Resistance and bioaccumulation of Cd2+, Cu2+, Co2+ and Mn2+ by thermophilic bacteria, Geobacillus thermantarcticus and Anoxybacillus amylolyticus

In this study, bioaccumulation and heavy metal resistance of Cd²⁺, Cu²⁺, Co²⁺ and Mn²⁺ ions by thermophilic Geobacillus thermantarcticus and Anoxybacillus amylolyticus was investigated. The bacteria, in an order with respect to metal resistance from the most resistant to the most sensitive, was found to be Mn²⁺ > Co²⁺ > Cu²⁺ > Cd²⁺ for both G. thermantarcticus and A. amylolyticus. It was determined that the highest metal bioaccumulation was performed by A. amylolyticus in Mn²⁺ (28,566 μg/g dry weight), and the lowest metal bioaccumulation was performed by A. amylolyticus in Co²⁺ (327.3 μg/g dry weight). The highest Cd²⁺ capacities of dried cell membrane was found to be 36.07 and 39.55 mg/g membrane for G. thermantarticus and A. amylolyticus, respectively, and the highest Cd²⁺ capacities of wet cell membrane was found to be 14.36 and 12.39 mg/g membrane for G. thermantarcticus and A. amylolyticus, respectively.     

Purification,biochemical characterization and gene sequencing of a thermostable raw starch digesting α-amylase from Geobacillus thermoleovorans subsp. stromboliensis subsp. nov.

This study reports the purification and biochemical characterization of a raw starch-digesting α-amylase from Geobacillus thermoleovorans subsp. stromboliensis subsp. nov. (strain Pizzo^T). The molecular weight was estimated to be 58 kDa by SDS–PAGE. The enzyme was highly active over a wide range of pH from 4.0–10.0. The optimum temperature of the enzyme was 70°C. It showed extreme thermostability in the presence of Ca²⁺, retaining 50% of its initial activity after 90 h at 70°C. The enzyme efficiently hydrolyzed 20% (w/v) of raw starches, concentration normally used in starch industries. The α-amylase showed an high stability in presence of many organic solvents. In particular the residual activity was of 73% in presence of 15% (v/v) ethyl alcohol, which corresponds to ethanol yield in yeast fermentation process. By analyzing its complete amyA gene sequence (1,542 bp), the enzyme was proposed to be a new α-amylase.     

Production and characterization of exopolysaccharides by Geobacillus thermodenitrificans ArzA-6 and Geobacillus toebii ArzA-8 strains isolated from an Armenian geothermal spring

The thermal ecosystems, including geothermal springs, are proving to be source of thermophiles able to produce extracellular polysaccharides (EPSs). Among the sixteen thermophilic bacilli isolated from sediment sampled from Arzakan geothermal spring, Armenia, two best EPSs producer strains were identified based on 16S rRNA gene sequence analysis and phenotypic characteristics, and designated as Geobacillus thermodenitrificans ArzA-6 and Geobacillus toebii ArzA-8 strains. EPSs production was investigated under different time, temperature and culture media’s composition. The highest specific EPSs production yield (0.27 g g⁻¹ dry cells and 0.22 g g⁻¹ dry cells for strains G. thermodenitrificans ArzA-6 and G. toebii ArzA-8, respectively) was observed after 24 h when fructose was used as sole carbon source at 65 °C and pH 7.0. Purified EPSs displayed a high molecular mass: 5 × 10⁵ Da for G. thermodenitrificans ArzA-6 and 6 × 10⁵ Da for G. toebii ArzA-8. Chemical composition and structure of the biopolymers, determined by GC–MS, HPAE-PAD and NMR, showed that both the two EPSs are heteropolymers composed by mannose as major monomer unit. Optical rotation values [α] ^25 °C_D of the two EPSs (2 mg ml⁻¹ H₂O) were − 142,135 and − 128,645 for G. thermodenitrificans ArzA-6 and G. toebii ArzA-8, respectively.

