Facultative Thermophile Research Articles

Identifying heat shock response systems from the genomic assembly of Ureibacillus thermophilus LM102 using protein-protein interaction networks

Ureibacillus thermophilus strain LM102 is a facultative thermophile with growth in range 37 °C–60 °C. Upon identification using the 16S rRNA marker, it showed highest similarity of 99.8% with U. thermophilus strain HC148. A phylogenetic analysis revealed U. suwonensis strain 6 T19 to be the closest species to strain LM102. Our aim was to determine the unique thermotolerant properties of LM102 by identifying thermostablity of its proteins and the interactions existing in its heat shock response systems (HSRS). The strain was sequenced, assembled and the draft genome (3,017,325 bp) was analyzed. Post-annotation, we randomly selected a set of hundred proteins and computed the percentage distribution of 12 amino acids which have been substantially studied for their role in thermostability. The protein homologues were searched and the residues of LM102 were compared with Bacillus subtilis and Thermus thermophilus, a mesophile and hyperthermophile respectively. Within the 95% confidence limit, a Z-score of −0.61 was observed between LM102 and B. subtilis. However, a significantly lower value of −8.84 was observed for the pair LM102 and T. thermophilus. The amino acid distribution did not appear to influence the protein thermostability. Further, we investigated the role of Protein-Protein interactions by building networks for heat shock responses, namely DNA repair, transcriptional regulation, and activation of heat shock proteins. Interaction data retrieved from the STRING database for more than 50 species were used to build these networks. Highly clustered MCODE results notably revealed RNA 3′-5′ exonuclease, CshA and HemW previously unreported, in association with other proteins. Additionally, these and other proteins estimated from the HSRS networks were found in both mesophiles and thermophiles, suggesting a crucial role of gene regulatory networks in the cellular viability of LM102 at high temperatures.

Biofilm Formation of the Facultative Thermophile Bacillus pumilus D194A and Affects of Sanitation Agents on Its Biofilms

Bacillus pumilus D194a formed a strong biofilm on 96-well polystyrene microtiter plates and stainless-steel surfaces. Its optimum environmental factors for growth were determined as 50°C, pH 6.0, 1.5% NaCl for 48 h, whereas, the optimum biofilm formation was measured at 50°C, pH 8.0, 0% NaCl. The isolate was observed to have peritrichous flagella and it produced rigid pellicle. Extracellular polymeric substances (EPSs) of this strain were also determined to be; carbohydrate (33.5 µg/mL), protein (790 µg/mL) and extracellular DNA (eDNA) (18.5 kb). D194a cells formed biofilms on many abiotic surfaces, such as glass, polystyrene, stainless steel, polyvinyl chloride, polycarbonate, polypropylene, wherein polycarbonate (6.23 log/cm2) and polypropylene (6.14 log/cm2) were the generally preferred ones. Biofilm samples were treated with 15 different sanitation agents, including sodium metaperiodate (96.83%), nisin (93.82%), trichloroacetic acid (93.66%) and lysozyme (94.1%) for sanitation purposes. The elimination of eDNA in its 2 h-biofilm with DNase I was 98.48%, while this results was found as 96.06% for its 12 h-mature biofilm. In conclusion, this research demonstrated that B. pumilus D194a is a strong biofilm producer with a rigid structure harboring high protein and eDNA content, which cannot be easily destroyed by various chemical agents.

