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Isolation of Lipase Gene of the Thermophilic Geobacillus stearothermophilus Strain-5
M. Sifour,H.M. Saeed,T.I. Zaghloul,M.M. Berekaa
Biotechnology , 2010,
Abstract: In earlier study a new thermophilic strain Geobacillus stearothermophilus strain-5 producing thermostable lipase was isolated and identified based on 16S rRNA sequencing. Phylogenetic analysis revealed its closeness to geobacilli especially the thermophilic Geobacillus stearothermophilus with optimal growth and lipolytic enzyme activity at 60°C and pH 7.0. In this study thermostable lipase gene from this bacterium was isolated by PCR using degenerate primers. The DNA fragment coding for lipase gene was cloned in the pCR 4-TOPO plasmid and the ligation products were transformed into Escherichia coli XL1-blue cells. Partial sequencing of the gene was carried out (accession number DQ923401). Analysis by BLAST program showed some sequence similarity to that, of several lipase genes from thermophilic Geobacillus and Bacillus submitted to Genbank.
Isolation of the phe-operon from G. stearothermophilus comprising the phenol degradative meta-pathway genes and a novel transcriptional regulator
Bastian Omokoko, Uwe K J?ntges, Martin Zimmermann, Monika Reiss, Winfried Hartmeier
BMC Microbiology , 2008, DOI: 10.1186/1471-2180-8-197
Abstract: A 20.2 kb DNA fragment was isolated as a result of the DNA walk. Fifteen open reading frames residing on a low-copy megaplasmid were identified. Eleven genes are co-transcribed in one polycistronic mRNA as shown by reverse transcription-PCR. Ten genes encode proteins, that are directly linked with the meta-cleavage pathway. The deduced amino acid sequences display similarities to a two-component phenol hydroxylase, a catechol 2,3-dioxygenase, a 4-oxalocrotonate tautomerase, a 2-oxopent-4-dienoate hydratase, a 4-oxalocrotonate decarboxylase, a 4-hydroxy-2-oxovalerate aldolase, an acetaldehyde dehydrogenase, a plant-type ferredoxin involved in the reactivation of extradiol dioxygenases and a novel regulatory protein. The only enzymes missing for the complete mineralization of phenol are a 2-hydroxymuconic acid-6-semialdehyde hydrolase and/or 2-hydroxymuconic acid-6-semialdehyde dehydrogenase.Research on the bacterial degradation of aromatic compounds on a sub-cellular level has been more intensively studied in gram-negative organisms than in gram-positive bacteria. Especially regulatory mechanisms in gram-positive (thermophilic) prokaryotes remain mostly unknown. We isolated the first complete sequence of an operon from a thermophilic bacterium encoding the meta-pathway genes and analyzed the genetic organization. Moreover, the first transcriptional regulator of the phenol metabolism in gram-positive bacteria was identified. This is a first step to elucidate regulatory mechanisms that are likely to be distinct from modes described for gram-negative bacteria.Geobacillus stearothermophilus, formerly Bacillus stearothermophilus, is a thermophilic, gram-positive, spore-forming bacterium. The rod-shaped organism has an optimal growth temperature of 50–60°C and is widely distributed in soil and sediments. The first strain able to metabolize various phenols was isolated by Buswell and Twomey [1]. Other phenol-degrading bacteria of the same genus isolated since include G. the
Geobacillus zalihae sp. nov., a thermophilic lipolytic bacterium isolated from palm oil mill effluent in Malaysia
Raja Rahman, Thean Leow, Abu Salleh, Mahiran Basri
BMC Microbiology , 2007, DOI: 10.1186/1471-2180-7-77
Abstract: Twenty-nine putative lipase producers were screened and isolated from palm oil mill effluent in Malaysia. Of these, isolate T1T was chosen for further study as relatively higher lipase activity was detected quantitatively. The crude T1 lipase showed high optimum temperature of 70°C and was also stable up to 60°C without significant loss of crude enzyme activity. Strain T1T was a Gram-positive, rod-shaped, endospore forming bacterium. On the basic of 16S rDNA analysis, strain T1T was shown to belong to the Bacillus rRNA group 5 related to Geobacillus thermoleovorans (DSM 5366T) and Geobacillus kaustophilus (DSM 7263T). Chemotaxonomic data of cellular fatty acids supported the affiliation of strain T1T to the genus Geobacillus. The results of physiological and biochemical tests, DNA/DNA hybridization, RiboPrint analysis, the length of lipase gene and protein pattern allowed genotypic and phenotypic differentiation of strain T1T from its validly published closest phylogenetic neighbors. Strain T1T therefore represents a novel species, for which the name Geobacillus zalihae sp. nov. is