     

Tolerance and bioaccumulation of heavy metals in five populations of Cistus ladanifer L. subsp. ladanifer

The effects of heavy metals on the growth, mineral composition (P, K, Fe and Mn) and metal accumulation of five populations of Cistus ladanifer subsp. ladanifer from NE Portugal were investigated in hydroponic experiments. Plants were exposed to increasing concentrations (0–2000 M) of one of eight heavy metals: Cd, Co, Cr, Cu, Mn, Ni, Pb or Zn. Populations of C. ladanifer, whose origin was ultramafic soils (S and UB) or soils developed on basic rocks (B), showed a higher tolerance to the metals Cd, Co, Cr, and Mn, and a considerable degree of tolerance to Ni. In contrast, populations originating on acid-rock soils (M and SC) showed higher tolerance to the metals Cu and Zn. Populations showed different patterns of metal accumulation and distribution in the plant parts, suggesting different mechanisms of metal tolerance are used. The more Cd-, Co- and Mn-tolerant populations (S, UB, B and SC (Cd)) showed accumulation of these three metals in the shoots (shoot:root metal concentration ratios (S:R) > 1). Shoot concentrations of up to 309 g Cd g^–1, 2667 g Co g^–1 and 6214 g Mn g^–1 were found in these populations. The populations, UB and M, showed considerable tolerance to Ni and Zn, respectively. These populations accumulated up to 4164 g Ni g^–1 and 7695 g Zn g^–1 in their shoot tissues, and these metals were efficiently transported from the roots to aerial parts (S:R > 3 (Ni), S:R > 1 (Zn)). In contrast, the S and SC populations maintained higher growth rates in the presence of Ni and Zn, respectively, but showed exclusion mechanisms of metal tolerance: reduced Ni and Zn transport to shoots (S:R < 1). Cistus ladanifer was not able to efficiently transport Cr, Cu or Pb from its roots to its aerial parts (S:R ranged from 0–0.4). The more Cu-tolerant populations, M and SC, showed a greater restriction of Cu transport to the shoots than the ultramafic- or basic-rock populations. Significant changes in the plant mineral composition were found, however, concentrations were generally above mineral deficiency levels. Based on these preliminary results the possible usefulness of this plant for phytoremediation technologies is discussed. However, further investigations are necessary to evaluate its growth and metal accumulation under soil and field conditions.     

Purification and characterization of carboxymethylcellulase isolated from a marine bacterium, Bacillus subtilis subsp. subtilis A-53

The microorganism hydrolyzing carboxymethylcellulose (CMC) was isolated from seawater, identified as Bacillus subtilis subsp. subtilis by analyses of 16S rDNA and partial sequences of the gyrA gene, and named as B. subtilis subsp. subtilis A-53. The molecular weight of the purified carboxymethylcellulase (CMCase) was estimated to be about 56 kDa with the analysis of SDS-PAGE. The purified CMCase hydrolyzed carboxymethylcellulose (CMC), cellobiose, filter paper, and xylan, but not avicel, cellulose, and p-nitrophenyl-β-d-glucospyranoside (PNPG). Optimal temperature and pH for the CMCase activity were determined to be 50 °C and 6.5, respectively. More than 70% of original CMCase activity was maintained at relative low temperatures ranging from 20 to 40 °C after 24 h incubation at 50 °C. The CMCase activity was enhanced by EDTA and some metal ions in order of EDTA, K⁺, Ni²⁺, Sr²⁺, Pb²⁺, and Mn²⁺, but inhibited by Co²⁺ and Hg²⁺.     