PRODUCTIVE BIOFILMS FROM MESOPHILIC TO THERMOPHILIC ENDOSPORE-FORMING BACILLI FOR INDUSTRIAL APPLICATIONS

The basic biofilm characteristics and extracellular polymeric substances (EPSs) production of Bacillus licheniformis E114 (mesophile), Bacillus pumilus D75b, Brevibacillus thermoruber B93 (facultative thermophile) and Anoxybacillus caldiproteolyticus D621 (thermophile) isolates were evaluated in this study. They were initially investigated in terms of their pellicle formation at the air-liquid interface, complex carbohydrate production in TSA (Tryptic Soy Agar) medium supplemented with calcofluor dye, and also colony morphologies in TSA medium supplemented with Congo Red dye. Their biofilm productions on abiotic surfaces such as polystyrene and stainless steel were also quantified by crystal violet binding assay and viable cell enumerations. The ideal way for the mechanical removal of the adherent cells from the steel surface such as bead vortexing or scraping were also tested for each isolate. The optimal growth and biofilm production parameters of E114, D75b, B93, and D621 in terms of pH, NaCl, and temperature were determined. Under optimal conditions, their EPSs components such as carbohydrate, protein, and eDNA were also measured quantitatively. The isolates evaluated in this study presented colony morphotypes in different characteristics on Congo Red agar that display also unique biofilm forming capacities for each isolate. Also, useful optimization approaches were determined that might be adapted to potential industrial applications for the biofilm and EPSs producers. According to these findings, four different endospore-forming bacilli exhibiting mesophilic to thermophilic growth, and biofilm production, possessing various EPSs contents which may be novel were elucidated that could be important in many industrial processes.

Determination of the biofilm production capacities and characteristics of members belonging to Bacillaceae family.

The biofilm characteristics of many endospore-forming 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 °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.

DISTRIBUTION OF CYANOBACTERIA IN TWO SIRCH HOT SPRINGS WITH REGARDS TO THE PHYSICOCHEMICAL TRAITS OF WATER

Cyanobacteria live in diverse ecosystems including hot springs. Life in hot spring condition is like the Earth's beginning conditions in which cyanobacteria emerged. In the present study, cyanobacterial flora of two hot springs located in Kerman, Iran was investigated for the first time. After sample collection, species were identified based on their morphological traits. To understand the fundamental patterns of species distribution, Physico-chemical environmental factors and species frequency were measured using standard methods and thereafter Canonical Correspondence Analysis (CCA) of species frequency and environmental factors was carried out by Canoco program. Twenty one cyanobacteria were identified. Results showed an ordered distribution of the species along the environmental factors. Results also showed the presence of mesothermophilous and facultative thermophile cyanobacteria in types of unicellular and filamentous in the two studied springs. Results also suggest that some species are adapted and resistant forms to sulfide. Diazotrophic strains were found to align in lowest compound nitrogen level, where others were removed from competition.

The effects of temperature and light intensity on growth, reproduction and EPS synthesis of a thermophilic strain related to the genus Graesiella.

Tunisian microalgae are diverse and rarely been studied. This study reports a first investigation of thermophile Chlorophyta isolated from mats community colonizing the geothermal springs in the north of Tunisia at water temperature 60 °C. In the study, the combined effect of temperature and light intensity was investigated on the cell growth, the mother and daughter cells abundance and the extracellular polymeric substances synthesis in batch culture of the isolated species. Three levels were tested for each factor, 20, 30, 40 °C for temperature; and 20, 70, 120 μmol photons m(-2) s(-1) for light intensity, using full factorial design and response surface methodology. The thermophile strain was identified as a genus Graesiella and showed 99.8% similarity with two Graesiella species: Graesiella emersonii and Graesiella vacuolata based on the 18S rDNA molecular identification. The optimal growth condition was found at 30 °C and 120 µmol photons m(-2) s(-1) (7 MC mL(-1) day(-1)), with the abundance of vegetative cells (daughter cells). In contrast, the number of mother cells increased significantly as the growth decreased; consequently, the highest ratio of auto spore mother cells versus daughter cells (19.4) was obtained at 20 °C and 20 µmol photons m(-2) s(-1). The highest yield of EPS production (11.7 mg L(-1) day(-1)) was recorded at the highest temperature (40 °C) and lowest light intensity (20 µmol photons m(-2)s(-1)). These results revealed how the species respond to high and low temperatures and suggest that the species should be considered as facultative thermophile.

Transformable facultative thermophile Geobacillus stearothermophilus NUB3621 as a host strain for metabolic engineering.