proposed, with the type strain T1T (=DSM 18318T; NBRC 101842T).Strain T1T was able to secrete extracellular thermostable lipase into culture medium. The strain T1T was identified as Geobacillus zalihae T1T as it differs from its type strains Geobacillus kaustophilus (DSM 7263T) and Geobacillus thermoleovorans (DSM 5366T) on some physiological studies, cellular fatty acids composition, RiboPrint analysis, length of lipase gene and protein profile.The Bacillus rRNA group 5 which comprised thermophilic Bacillus strains was transferred into new genus Geobacillus which represented a phenotypically and phylogenetically coherent group of thermophilic bacilli with high levels of 16S rRNA sequence similarity (96.5–99.2%) [1]. The members of this genus are widespread in various thermophilic and mesophilic geographic areas on the earth such as oilfields, hay compost, hydrothermal vent or soils [1-5]
Alkane inducible proteins in Geobacillus thermoleovorans B23
Tomohisa Kato, Asuka Miyanaga, Shigenori Kanaya, Masaaki Morikawa
BMC Microbiology , 2009, DOI: 10.1186/1471-2180-9-60
Abstract: An extremely thermophilic and alkane degrading Geobacillus thermoleovorans B23 was previously isolated from a deep subsurface oil reservoir in Japan. In the present study, we identified novel membrane proteins (P16, P21) and superoxide dismutase (P24) whose production levels were significantly increased upon alkane degradation. Unlike other bacteria acyl-CoA oxidase and catalase activities were also increased in strain B23 by addition of alkane.We first suggested that peroxisomal β-oxidation system exists in bacteria. This eukaryotic-type alkane degradation pathway in thermophilic bacterial cells might be a vestige of primitive living cell systems that had evolved into eukaryotes.Thermophilic bacteria offer crucial advantages over mesophilic or psychrophilic bacteria, especially when they are applied to ex-situ bioremediation processes. Limited biodegradation of hydrophobic substrates caused by low water solubility at moderate temperature conditions can be overcome if the reaction temperature could be increased enough. We previously isolated an extremely thermophilic alkane-degrading bacterium, Goebacillus thermoleovorans (previously Bacillus thermoleovorans) B23, from a deep-subsurface oil reservoir in Japan [1,2]. Strain B23 effectively degraded alkanes at 70°C with the carbon chain longer than twelve, dodecane. Since tetradecanoate and hexadecanoate or pentadecanoate and heptadecanoate were accumulated as degradation intermediates of hexadecane or heptadecane, respectively, it was indicated that the strain B23 degraded alkanes by a terminal oxidation pathway, followed by β-oxidation pathway. Recently, another long-chain alkane degrading Geobacillus thermodenitrificans NG80-2 was also isolated from a deep-subsurface oil reservoir [3] and its complete genome sequence was determined [4].Besides their biotechnological importance, thermophilic microorganisms maintain interesting features useful for studying evolution of life. Microorganisms living under extremely high
Thermophilic fermentation of acetoin and 2,3-butanediol by a novel Geobacillus strain  [cached]
Xiao Zijun,Wang Xiangming,Huang Yunling,Huo Fangfang
Biotechnology for Biofuels , 2012, DOI: 10.1186/1754-6834-5-88
Abstract: Background Acetoin and 2,3-butanediol are two important biorefinery platform chemicals. They are currently fermented below 40°C using mesophilic strains, but the processes often suffer from bacterial contamination. Results This work reports the isolation and identification of a novel aerobic Geobacillus strain XT15 capable of producing both of these chemicals under elevated temperatures, thus reducing the risk of bacterial contamination. The optimum growth temperature was found to be between 45 and 55°C and the medium initial pH to be 8.0. In addition to glucose, galactose, mannitol, arabionose, and xylose were all acceptable substrates, enabling the potential use of cellulosic biomass as the feedstock. XT15 preferred organic nitrogen sources including corn steep liquor powder, a cheap by-product from corn wet-milling. At 55°C, 7.7 g/L of acetoin and 14.5 g/L of 2,3-butanediol could be obtained using corn steep liquor powder as a nitrogen source. Thirteen volatile products from the cultivation broth of XT15 were identified by gas chromatography–mass spectrometry. Acetoin, 2,3-butanediol, and their derivatives including a novel metabolite 2,3-dihydroxy-3-methylheptan-4-one, accounted for a total of about 96% of all the volatile products. In contrast, organic acids and other products were minor by-products. α-Acetolactate decarboxylase and