Optimization of thermostable lipase production from a thermophilic Geobacillus sp. using Box-Behnken experimental design

Thermostable lipase production by Geobacillus thermoleovorans was optimized in shake-flask cultures using Box-Behnken experimental design. An empirical model was developed through response surface methodology to describe the relationship between tested variables (Tween 80, olive oil, temperature and pH) and enzyme activity. Maximum enzyme activity (495 U l^–1) was attained with Tween 80 at 5 g l^–1; olive oil at 60 g l^–1; 70 °C and pH 9. Experimental verification of the model showed a validation of 95%, which is more than 4-fold increase compared to the basal medium.     

Biosorption of Heavy Metals (Cd2+, Cu2+, Co2+, and Mn2+) by Thermophilic Bacteria,Geobacillus thermantarcticus and Anoxybacillus amylolyticus: Equilibrium and Kinetic Studies

ABSTRACT

Two strains of thermophilic bacteria, Geobacillus thermantarcticus and Anoxybacillus amylolyticus, were employed to investigate the biosorption of heavy metals including Cd²⁺, Cu²⁺, Co²⁺, and Mn²⁺ ions. The effects of different biosorption parameters such as pH (2.0–10.0), initial metal concentrations (10.0–300.0 mg L^?1), amount of biomass (0.25–10 g L^?1), temperature (30–80°C), and contact time (15–120 min) were investigated. Concentrations of metal ions were determined by using an inductively coupled plasma optical emission spectrometry (ICP-OES). Optimum pHs for Cd²⁺, Cu²⁺, Co²⁺, and Mn²⁺ biosorption by Geobacillus thermantarcticus were found to be 4.0, 4.0, 5.0, and 6.0, respectively. For Anoxybacillus amylolyticus, the optimum pHs for Cd²⁺, Cu²⁺, Co²⁺, and Mn²⁺ biosorption were found to be 5.0, 4.0, 5.0, and 6.0, respectively. The Cd²⁺, Cu²⁺, Co²⁺, and Mn²⁺ removals at 50 mg L^?1 in 60 min by 50 mg dried cells of Geobacillus thermantarcticus were 85.4%, 46.3%, 43.6%, and 65.1%, respectively, whereas 74.1%, 39.8%, 35.1%, and 36.6%, respectively, for Anoxybacillus amylolyticus. The optimum temperatures for heavy metal biosorption were near the optimum growth temperatures for both strains. Scatchard plot analysis was employed to obtain more compact information about the interaction between metal ions and biosorbents. The plot results were further studied to determine if they fit Langmuir and Freundlich models.     

Isolation and Characterization of a Bacteriocin-Like Substance Produced by <Emphasis Type="Italic">Geobacillus toebii</Emphasis> Strain HBB-247

A total of 201 thermophilic bacteria isolated from various thermal spring, mud and soil were tested for their antibacterial activity. Among the mostly active isolates, Geobacillus toebii HBB-247 was further examined. Bacteriocin-like inhibitory substance (BLIS) produced by strain HBB-247 was found to be stable up to 60°C, sensitive to proteolytic enzymes and effective against Enterococcus faecalis, Listeria sp., E. avium, Clostridium pasteurianum, Cellulomonas fimi and some thermophilic strains isolated and identified in this study. As a result of Tricine-SDS-PAGE molecular weight of BLIS was estimated about 38 kDa. Production studies showed that G. toebii HBB-247 starts to produce antibacterial substance at early logarithmic phase of growth and maximum production was detected at the end of the logarithmic phase.     

Characteristics and applications of recombinant thermostable amylopullulanase of Geobacillus thermoleovorans secreted by Pichia pastoris

The 3′-deleted amylopullulanase gene from the extreme thermophile Geobacillus thermoleovorans (Gt-apuΔC) was expressed extracellularly in Pichia pastoris under both methanol-inducible AOX1 and constitutive GAP promoters. The expression of the gene (Gt-apuΔC) was higher under GAP promoter (36.2 U ml⁻¹, α-amylase; 33.5 U ml⁻¹, pullulanase) than that under AOX1 promoter (32.5 and 28.6 U ml⁻¹). The heavily glycosylated Gt-apuΔC from the recombinant P. pastoris displays higher substrate specificity, thermal stability and starch saccharification efficiency than that expressed in Escherichia coli. The enzyme hydrolyses maltotriose and maltotetraose unlike that expressed in E. coli. The enzyme action on wheat bran liberates maltose and glucose without detectable amount(s) of maltooligosaccharides. The sugars released from wheat bran (glucose and maltose) could be fractionated by ultrafiltration, as confirmed by TLC and HPLC analysis. This is the first report on the production of recombinant amylopullulanase extracellularly in P. pastoris.