Metabolic engineers develop inexpensive enantioselective syntheses of high-value compounds, but their designs are sometimes confounded by the misfolding of heterologously expressed proteins. Geobacillus stearothermophilus NUB3621 is a readily transformable facultative thermophile. It could be used to express and properly fold proteins derived from its many mesophilic or thermophilic Bacillaceae relatives or to direct the evolution of thermophilic variants of mesophilic proteins. Moreover, its capacity for high-temperature growth should accelerate chemical transformation rates in accordance with the Arrhenius equation and reduce the risks of microbial contamination. Its tendency to sporulate in response to nutrient depletion lowers the costs of storage and transportation. Here, we present a draft genome sequence of G. stearothermophilus NUB3621 and describe inducible and constitutive expression plasmids that function in this organism. These tools will help us and others to exploit the natural advantages of this system for metabolic engineering applications.

The genetic diversity of genus Bacillus and the related genera revealed by 16S rRNA gene sequences and ardra analyses isolated from geothermal regions of turkey

Previously isolated 115 endospore-forming bacilli were basically grouped according to their temperature requirements for growth: the thermophiles (74%), the facultative thermophiles (14%) and the mesophiles (12%). These isolates were taken into 16S rRNA gene sequence analyses, and they were clustered among the 7 genera: Anoxybacillus, Aeribacillus, Bacillus, Brevibacillus, Geobacillus, Paenibacillus, and Thermoactinomycetes. Of these bacilli, only the thirty two isolates belonging to genera Bacillus (16), Brevibacillus (13), Paenibacillus (1) and Thermoactinomycetes (2) were selected and presented in this paper. The comparative sequence analyses revealed that the similarity values were ranged as 91.4-100 %, 91.8- 99.2 %, 92.6- 99.8 % and 90.7 - 99.8 % between the isolates and the related type strains from these four genera, respectively. Twenty nine of them were found to be related with the validly published type strains. The most abundant species was B. thermoruber with 9 isolates followed by B. pumilus (6), B. lichenformis (3), B. subtilis (3), B. agri (3), B. smithii (2), T. vulgaris (2) and finally P. barengoltzii (1). In addition, isolates of A391a, B51a and D295 were proposed as novel species as their 16S rRNA gene sequences displayed similarities ≤ 97% to their closely related type strains. The AluI-, HaeIII- and TaqI-ARDRA results were in congruence with the 16S rRNA gene sequence analyses. The ARDRA results allowed us to differentiate these isolates, and their discriminative restriction fragments were able to be determined. Some of their phenotypic characters and their amylase, chitinase and protease production were also studied and biotechnologically valuable enzyme producing isolates were introduced in order to use in further studies.

The genetic diversity of genus Bacillus and the related genera revealed by 16s rRNA gene sequences and ardra analyses isolated from geothermal regions of turkey.

Previously isolated 115 endospore-forming bacilli were basically grouped according to their temperature requirements for growth: the thermophiles (74%), the facultative thermophiles (14%) and the mesophiles (12%). These isolates were taken into 16S rRNA gene sequence analyses, and they were clustered among the 7 genera: Anoxybacillus, Aeribacillus, Bacillus, Brevibacillus, Geobacillus, Paenibacillus, and Thermoactinomycetes. Of these bacilli, only the thirty two isolates belonging to genera Bacillus (16), Brevibacillus (13), Paenibacillus (1) and Thermoactinomycetes (2) were selected and presented in this paper. The comparative sequence analyses revealed that the similarity values were ranged as 91.4–100 %, 91.8- 99.2 %, 92.6- 99.8 % and 90.7 - 99.8 % between the isolates and the related type strains from these four genera, respectively. Twenty nine of them were found to be related with the validly published type strains. The most abundant species was B. thermoruber with 9 isolates followed by B. pumilus (6), B. lichenformis (3), B. subtilis (3), B. agri (3), B. smithii (2), T. vulgaris (2) and finally P. barengoltzii (1). In addition, isolates of A391a, B51a and D295 were proposed as novel species as their 16S rRNA gene sequences displayed similarities ≤ 97% to their closely related type strains. The AluI-, HaeIII- and TaqI-ARDRA results were in congruence with the 16S rRNA gene sequence analyses. The ARDRA results allowed us to differentiate these isolates, and their discriminative restriction fragments were able to be determined. Some of their phenotypic characters and their amylase, chitinase and protease production were also studied and biotechnologically valuable enzyme producing isolates were introduced in order to use in further studies.

