24 research outputs found
Introduction of novel thermostable alpha-amylases from genus Anoxybacillus and proposing to group the Bacillaceae related alpha-amylases under five individual GH13 subfamilies
No full textAmong the thermophilic Bacillaceae family members, alpha-amylase production of 15 bacilli from genus Anoxybacillus was investigated, some of which are biotechnologically important. These Anoxybacillus alpha-amylase genes displayed ae 91.0% sequence similarities to Anoxybacillus enzymes (ASKA, ADTA and GSX-BL), but relatively lower similarities to Geobacillus (ae 69.4% to GTA, Gt-amyII), and Bacillus aquimaris (ae 61.3% to BaqA) amylases, all formerly proposed only in a Glycoside Hydrolase 13 (GH13) subfamily. The phylogenetic analyses of 63 bacilli-originated protein sequences among 93 alpha-amylases revealed the overall relationships within Bacillaceae amylolytic enzymes. All bacilli alpha-amylases formed 5 clades different from 15 predefined GH13 subfamilies. Their phylogenetic findings, taxonomic relationships, temperature requirements, and comparisonal structural analyses (including their CSR-I-VII regions, 12 sugar- and 4 calcium-binding sites, presence or absence of the complete catalytic machinery, and their currently unassigned status in a valid GH13 subfamiliy) revealed that these five GH13 alpha-amylase clades related to familly share some common characteristics, but also display differentiative features from each other and the preclassified ones. Based on these findings, we proposed to divide Bacillaceae related GH13 subfamilies into 5 individual groups: the novel a2 subfamily clustered around alpha-amylase B2M1-A (Anoxybacillus sp.), the a1, a3 and a4 subfamilies (including the representatives E184aa-A (Anoxybacillus sp.), ATA (Anoxybacillus tepidamans), and BaqA,) all of which were composed from the division of the previously grouped single subfamily around alpha-amylase BaqA, and the undefinite subfamily formerly defined as xy including Bacillus megaterium NL3
Taxonomic Classification of Anoxybacillus Isolates from Geothermal Regions in Turkey by 16S rRNA Gene Sequences and ARDRA, ITS-PCR, Rep-PCR Analyses
No full textA total of 115 endospore-forming bacilli were taken for 16S rRNA gene sequence analyses and clustered among 7 genera. In this paper, the most abundant thermophiles belonging to genus Anoxybacillus with its 53 isolates are presented. The Anoxybacillus species, some of which were producing biotechnologically valuable enzymes, mostly displayed amylolytic and glucosidic activities and the ability of carbohydrate degradation made them superior in number among the other bacilli in these extreme habitats. In comparative sequence analyses, similarities ranged from 91.1% to 99.9% between the isolates and the type strains. Isolates were clustered into eight phylogenetic lineages within the type strains of A. kamchatkensis, A. flavithermus, A. kamchatchensis subsp. asaccharedens, and A. salavatliensis. In addition, C161ab and A321 were proposed as novel species which displayed < 97.0% similarities to their closest relatives. Moreover, their individual AluI, HaeIII, and TaqI ARDRA restriction patterns, ITS-, (GTG)(5)-, and BOX-PCR fingerprintings generated 27, 28, 31, 35, 40, and 41 clusters, respectively. The twelve type strains and 35 of the isolates showed unique distinctive patterns from all the others at least in two of these analyses. These phenotypic and genomic characters allowed us to differentiate their genotypic diversity from the reference strains
<i>Anoxybacillus salavatliensis</i>sp. nov., an α-glucosidase producing, thermophilic bacterium isolated from Salavatli, Turkey
No full textA novel moderately thermophilic, Gram-positive staining, rod-shaped, spore-forming, motile, facultative anaerobic, and a-glucosidase producing strain A343(T), was isolated from a high temperature well-pipeline sediment sample in Salavatli province of Aydin, Turkey. Growth was observed at 37-69 degrees C (optimum 60 degrees C), at pH 5.5-9.5 (optimum 8.0-9.0) and at salinities from 0 to 4.5% (w/v) (optimum 2%). Strain A343(T) was able to grow on a wide range of carbon sources. Gelatin and starch utilization, amylase, catalase and oxidase activities, reduction of nitrate to nitrite were all positive. The G+C content of the genomic DNA was 45.1 mol%. The major menaquinone was MK-7. The dominant cellular fatty acids were: iso-C15:0, C16:0, and iso-C17:0. The phylogenetic analysis based on the 16S rRNA gene sequence revealed that the strain A343(T) belonged to the genus Anoxybacillus. The 16S rRNA gene sequence similarity between strain A343(T) and the type strains of recognized Anoxybacillus species was ranged from 95.8 to 99.4%. DNA-DNA hybridization revealed low homology with its closest relative Anoxybacillus kamchatkensis (49.7%). In addition to the total cell protein profiles, the Rep-PCR and the intergenic 16S-23S rRNA gene fingerprinting profiles differentiated strain A343(T) from all of the reference Anoxybacillus species used. Based upon phenotypic, phylogenetic and chemotaxonomic evidence, strain A343(T) was assigned to a new species within the genus Anoxybacillus, A. salavatliensis sp. nov. (The type strain A343(T) = DSM 22626(T) = NCIMB 14579(T)). The 16S rRNA gene nucleotide sequence of strain A343(T) is available in the GenBank database under the accession number - EU326496