     

Heavy metal resistant <Emphasis Type="Italic">Distigma proteus</Emphasis> (Euglenophyta) isolated from industrial effluents and its possible role in bioremediation of contaminated wastewaters

Summary The alga, Distigma proteus, isolated from industrial wastewater showed tolerance against Cd²⁺ (8.0 μg/ml), Cr⁶⁺ (12 μg/ml), Pb²⁺ (15 μg/ml) and Cu²⁺ (10 μg/ml). The metal ions slowed down the growth of the organism after 4–5 days of exposure. The reduction in cell population was 90% for Cu²⁺, 84% for Cd²⁺, 71% for Cr⁶⁺, and 63% for Pb²⁺ after 8 days of metal stress. The order of resistance to heavy metal, in terms of reduction in the cellular population, was Cu²⁺ > Cd²⁺ > Cr⁶⁺ > Pb²⁺. Chromium- and cadmium-processing capabilities of the alga were worked out for its potential use as a bioremediator of wastewater. The reduction in the amount of Cr⁶⁺ after 2, 4, 6 and 8 days of algal culture containing 5.0 μg Cr⁶⁺ ml⁻¹ of culture medium was 77, 85, 92 and 97%, respectively. Distigma could also remove 48% Cd²⁺after 2 days, 68% after 4 days, 80% after 6 days and 90% after 8 days from the medium. The heavy metal uptake ability of Distigma can be exploited for metal detoxification and environmental clean-up operations.     

Purification and characterization of hyperthermotolerant leucine aminopeptidase from Geobacillus thermoleovorans 47b

A thermophilic bacterium, which we designated as Geobacillus thermoleovorans 47b was isolated from a hot spring in Beppu, Oita Prefecture, Japan, on the basis of its ability to grow on bitter peptides as a sole carbon and nitrogen source. The cell-free extract from G. thermoleovorans 47b contained leucine aminopeptidase (LAP; EC 3.4.11.10), which was purified 164-fold to homogeneity in seven steps, using ammonium sulfate fractionation followed by the column chromatography using DEAE-Toyopearl, hydroxyapatite, MonoQ and Superdex 200 PC gel filtration, followed again by MonoQ and hydroxyapatite. The enzyme was a single polypeptide with a molecular mass of 42,977.2 Da, as determined by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry, and was found to be thermostable at 90°C for up to 1 h. Its optimal pH and temperature were observed to be 7.6–7.8 and 60°C, respectively, and it had high activity towards the substrates Leu-p-nitroanilide (p-NA)(100%), Arg-p-NA (56.3%) and LeuGlyGly (486%). The K_m and V_max values for Leu-p-NA and LeuGlyGly were 0.658 mM and 25.0 mM and 236.2 mol min^–1 mg^–1 protein and 1,149 mol min^–1 mg^–1 protein, respectively. The turnover rate (k_cat) and catalytic efficiency (k_cat/ K_m) for Leu-p-NA and LeuGlyGly were 10,179 s^–1 and 49,543 s^–1 and 15,470 mM^–1 s^–1 and 1981.7 mM^–1 s^–1, respectively. The enzyme was strongly inhibited by EDTA, 1,10-phenanthroline, dithiothreitol, -mercaptoethanol, iodoacetate and bestatin; and its apoenzyme was found to be reactivated by Co²⁺ .     