Experimental Evolution of a Facultative Thermophile from a Mesophilic Ancestor

Experimental evolution via continuous culture is a powerful approach to the alteration of complex phenotypes, such as optimal/maximal growth temperatures. The benefit of this approach is that phenotypic selection is tied to growth rate, allowing the production of optimized strains. Herein, we demonstrate the use of a recently described long-term culture apparatus called the Evolugator for the generation of a thermophilic descendant from a mesophilic ancestor (Escherichia coli MG1655). In addition, we used whole-genome sequencing of sequentially isolated strains throughout the thermal adaptation process to characterize the evolutionary history of the resultant genotype, identifying 31 genetic alterations that may contribute to thermotolerance, although some of these mutations may be adaptive for off-target environmental parameters, such as rich medium. We undertook preliminary phenotypic analysis of mutations identified in the glpF and fabA genes. Deletion of glpF in a mesophilic wild-type background conferred significantly improved growth rates in the 43-to-48°C temperature range and altered optimal growth temperature from 37°C to 43°C. In addition, transforming our evolved thermotolerant strain (EVG1064) with a wild-type allele of glpF reduced fitness at high temperatures. On the other hand, the mutation in fabA predictably increased the degree of saturation in membrane lipids, which is a known adaptation to elevated temperature. However, transforming EVG1064 with a wild-type fabA allele had only modest effects on fitness at intermediate temperatures. The Evolugator is fully automated and demonstrates the potential to accelerate the selection for complex traits by experimental evolution and significantly decrease development time for new industrial strains.

Kinetics of autothermal thermophilic aerobic digestion – application and extension of Activated Sludge Model No 1 at thermophilic temperatures

The application of an ASM1-based mathematical model for the modeling of autothermal thermophilic aerobic digestion is demonstrated. Based on former experimental results the original ASM1 was extended by the activation of facultative thermophiles from the feed sludge and a new component, the thermophilic biomass was introduced. The resulting model was calibrated in the temperature range of 20-60 degrees C. The temperature dependence of the growth and decay rates in the model is given in terms of the slightly modified Arrhenius and Topiwala-Sinclair equations. The capabilities of the calibrated model in realistic ATAD scenarios are demonstrated with a focus on autothermal properties of ATAD systems at different conditions.

Diversity of thermophilic populations during thermophilic aerobic digestion of potato peel slurry

To study the diversity of thermophiles during thermophilic aerobic digestion (TAD) of agro-food waste slurries under conditions similar to full-scale processes. Population diversity and development in TAD were studied by standard microbiological techniques and the processes monitored by standard fermentation procedures. Facultative thermophiles were identified as Bacillus coagulans and B. licheniformis, while obligate thermophiles were identified as B. stearothermophilus. They developed rapidly to peaks of 10(7) to 10(8) in <or=48 h. Stability of obligate thermophiles increased with process temperatures. Thermophiles were unstable at process pH above or below neutral, but developed rapidly at all aeration rates. Peak populations were higher in the median than at extremes of aeration rates. Obligate thermophiles were unstable at low aeration rates. Process self-heating was higher at lower than at higher aeration rate. Beyond 96 h most thermophiles were present as spores. Limited range of indigenous thermophiles drives TAD of slurry. They develop rapidly and are stable at most digestion conditions. Development and stability of thermophiles in TAD suggest that the process may be operated in a wide range of conditions; and even at short HRT in continuous processes without compromising efficiency.

Crystal structure of 3-isopropylmalate dehydrogenase from the moderate facultative thermophile, Bacillus coagulans: two strategies for thermostabilization of protein structures.