Mathematical Models for the Biofilm Formation of Geobacillus and Anoxybacillus on Stainless Steel Surface in Whole Milk
No full textBiofilm formation of Geobacillus thermodenitrificans, Geobacillus thermoglucosidans and Anoxybacillus flavithermus in milk on stainless steel were monitored at 55 degrees C, 60 degrees C, and 65 degrees C for various incubation times. Although species of Geobacillus showed a rapid response and produced biofilm within 4 h on stainless steel, a delay (lag time) was observed for Anoxybacillus. A hyperbolic equation and a hyperbolic equation with lag could be used to describe the biofilm formation of Geobacillus and Anoxybacillus, respectively. The highest biofilm formation amount was obtained at 60 degrees C for both Geobacillus and Anoxybacillus. However, the biofilm formation rates indicated that the lowest rates of formation were obtained at 60 degrees C for Geobacillus. Moreover, biofilm formation rates of G. thermodenitrificans (1.2-1.6 Log(10)CFU/mL.h) were higher than G. thermoglucosidans (0.4-0.7 Log(10)CFU/mL.h). Although A. flavithermus had the highest formation rate values (2.7-3.6 Log(10)CFU/mL.h), this was attained after the lag period (4 or 5 h). This study revealed that modeling could be used to describe the biofilm formation of thermophilic bacilli in milk
Purification and characterization of intracellular and extracellular α-glucosidases from Geobacillus toebii strain E134
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Anoxybacillus and Geobacillus biofilms in the dairy industry: effects of surface material, incubation temperature and milk type
No full textAnoxybacillus (A. flavithermus, A. kamchatkensis subsp. asachharedens, A. caldiproteolyticus and A. tepidamans) and Geobacillus (two strains of G. thermodenitrificans, G. thermoglucosidans and G. vulcanii) isolates and reference strains in whole milk were evaluated for their biofilm production on six different abiotic surfaces. G. thermodenitrificans DSM 465(T) had the highest cell counts (>4 log(10) CFU cm(-2)) on glass and stainless steel (SS) at 55 and 65 degrees C, respectively. G. thermodenitrificans D195 had the highest counts on SS at 55 degrees C (>5 log(10) CFU cm(-2)) and polyvinyl chloride (PVC) at 65 degrees C (>4 log(10) CFU cm(-2)), indicating the existence of strain variation. The ideal surfaces for all strains were SS and glass at 55 degrees C, but their preferences were polystyrene and SS at 65 degrees C. Moreover, Anoxybacillus members were more prone to form biofilms in skim milk than in semi-skim and whole milk, whereas the results were the opposite for Geobacillus. Both the attachment and sporulation of Geobacillus in whole milk was higher than in semi-skim or skim milk. This study proposes that the surface material, temperature and milk type had a cumulative effect on biofilm formation
Determination of the biofilm production capacities and characteristics of members belonging to Bacillaceae family
No full textThe biofilm characteristics of many endosporeforming bacilli, especially the thermophiles are still unclear. In this study, a detailed identification and description of biofilm production characteristics of totally 145 isolates and reference strains belonging to Bacillaceae family, displaying thermophilic (n = 115), facultative thermophilic (n = 24) and mesophilic (n = 6) growth from genera Anoxybacillus, Geobacillus, Thermolongibacillus, Aeribacillus, Brevibacillus, Paenibacillus and Bacillus were presented. The incubation temperatures were adjusted to 37, 45 and 55-65 degrees C for mesophiles, facultative thermophiles, and thermophiles, respectively. The bacilli were evaluated based on their colony morphotypes on Congo red (CR) agar, their complex exopolysaccharide production on calcofluor supplemented tryptic soy agar, and as well as their pellicle formation at the liquid-air surface in tryptic soy broth cultures. Their biofilm production capabilities were also tested on abiotic surfaces of both polystyrene and stainless steel by crystal violet binding assay and viable biofilm cell enumerations, respectively. As a result, the biofilm production capacities of Bacillaceae members from genera to species level, the effects of osmolarity, temperature, incubation time and abiotic surfaces on biofilm formation as well as the CR morphotypes associated with the biofilm production were able to reveal in a wide group of bacilli. Besides, general enrichment-inoculation approaches and methodologies were also offered, which allow and facilitate the screening and determining the biofilm producing endospore forming bacilli