acetoin:2,6-dichlorophenolindophenol oxidoreductase in XT15, the two key enzymes in acetoin metabolic pathway, were found to be both moderately thermophilic with the identical optimum temperature of 45°C. Conclusions Geobacillus sp. XT15 is the first naturally occurring thermophile excreting acetoin and/or 2,3-butanediol. This work has demonstrated the attractive prospect of developing it as an industrial strain in the thermophilic fermentation of acetoin and 2,3-butanediol with improved anti-contamination performance. The novel metabolites and enzymes identified in XT15 also indicated its strong promise as a precious biological resource. Thermophilic fermentation also offers great prospect for improving its yields and efficiencies. This remains a core aim for future work.
Structural Analysis of Xylanase from Marine Thermophilic Geobacillus stearothermophilus in Tanjung Api, Poso, Indonesia
HAYATI Journal of Biosciences , 2010,
Abstract: A xylanase gene, xynA, has been cloned from thermophilic strain Geobacillus stearothermophilus, which was isolated from marine Tanjung Api, Indonesia. The polymerase chain reaction product of 1266 bp of xynA gene consisted of 1221 bp open reading frame and encoded 407 amino acids including 30 residues of signal peptide. The sequence exhibited highest identity of 98.7% in the level of amino acid, with an extracellular endo-1,4-β-xylanase from G. stearothermophilus T-6 (E-GSX T-6) of the glycoside hydrolase family 10 (GH10). A comparative study between the local strain G. stearothermophilus (GSX L) and E-GSX T-6 on homology of amino acid sequence indicated five differents amino acids in the gene. They were Threonine/Alanine (T/A), Asparagine/Aspartate (N/D), Lysine/Asparagine (K/N), Isoleucine/Methionine (I/M), Serine/Threonine (S/T) at the position 220, 227, 228, 233, and 245, respectively. Protein structural analysis of those differences suggested that those amino acids may play role in biochemical properties such as enzyme stability, in particular its thermostability.
Biochemical characterization of thermophilic lignocellulose degrading enzymes and their potential for biomass bioprocessing  [PDF]
Vasudeo Zambare, Archana Zambare, Kasiviswanath Muthukumarappan, Lew P. Christopher
International Journal of Energy and Environment , 2011,
Abstract: A thermophilic microbial consortium (TMC) producing hydrolytic (cellulolytic and xylanolytic) enzymes was isolated from yard waste compost following enrichment with carboxymethyl cellulose and birchwood xylan. When grown on 5% lignocellulosic substrates (corn stover and prairie cord grass) at 600C, the thermophilic consortium produced more xylanase (up to 489 U/l on corn stover) than cellulase activity (up to 367 U/l on prairie cord grass). Except for the carboxymethyl cellulose-enriched consortium, thermo-mechanical extrusion pretreatment of these substrates had a positive effect on both activities with up to 13% and 21% increase in the xylanase and cellulase production, respectively. The optimum temperatures of the crude cellulase and xylanase were 600C and 700C with half-lives of 15 h and 18 h, respectively, suggesting higher thermostability for the TMC xylanase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the crude enzyme exhibited protein bands of 25-77 kDa with multiple enzyme activities containing 3 cellulases and 3 xylanases. The substrate specificity declined in the following descending order: avicel>birchwood xylan>microcrystalline cellulose>filter paper>pine wood saw dust>carboxymethyl cellulose. The crude enzyme was 77% more active on insoluble than soluble cellulose. The Km and Vmax values were 36.49 mg/ml and 2.98 U/mg protein on avicel (cellulase), and 22.25 mg/ml and 2.09 U/mg protein, on birchwood xylan (xylanase). A total of 50 TMC isolates were screened for cellulase and xylanase secretion on agar plates. All single isolates showed significantly lower enzyme activities when compared to the thermophilic consortia. This is indicative of the strong synergistic interactions that exist within the thermophilic microbial consortium and enhance its hydrolytic capabilities. It was further demonstrated that the thermostable enzyme-generated lignocellulosic hydrolyzates can be fermented to bioethanol by a recombinant strain of Escherichia coli. This could have important implications in the enzymatic breakdown of lignocellulosic biomass for the establishment of a robust and cost-efficient process for production of cellulosic ethanol. To the best of our knowledge, this work represents the first report in literature on biochemical characterization of lignocellulose-degrading enzymes from a thermophilic microbial consortium.