The crystal structure of 3-isopropylmalate dehydrogenase from the moderate facultative thermophile Bacillus coagulans (BcIPMDH) has been determined by the X-ray method. BcIPMDH is a dimeric enzyme composed of two identical subunits, each of which takes an open alpha/beta structure with 11 alpha-helices and 14 beta-strands. The polypeptide is folded into two domains. The first domain is composed of residues 1-101 and 257-356, and the second domain, of residues 102-256. The latter domains of the two subunits are associated with one another by a dyad axis to make the dimer, locally forming a beta-sheet and a four-helix bundle. As compared with the structure of the enzyme from the extreme thermophile Thermus thermophilus (TtIPMDH), a new short beta-sheet (residues 329-330 and 340-341) absent in TtIPMDH is formed by the insertion of 5 residues in BcIPMDH. In terms of determinants for thermostabilization, both consistent and inconsistent changes were found between the two enzymes. The regions including inconsistent changes are formed by different usages of the determinants for stabilizing the loops at different levels. Those in BcIPMDH contain some structural redundancies in length of amino acid sequence and flexibility of residues, which seem to be unnecessary for the enzymatic reaction. Such redundancies are also found in the primary structure of the enzyme of the mesophile Bacillus subtilis, but these parts are more stabilized in BcIPMDH by hydrogen bonds and salt bridges. On the other hand, TtIPMDH is stabilized by reducing such redundant parts. This contrast suggests that different strategies may be preferred for thermostabilization, depending on temperature.

Bacillus thermoamyloliquefaciens KP1071 alpha-glucosidase II is a thermostable M(r) 540,000 homohexameric alpha-glucosidase with both exo-alpha-1,4-glucosidase and oligo-1,6-glucosidase activities.

alpha-Glucosidase II of the facultative thermophile Bacillus thermoamyloliquefaciens KP1071 (FERM-P8477; growth over 30-66 degrees C) was purified to a homogeneous state. Its M(r) was estimated as 90000 by SDS/PAGE. However, the enzyme behaved as an active Mr 540000 protein on gel filtration with each of two gels of different matrices as well as on gel electrophoresis under native conditions. The enzyme was not glycosylated. Its isoelectric point was estimated as 5.7. The N-terminal sequence of 20 residues was determined asAla1-Ile-Gln-Pro-Glu-Gln-Asp-Asp-Lys-Thr-Gln-Glu-Asp-Gly- Tyr-Ile-Asp-Ile-Gly-Asn20. The sequence did not resemble those of procaryotic and eucaryotic proteins hitherto reported including the monomeric exo-alpha-1,4-glucosidase and the monomeric oligo-1,6-glucosidase from the same microorganism. The alpha-glucosidase II had no antigenic group shared with the latter two enzymes. Analysis of substrate specificity showed that the alpha-glucosidase II has dual activity towards oligo-1,6-glucosidases and exo-alpha-1,4-glucosidases, but its preference is for non-reducing terminal alpha-1,4 glucosidic bonds in substrates. Kinetic studies proved that both activities are attributed to the same catalytic site. The enzyme was most active at 81 degrees C and pH 7.0. Its half-life at pH 6.8 was 10 min at 81 degrees C, and 5 h at 55 degrees C in 6.4 M urea, 26% ethanol or 2.5% SDS. We suggest that the alpha-glucosidase II is a thermostable, homohexameric enzyme of origin distinct from the exo-alpha-1,4-glucosidase and the oligo-1,6-glucosidase present in the same strain.

Bi-phasic production of a-amylase of Bacillus flavothermusin batch fermentation

The facultative thermophile, Bacillus flavothermus, produced highest a-amylase activity (28.6 units/ml) with lactose (4%, w/v) and Yeatex (2%, w/v) and an initial pH 6.0 at 55°C. In batch fermentation, biomass and a-amylase activity peaked twice. In the first of these growth cycles the organism utilised the nitrogen source, Yeatex, and maximum enzyme was associated with cell lysis. In the second growth phase the carbohydrate source, lactose, was utilised and enzyme peaked in the early stages of growth.

Crystallization and preliminary X-ray analysis of 3-isopropylmalate dehydrogenase from the moderate facultative thermophile Bacillus coagulans.

Three crystalline forms of 3-isopropylmalate dehydrogenase from the moderate facultative thermophile Bacillus coagulans were obtained by hanging-drop vapor-diffusion methods. One of them, which had crystallized under slightly milder conditions than the others, was suitable for X-ray analysis. Its asymmetric unit contains one dimeric molecule and the solvent content is higher than in other protein crystals. The crystal structure was solved in a preliminary manner by the molecular-replacement technique.