Phylogenetic diversity of isolates belonging to genera Geobacillus and Aeribacillus isolated from different geothermal regions of Turkey
No full textThe phylogenetic diversity of 31 thermophilic bacilli belonging to genera Geobacillus and Aeribacillus were investigated which were isolated from various geothermal sites of Turkey. Twenty-seven of these isolates were found to be belonged within the genus Geobacillus, whereas 4 of them were identified as Aeribacillus pallidus. The comparative 16S rRNA gene sequence analyses revealed that the A. pallidus isolates displayed sequence similarity values from 98.0 to 99.6% to their closest relative. Furthermore, Geobacillus isolates showed sequence similarity values from 88.9 to 99.8% with the reference type strains. According to the phylogenetic analysis, isolates belonging to genus Geobacillus were diverged into nine clusters and among these isolates, 19 of them were identified as strains related to G. caldoproteolyticus, G. thermodenitrificans, G. stearothermophilus, G. thermoglucosidasius and G. toebii with the most abundant 13 isolates from G. caldoproteolyticus. Four of the Geobacillus isolates were named as unidentified mix group, as they found to be genetically very homogenous like their closely related type species: G. thermoleovorans, G. vulcani, G. lituanicus, G. kaustophilus, G. caldovelox, G. caldotenax, and G. uralicus. Moreover, the sequence comparisons of E173a, E265, C161ab and A142 isolates demonstrated that they represented novel species among genus Geobacillus as they shared lower than 96.7% sequence similarity to all the described type species. The AluI-, HaeIII- and TaqI-ARDRA results were in congruence with the 16S rRNA gene sequence analyses. By ARDRA results, the isolates were able to be differentiated and clustered, the discriminative restriction fragments of these isolates and type species were determined and the novelty of E173, E265, C161ab and A142 isolates could be displayed. Some differentiating phenotypic characters and the ability of amylase, glucosidase and protease production of these bacilli were also studied and biotechnologically valuable thermostable enzyme producing isolates were introduced in order to use in further studies
Introduction of novel thermostable α-amylases from genus Anoxybacillus and proposing to group the Bacillaceae related α-amylases under five individual GH13 subfamilies
No full textAmong the thermophilic Bacillaceae family members, alpha-amylase production of 15 bacilli from genus Anoxybacillus was investigated, some of which are biotechnologically important. These Anoxybacillus alpha-amylase genes displayed ae 91.0% sequence similarities to Anoxybacillus enzymes (ASKA, ADTA and GSX-BL), but relatively lower similarities to Geobacillus (ae 69.4% to GTA, Gt-amyII), and Bacillus aquimaris (ae 61.3% to BaqA) amylases, all formerly proposed only in a Glycoside Hydrolase 13 (GH13) subfamily. The phylogenetic analyses of 63 bacilli-originated protein sequences among 93 alpha-amylases revealed the overall relationships within Bacillaceae amylolytic enzymes. All bacilli alpha-amylases formed 5 clades different from 15 predefined GH13 subfamilies. Their phylogenetic findings, taxonomic relationships, temperature requirements, and comparisonal structural analyses (including their CSR-I-VII regions, 12 sugar- and 4 calcium-binding sites, presence or absence of the complete catalytic machinery, and their currently unassigned status in a valid GH13 subfamiliy) revealed that these five GH13 alpha-amylase clades related to familly share some common characteristics, but also display differentiative features from each other and the preclassified ones. Based on these findings, we proposed to divide Bacillaceae related GH13 subfamilies into 5 individual groups: the novel a2 subfamily clustered around alpha-amylase B2M1-A (Anoxybacillus sp.), the a1, a3 and a4 subfamilies (including the representatives E184aa-A (Anoxybacillus sp.), ATA (Anoxybacillus tepidamans), and BaqA,) all of which were composed from the division of the previously grouped single subfamily around alpha-amylase BaqA, and the undefinite subfamily formerly defined as xy including Bacillus megaterium NL3