Isolation and characterization of phenol degrading Xanthobacter flavus
A Nagamani, R Soligalla, M Lowry
African Journal of Biotechnology , 2009,
Abstract: A soil bacterium isolated from a contaminated site degraded phenol as the sole carbon and energy source was identified as Xanthobacter flavus MTCC 9130. This microbial strain was able to tolerate phenol up to 1100 mg/l concentration. The lag phase increased with the increase in phenol concentration. The optimum growth temperature was 37°C. The organism could degrade completely within 120 h when initial concentration was less than 600 mg/l. Enzyme assay through cell free extract showed the presence of catechol-1,2-dioxygenase. The specific activity was 0.146 ìmol/min/mg protein. However higher concentrations of phenol in the medium showed a negative effect on the growth of the bacterium. Hence X. flavus can be effectively used for bioremediation of phenol-contaminated sites.
Combination of Oxyanion Gln114 Mutation and Medium Engineering to Influence the Enantioselectivity of Thermophilic Lipase from Geobacillus zalihae  [PDF]
Roswanira Abdul Wahab,Mahiran Basri,Mohd Basyaruddin Abdul Rahman,Raja Noor Zaliha Raja Abdul Rahman,Abu Bakar Salleh,Thean Chor Leow
International Journal of Molecular Sciences , 2012, DOI: 10.3390/ijms130911666
Abstract: The substitution of the oxyanion Q114 with Met and Leu was carried out to investigate the role of Q114 in imparting enantioselectivity on T1 lipase. The mutation improved enantioselectivity in Q114M over the wild-type, while enantioselectivity in Q114L was reduced. The enantioselectivity of the thermophilic lipases, T1, Q114L and Q114M correlated better with log p as compared to the dielectric constant and dipole moment of the solvents. Enzyme activity was good in solvents with log p < 3.5, with the exception of hexane which deviated substantially. Isooctane was found to be the best solvent for the esterification of ( R, S)-ibuprofen with oleyl alcohol for lipases Q114M and Q114L, to afford E values of 53.7 and 12.2, respectively. Selectivity of T1 was highest in tetradecane with E value 49.2. Solvents with low log p reduced overall lipase activity and dimethyl sulfoxide ( DMSO) completely inhibited the lipases. Ester conversions, however, were still low. Molecular sieves employed as desiccant were found to adversely affect catalysis in the lipase variants, particularly in Q114M. The higher desiccant loading also increased viscosity in the reaction and further reduced the efficiency of the lipase-catalyzed esterifications.
Culture and Biodegradation Performance for Phenol-Degrading Bacterium in High Phenol Concentration

Lv Rong-hu,FU Qiang,

环境科学 , 2005,
Abstract: The activated sludge was acclimated by the inorganic salt medium containing phenol as the sole carbon source. The phenol-degrading bacterium obtained now can degrade completely phenol at initial concentration of 1 700mg/L at 30 for 15h, and COD can be degraded by 96.9%. Some factors effecting degradation of phenol and COD were studied, which include initial phenol concentration, quantity of bacterium, temperature and pH. Four strains of bacterium were isolated from the mixed phenol-degrading bacterium. It is shown that the degradation effect of mixed bacterium is superior to that of sole strain.
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