Assignment of Bacillus thermoamyloliquefaciens KP1071 alpha-glucosidase I to an exo-alpha-1,4-glucosidase, and its striking similarity to bacillary oligo-1,6-glucosidases in N-terminal sequence and in structural parameters calculated from the amino acid composition.

alpha-Glucosidase I of Bacillus thermoamyloliquefaciens KP1071 (FERM P8477, facultative thermophile) was purified to homogeneity. The relative molecular mass was estimated to be 62,000 Da. From its catalytic properties, the enzyme has been assigned to an exo-alpha-1,4-glucosidase. The enzyme shares its antigenic groups in part with Bacillus stearothermophilus ATCC12016 (obligate thermophile) exo-alpha-1,4-glucosidase. These exo-alpha-1,4-glucosidases strikingly resemble oligo-1,6-glucosidases from B. thermoamyloliquefaciens KP1071 and from Bacillus cereus ATCC7064 in the molecular properties tested, including relative molecular mass, N-terminal sequence of 15 residues, amino acid composition and structural parameters calculated from amino acid composition. We have suggested that bacillary exo-alpha-1,4-glucosidases take the same folded conformation, i.e. an (alpha/beta)8-barrel super-secondary structure in its N-terminal domain, as bacillary oligo-1,6-glucosidases.

A relationship between efficiency of isomaltosaccharide hydrolysis and thermostability of six Bacillus oligo-1,6-glucosidases

A p-nitrophenyl-α-d-glucopyranosidase from Bacillus thermoamyloliquefaciens KP 1171 capable of growing at 30°–66°C was assigned to an oligo-1,6-glucosidase (dextrin 6-α-d-glucanohydrolase, EC 3.2.1.10). The enzyme was compared with its homologous counterparts from B. cereus NY-14, B. cereus ATCC 7064 (each mesophile), B. coagulans ATCC 7050 (facultative thermophile), B. thermoglucosidasius KP 1006 (DSM 2542, obligate thermophile) and B. flavocaldarius KP 1228 (extreme thermophile) in thermostability and kinetic parameters at suboptimal temperatures for isomaltosaccharides (2–6 glucose units). This analysis showed that the efficiency of each isomaltosaccharide hydrolysis changes in a convex manner with increasing thermostability on the transition, NY-14 → ATCC 7064 → ATCC 7050 → KP 1071 → KP 1006 → KP 1228 enzymes, with a maximum at KP 1071 or ATCC 7050 enzyme.

Distribution of spermine in bacilli and lactic acid bacteria.

Obligate moderately thermophilic bacilli and obligate moderately thermoacidophilic bacilli contained spermine as the major polyamine in addition to putrescine and spermidine. The identity of spermine was confirmed by thin-layer chromatography and high-performance liquid chromatography before and after treatment with putrescine oxidase. Using these methods, thermospermine and spermine can be separated; thermospermine was not present in these organisms. On the other hand, various facultative thermophiles and mesophilic strains of the genus Bacillus, including alkalophiles and halophiles, lack spermine and other tetraamines. No spermine was detected in several strains of mesophilic or facultative slightly thermophilic lactic acid bacteria, Lactobacillus and Streptococcus.

A strong correlation between the increase in number of proline residues and the rise in thermostability of five Bacillus oligo-1,6-glucosidases

A p-nitrophenyl-α-d-glucopyranoside-hydrolysing oligo-1,6-glucosidase (dextrin 6-α-d-glucanohydrolase, EC 3.2.1.10) of Bacillus sp. KP 1071 capable of growing at 30°–66°C was purified to homogeneity. The molecular weight was estimated to be 62,000. The amino-terminal amino acid was methionine. The enzyme shared its antigenic groups in part with its homologous counterpart from Bacillus thermoglucosidasius KP 1006 (obligate thermophile), but did not at all with any one of oligo-1,6-glucosidases from Bacillus cereus ATCC 7064 (mesophile), Bacillus coagulans ATCC 7050 (facultative thermophile) and Bacillus flavocaldarius KP 1288 (extreme thermophile). A comparison of amino acid composition showed that the proline content increased greatly in a linearity with the rise in thermostability in the order, mesophile → facultative thermophile → KP 1071 → obligate thermophile → extreme thermophile enzymes.