Geobacillus toebii subsp. decanicus subsp. nov., a hydrocarbon-degrading, heavy metal resistant bacterium from hot compost

A thermophilic, spore-forming bacterial strain L1(T) was isolated from hot compost "Pomigliano Environment" s.p.a., Pomigliano, Naples, Italy. The strain was identified by using a polyphasic taxonomic approach. L1(T) resulted in an aerobic, gram-positive, rod-shaped, thermophilic with an optimum growth temperature of 68 degrees C chemorganotrophic bacterium which grew on hydrocarbons as unique carbon and energy sources and was resistant to heavy metals. The G+C DNA content was 43.5 mol%. Phylogenetic analysis of 16S rRNA gene sequence and Random Amplified Polymorphic DNA-PCR (RAPD-PCR) analysis of L1(T) and related strains showed that it forms within Geobacillus toebii, a separate cluster in the Geobacillus genus. The composition of cellular fatty acids analyses by Gas-Mass Spectroscopy differed from that typical for the genus Geobacillus in that it is lacking in iso-C15 fatty acid, while iso-C16 and iso-C17 were predominant. Isolates grew on a rich complex medium at temperatures between 55-75 degrees C and presented a doubling time (t(d)) of 2 h and 6 h using complex media and hydrocarbon media, respectively. Among hydrocarbons tested, n-decane (2%) was the more effective to support the growth (1 g/L of wet cells). The microorganism showed resistance to heavy metal tested during the growth. Furthermore, intracellular alpha-galactosidase and alpha-glucosidase enzymatic activities were detectable in the L1(T) strain. Based on phenotypic, phylogenetic, fatty acid analysis and results from DNA-DNA hybridization, we propose assigning a novel subspecies of Geobacillus toebii, to be named Geobacillus toebii subsp. decanicus subsp. nov., with the type strain L1(T) (=DSM 17041=ATCC BAA 1004).     

Optimization of flow rate,initial metal ion concentration and biomass density for maximum removal of Cu2+ by immobilized Microcystis

The potential of alginate-immobilized Microcystis packed in a column for maximum removal of Cu²⁺ at different flow rates, biomass, and initial metal ion concentration was assessed in a continuous flow system. Although Cu²⁺ removal did occur at all the flow rates tested, it was maximum (54%) at 0.75-ml min⁻¹ flow rate, 30 μg ml⁻¹ initial metal ion concentration and 0.016 g biomass. Cu²⁺ removal was influenced by inlet metal ion concentration and biomass density. An increase in the biomass concentration from 0.016 to 0.128 g resulted in an apparent increase in percentage removal but the Cu²⁺ adsorbed per unit dry wt. declined. When the flow rate (0.75 ml min⁻¹) and biomass density (0.064 g) were kept constant and the inlet metal ion concentration was varied from 10 to 150 μg ml⁻¹, a 68% removal of Cu²⁺ was obtained at 50 μg ml⁻¹ initial concentration in a time duration of 15 min. The metal-laden columns were efficiently desorbed and regenerated following elution with double distilled water (DDW) (pH 2) (89%). This was followed by 1 mm EDTA > 1 mm NTA > 0.1 mm EDTA > 1 mm HCl > 1 mm HNO₃ > 5 mm CaCl₂ > DDW (pH 7.0) > 1 mm NaHCO₃ > 1 mm CaCl₂. Of the total (2.83 mg) adsorbed Cu²⁺, 1.89 mg (67%) was desorbed by DDW (pH 2) within the first 20 min of elution time. Thereafter the desorption rate slowed down and only 22% (0.632 mg) desorption was obtained in the last 20 min. In contrast to water pH 2, the desorption of Cu²⁺ by 1 mm EDTA was very slow, the maximum being 8% after 40 min of elution. This revised version was published online in July 2006 with corrections to the Cover Date.     

