Anoxybacillus sp. SO B1–Immobilized Amberlite XAD-16 for Solid-Phase Preconcentration of Cu(II) and Pb(II) and Their Determinations by Flame Atomic Absorption Spectrometry

A new method for the determination of Cu(II) and Pb(II) by flame atomic absorption spectrometry (FAAS) after preconcentrating on a column containing Anoxybacillus sp. SO B1–immobilized Amberlite XAD-16 was developed. The functional groups of Anoxybacillus sp. SO B1 immobilized on Amberlite XAD-16 were characterized in KBr tablets by Fourier transform infrared (FT-IR) spectrometry. Various parameters such as pH, amount of the adsorbent, eluent type and volume, and flow rate of the sample solution were studied. The optimum pH values of quantitative sorption for Cu(II) and Pb(II) were found to be pH 7.0 and 5.0 and Cu(II) and Pb(II) ions could be quantitatively eluted with 5.0 ml of 1.0 mol L^?1 HCI and 10.0 ml of 0.25 mol L^?1 HNO₃, respectively. Recoveries of Cu(II) and Pb(II) were found to be 100.9 ± 1.57% and 100.3 ± 0.49% (N = 5), the limits of detection of Cu(II) and Pb(II) in the determination by FAAS (3 s, N = 10) were found to be 0.8 and 1.6 μg L^?1, respectively. The proposed enrichment method was applied for metal ion determination from water samples such as two parts of Tigris River water in Diyarbak?r and Elaz??, Lake of Hazar in Elaz??, and tap water in Diyarbak?r. Furthermore, the accuracy of the proposed method was verified by studying the analytical recovery and by analyzing certified reference material (NCS-DC 73350 leaves of poplar).     

Immunostimulatory activities of specific bacterial secondary metabolite of Anoxybacillus flavithermus strain SX‐4 on carp,Cyprinus carpio

Aims: To determine the capacity of secondary metabolite of strain SX‐4, to enhance the nonspecific immunity and survival of carp (Cyprinus carpio), and to identify the constituents that are responsible. Methods and Results: A thermophilic strain SX‐4 that is able to produce immunostimulatory metabolite was isolated from sludge sample of hot spring and identified by comparison with 16S rRNA sequences (99% of homology) as Anoxybacillus flavithermus. Bioactivity‐guided fractionation of methanol extract from its cell‐free culture, one bacterial peptide with the capacity of improving the nonspecific immune responses and disease resistance (relative per cent survival = 66·67%) was obtained and the compound was characterized as cyclo‐(L‐Pro‐Gly) by IR, ESI‐MS, ¹H NMR and ¹³C NMR spectroscopic analyses. After intraperitoneal administration of this peptide, selected innate immune parameters including phagocytic activity, superoxide anion production, serum lysozyme activity and serum SOD activity, along with immune‐related genes expression (i.e. interleukin‐1β and inducible nitric oxide synthase), in the blood were found to be significantly increased. Conclusions: The bacterial peptide cyclo‐(L‐Pro‐Gly) significantly enhances nonspecific immunity and survival of carp. Significance and Impact of the Study: There is a possibility of using cyclo‐(L‐Pro‐Gly) as a better natural immunostimulant, which could have a promising role in aquaculture to prevent diseases and disease outbreaks.     

Isolation and characterization of novel denitrifying alkalithermophiles, AT-1 and AT-2

Two novel denitrifying alkalithermophilic bacteria, AT-1 and AT-2, were isolated from manure-amended soil. The isolates grew at 35–65°C with an optimum temperature at 50–60°C, and pH 6.5–10.0 with an optimum pH at 9.5. Both isolates were Gram-positive, facultative anaerobic, non-motile rod-shaped bacteria. A phylogenetic analysis based on 16S rRNA sequence data indicated that both AT-1 and AT-2 are members of the genus Anoxybacillus. DNA-DNA hybridization revealed moderate relatedness between AT-1 and AT-2 and one phylogenetically related strain, A. pushchinensis K1 (69.5 and 69.1%, respectively). Comparative analysis of morphology and biochemical characteristics of the two isolates also showed similarity to A. pushchinensis K1. Based on these results, we identified AT-1 and AT-2 as A. pushchinensis. To our knowledge, this is the first report of denitrifying bacterium isolated from alkalithermophilic Anoxybacillus spp.     

