Technology
[Show abstract][Hide abstract]ABSTRACT: Amino acid sequence alignment between ASKA, ADTA, and known α-amylases. Enzyme sources are abbreviated as follows: ASKA, strain SK3-4; ADTA, strain DT3-1; BLA, Bacillus licheniformis (P06278); BSTA, Bacillus stearothermophilus (P06279); TAKA, Aspergillus oryzae (P0C1B3); and AmyA, Anaerobranca gottschalkii (Q5I942). The seven highly conserved regions among amylases are boxed; the three active site residues are highlighted; and the different amino acids between ASKA and ADTA are shown in white text
[Show abstract][Hide abstract]ABSTRACT: Two genes that encode α-amylases from two Anoxybacillus species were cloned and expressed in Escherichia coli. The genes are 1,518 bp long and encode 506 amino acids. Both sequences are 98% similar but are distinct from other well-known α-amylases. Both of the recombinant enzymes, ASKA and ADTA, were purified using an α-CD-Sepharose column. They exhibited an optimum activity at 60°C and pH 8. Both amylases were stable at pH 6-10. At 60°C in the absence of Ca²⁺, negligible reduction in activity for up to 48 h was observed. The activity half-life at 65°C was 48 and 3 h for ASKA and ADTA, respectively. In the presence of Ca²⁺ ions, both amylases were highly stable for at least 48 h and had less than a 10% decrease in activity at 70°C. Both enzymes exhibited similar end-product profiles, and the predominant yield was maltose (69%) from starch hydrolysis. To the best of our knowledge, most α-amylases that produce high levels of maltose are active at an acidic to neutral pH. This is the first report of two thermostable, alkalitolerant recombinant α-amylases from Anoxybacillus that produce high levels of maltose and have an atypical protein sequence compared with known α-amylases.
Full-text · Article · Jan 2012 · Journal of Industrial Microbiology
[Show abstract][Hide abstract]ABSTRACT: Trehalose has similar chemical formula as maltose. In terms of price, trehalose is much more expensive than maltose. A pink-pigmented bacterium identified as Meiothermus sp. SK3-2 was found to be able to convert maltose to trehalose. Based on fatty acids analysis, the Meiothermus sp. SK3-2 may be a new strain that produce trehalose synthase (TreS). Meiothermus sp. SK3-2 achieved a better biomass growth in MM medium containing maltose. Besides that, TreS activity yield was also higher in MM medium, approximately 3.5, 1.8 and 0.3 fold than that in PY medium, thermophilic Bacillus medium and Castenholz medium, respectively. The optimum working temperature and pH for Meiothermus sp. SK3-2 TreS was 65°C and pH 6.0, respectively. Ammonium chloride at 10 mM increased the activity significantly, while calcium chloride at 5 mM decreased the activity by about 80% and the activity was fully retarded by 10 mM CaCl 2. It was found that the product specificity of this TreS was influenced by factors like temperature, pH and buffer system used. Analysis of the nucleotide sequence revealed the presence of an open reading frame of 2,890 bp which encoded a 963 amino acid protein. In conclusion, Meiothermus sp. SK3-2 TreS could serve as an alternative source to trehalose production.
No preview · Article · Oct 2011 · AFRICAN JOURNAL OF BIOTECHNOLOGY