Uptake of heavy metals by <Emphasis Type="Italic">Stylonychia mytilus</Emphasis> and its possible use in decontamination of industrial wastewater

The heavy metal resistant ciliate, Stylonychia mytilus, isolated from industrial wastewater has been shown to be potential bioremediator of contaminated wastewater. The ciliate showed tolerance against Zn²⁺ (30 μg/mL), Hg²⁺ (16 μg/mL) and Ni²⁺ (16 μg/mL). The metal ions slowed down the growth of the ciliate as compared with the culture grown without metal stress. The reduction in cell population was 46% for Cd²⁺, 38% for Hg²⁺, 23% for Zn²⁺, 39% for Cu²⁺ and 51% for Ni²⁺ after 8 days of metal stress. S. mytilus reduced 91% of Cd²⁺, 90% of Hg²⁺ and 98% of Zn²⁺ from the medium after 96 h of incubation in a culture medium containing 10 μg/mL of the respective metal ions. Besides this, the ciliate could also remove 88% of Cu²⁺ and 73% Ni²⁺ from the medium containing 5 μg/mL of each metal after 96 h. The ability of Stylonychia to take up variety of heavy metals from the medium could be exploited for metal detoxification and environmental clean-up operations.     

Synergistic or Antagonistic Effect of MTE plus TF or Icariin from <Emphasis Type="Italic">Epimedium koreanum</Emphasis> on the Proliferation and Differentiation of Primary Osteoblasts In Vitro

3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide test and alkaline phosphatase activity assay were employed to assess the effects of mixed trace elements including Zn²⁺, Ca²⁺, and Mn²⁺ plus total flavonoids or icariin from Epimedium koreanum on the proliferation and differentiation of primary osteoblasts in vitro. The results indicated that icariin (0.1, 1, and 10 μmol/L) and total flavonoids (0.06, 0.6, and 6 μg/mL) inhibited the proliferation and promoted the differentiation of primary osteoblasts. Mixed trace elements including Zn²⁺, Ca²⁺, and Mn²⁺ (0.1, 1, and 10 μmol/L) inhibited the proliferation and promoted the differentiation at 0.1 and 1 μmol/L, but inhibited the differentiation at 10 μmol/L. The effects of mixed trace elements including Zn²⁺, Ca²⁺, and Mn²⁺ plus total flavonoids or icariin from E. koreanum on the proliferation and differentiation of primary osteoblasts in vitro are complicated, and both synergistic and antagonistic effects are generated. The results suggest that there may be a potential cooperative action between flavonoids and trace metal elements on the proliferation and differentiation of primary osteoblasts by forming metal complexes. The combination model between flavonoids and trace metal elements is a pivotal factor for switching the biological effects from toxicity to activity, from damage to protection.     

Pseudomonas oleovorans subsp. lubricantis subsp. nov., and Reclassification of Pseudomonas pseudoalcaligenes ATCC 17440T as Later Synonym of Pseudomonas oleovorans ATCC 8062T

Isolate RS1^T isolated from used metalworking fluid was found to be a Gram-negative, motile, and non-spore forming rod. Based on phylogenetic analyses with 16S rRNA, isolate RS1^T was placed into the mendocina sublineage of Pseudomonas. The major whole cell fatty acids were C_18:1ω7c (32.6%), C_16:0 (25.5%), and C_15:0 ISO 2OH/C_16:1ω7c (14.4%). The sequence similarities of isolate RS1^T based on gyrB and rpoD genes were 98.9 and 98.0% with Pseudomonas pseudoalcaligenes, and 98.5 and 98.1% with Pseudomonas oleovorans, respectively. The ribotyping pattern showed a 0.60 similarity with P. oleovorans ATCC 8062^T and 0.63 with P. pseudoalcaligenes ATCC17440^T. The DNA G + C content of isolate RS1^T was 62.2 mol.%. The DNA–DNA relatedness was 73.0% with P. oleovorans ATCC 8062^T and 79.1% with P. pseudoalcaligenes ATCC 17440^T. On the basis of morphological, biochemical, and molecular studies, isolate RS1^T is considered to represent a new subspecies of P. oleovorans. Furthermore, based on the DNA–DNA relatedness (>70%), chemotaxonomic, and molecular profile, P. pseudoalcaligenes ATCC 17440^T and P. oleovorans ATCC 8062^T should be united under the same name; according to the rules of priority, P. oleovorans, the first described species, is the earlier synonym and P. pseudoalcaligenes is the later synonym. As a consequence, the division of the species P. oleovorans into two novel subspecies is proposed: P. oleovorans subsp. oleovorans subsp. nov. (type strain ATCC 8062^T = DSM 1045^T = NCIB 6576^T), P. oleovorans subsp. lubricantis subsp. nov. (type strain RS1^T = ATCC BAA-1494^T = DSM 21016^T).     