Characterization of BflI – A Thermostable,Co++-Requiring Isoschizomer of BsiYI from Anoxybacillus Flavithermus

A thermophile, isolated from geothermal areas in the northern Himalayan region of India, was identified by partial 16S rDNA sequence (GenBank accession # AF482430) analysis as Anoxybacillus flavithermus. The isolate produced BflI (REBASE # 4910), a Type II restriction endonuclease, which recognized the sequence 5′-CCNNNNN/NNGG-3′ and was the isoschizomer of BsiYI. The enzyme was purified to homogeneity by passing through Cibacron Blue F3GA agarose, DEAE-cellulose, heparin-agarose and MonoQ FPLC. The purified enzyme (MW 36 kDa) worked best at 60 °C in Promega's buffer C and preferentially required Co⁺⁺(0.4 mM) as cofactor followed by Mg⁺⁺(10 mM) and Mn⁺⁺(1 mM). The enzyme showed high specific activity and worked in the presence of high concentrations of β-mercaptoethanol (200 mM), Triton-X-100 (25%), urea (30%), formamide (6%) and guanidine (40 mM) and showed no star activity in the presence of 40% glycerol. In the absence of any stabilizing agent, BflI retained t _1/2 for at least 96 h at 37 °C, 6 h at 60 °C and 6 months at 4 °C. N-terminal sequencing showed that its first 10 amino acid residues were DFHEDKTIAR. This revised version was published online in July 2006 with corrections to the Cover Date.     

产木糖异构酶嗜热菌的筛选

取云南腾冲温泉水样在以木糖为唯一碳源的平板上进行培养,分离纯化得到11 株可以代谢木糖的菌株,分别进行液体摇床培养,并以半胱氨酸-咔唑法测定木糖异构酶活性,筛选得到一株产木糖异构酶活性较高的菌株RD-1,经形态学及16S rRNA 鉴定命名为Anoxybacillus .avithermus WL,该菌在24h 时显示最高酶活6.4 U/mL.     

Non-contiguous finished genome sequence of Anoxybacillus flavithermus subsp. yunnanensis type strain (E13T), a strictly thermophilic and organic solvent-tolerant bacterium

Anoxybacillus flavithermus subsp. yunnanensis is the only strictly thermophilic bacterium that is able to tolerate a broad range of toxic solvents at its optimal temperature of 55-60°C. The type strain E13^T was isolated from water-sediment slurries collected from a hot spring. This study presents the draft genome sequence of A. flavithermus subsp. yunnanensis E13^T and its annotation. The 2,838,393bp long genome (67 contigs) contains 3,035 protein-coding genes and 85 RNA genes, including 10 rRNA genes, and no plasmids. The genome information has been used to compare with the genomes from A. flavithermus subsp. flavithermus strains.     

一株产耐热普鲁兰酶菌株Anoxybacillus sp.LM14-2分离鉴定及酶学性质研究

从云南轮马热泉下游淤泥中筛选得到了一株产耐热普鲁兰酶菌株LM14-2.根据形态特征及16S rRNA序列同源性分析,初步判定为Anoxybacillus sp.LM14-2.该菌株发酵上清液中有耐热普鲁兰酶积累,其反应最适pH值为6.0,最适温度为70℃.利用染色体步移技术获得了完整的普鲁兰酶编码基因(HQ660582),经序列相似性进一步分析,确定该蛋白与Ⅰ型普鲁兰酶保守区b相吻合.通常的普鲁兰酶在高温下很快失活,难以满足淀粉加工,洗涤剂等相关工业的需求,而该新型的耐热普鲁兰醇的作用温度广泛,热稳定性较好,65℃保温55 h后达到其半衰期,具有广阔的开发应用前景.     

Anoxybacillus and Geobacillus biofilms in the dairy industry: effects of surface material,incubation temperature and milk type

Abstract

Anoxybacillus (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 log₁₀ CFU cm^?2) on glass and stainless steel (SS) at 55 and 65?°C, respectively. G. thermodenitrificans D195 had the highest counts on SS at 55?°C (>5 log₁₀ CFU cm^?2) and polyvinyl chloride (PVC) at 65?°C (>4 log₁₀ CFU cm^?2), indicating the existence of strain variation. The ideal surfaces for all strains were SS and glass at 55?°C, but their preferences were polystyrene and SS at 65?°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.     

Characterization of a xylanase from a thermophilic strain of <Emphasis Type="Italic">Anoxybacillus pushchinoensis</Emphasis> A8

A facultatively anaerobic, thermophilic, xylanolytic bacterium was isolated from a sample collected from the Diyadin Hot Springs, Turkey. According to morphological, biochemical and molecular identification, this new strain was suggested to be representative of the Anoxybacillus pushchinoensis and it was designated as Anoxybacillus pushchinoensis strain A8. It exhibited 97% similarity to 16S rRNA gene sequence of A. pushchinoensis and 77% DNA homology by DNA-DNA hybridization studies. Q-sepharose and CM-sepharose chromatography was used to purify an extracellular xylanase to >90% purity from this species. The enzyme had a molecular mass of approximately 83 kDa. The enzyme showed optimum activity at pH 6.5 and it was 96% stable over a broad pH range of 6.5–11 for 24 hours. The enzyme had optimum activity at 55°C and it was 100% stable at temperature between 50–60°C up to 24 hours. Kinetic characterization of the enzyme was performed at temperature optima (55°C) and V_max and K _m were found to be 59.88 U/mg protein and 0.909 mg/mL, respectively. Oat spelt xylan but not xylooligosaccharides was degraded by the enzyme and xylose was the only product detected from oat xylan degradation. This suggested that the enzyme was an exo-acting xylanase.     

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.