Chemical Composition and Antimicrobial Activity of the Essential Oil and Methanol Extract of Hypericum aegypticum subsp. webbii (Spach) N. Robson

Hypericum aegypticum subsp. webbii is an evergreen shrub spread in Mediterranean part of central and southeastern Europe. The chemical composition and antimicrobial activity of the essential oil and MeOH extract of H. aegypticum subsp. webbii were investigated. The monoterpenes α‐pinene (63.4 – 68.5%) and β‐pinene (16.9 – 17.0%) were main compounds in the volatile oil from aerial parts. In the cluster analysis, the essential oil of H. aegypticum subsp. webbii was separated and chemically different from the oil of other subspecies of H. aegypticum as well as other Hypericum species from Greece. SIMPER analysis revealed that α‐pinene (24.79%) was the component that contributed the most to differences between all oils. Also, there was extremely high overall dissimilarity between three subspecies of H. aegypticum. MeOH extract of aerial parts of H. aegypticum subsp. webbii contained flavonoids rutin (56.4 ± 0.9 mg/g), hyperoside and quercetin, and phenolic acids chlorogenic and caffeic acid, while naphthodianthrones were not detected. The antimicrobial activity of essential oil was moderate (MIC from 100 to >200 μg/ml), while MeOH extract inhibited the growth of Gram‐positive bacteria Bacillus subtilis, Enterococcus faecalis, Staphylococcus epidermidis, and Micrococcus luteus (MIC 50 – 100 μg/ml), more pronounced than the extract of H. perforatum (MIC 200 – >200 μg/ml).     

Biochemical and molecular characterization of recombinant acidic and thermostable raw-starch hydrolysing α-amylase from an extreme thermophile Geobacillus thermoleovorans

A gene encoding acidic, thermostable and raw starch hydrolysing α-amylase was cloned from an extreme thermophile Geobacillus thermoleovorans and expressed. The ORF of 1650 bp encodes a 515 amino acid protein (Gt-amy) with a signal peptide of 34 amino acids at the N-terminus. Seven conserved sequences of GH-13 family have been found in its sequence. The specific enzyme activity of recombinant Gt-amy is 1723 U mg⁻¹ protein with a molecular mass of 59 kDa. It is optimally active at pH 5.0 and 80 °C with t_1/2 values of 283, 184 and 56 min at 70, 80 and 90 °C, respectively. The activation energy required for its temperature deactivation is 84.96 kJ mol⁻¹. Ca²⁺ strongly inhibits Gt-amy at 10 mM concentration, and inhibition kinetics with Ca²⁺ reveals that inhibition occurs as a result of binding to a lower affinity secondary Ca²⁺ binding site in the active centre in a mixed-type inhibition manner. The K_m and k_cat of the Gt-amy are 0.315 mg mL⁻¹ and 2.62 × 10³ s⁻¹, respectively. Gt-amy is Ca²⁺-independent at the concentration used in industrial starch saccharification, and hydrolyses raw corn and wheat starches efficiently, and thus, is applicable in starch saccharification at the industrial sub-gelatinization temperatures.