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Isolation, identification and enzyme characterization of a thermophilic cellulolytic anaerobic bacterium
嗜热厌氧纤维素分解菌的分离、鉴定及其酶学特性

Yinping Zhao,Shichun M,Yingjie Sun,Yan Huang,Yu Deng,
赵银瓶
,马诗淳,孙颖杰,黄艳,邓宇

微生物学报 , 2012,
Abstract: Objective] To identify a thermophilic bacterium from horse manure to degrade cellulose efficiently,and to enrich microbial resources producing cellulolytic ethanol by co-culturing with thermophilic ethanol producing bacterium.Methods] We used Hungate anaerobic technique to isolate a strain named as HCp from horse manure mixed culture;its phylogeny was identified through 16S rDNA sequencing.Enzymatic assays were determined using DNS method.Results] The isolated HCp cells were straight with rods size of(0.35-0.50)μm ×(2.42-6.40)μm,in the form of single or paring.This strain belongs to a strictly anaerobic Gram-negative bacterium,it is able to form spores,shows motile ability and resistance to neomycin.The strain could degrade filter paper cellulose,cellulose powder,microcrystalline cellulose,cotton wool,rice straw and gelatin,and it was also able to utilize abundant saccharides as substrates such as cellobiose,glucose,xylose,xylan,raffinose,maltose,sorbose,fructose and galactose.The growth pH ranges from 6.5 to 8.5,temperature from 35 to 70℃ and concentration of NaCl on cellulose from 0% to 1.0%,while the optima of pH6.85,60℃ and 0.2% NaCl.Under the optimal growth conditions,the filter paper cellulose degradation rate was up to 90.40% after 10 days.The optimum temperatures for FPA,CMCase,β-glucosidase and xylanase were 70℃,70℃,70℃,and 60℃ respectively.CMCase activity was found with high thermal stability.The phylogenetic analysis based on partial 16S rDNA revealed that HCp was close to Acetivibrio cellulolyticus and A.cellulosolvens with 97.5% sequence similarities.Conclusion] Strain HCp is thermophilic,efficiently cellulolytic anaerobe.It is able to utilize vast substrates and produce highly thermostable enzymes.It is a potential bacterium that can be used for cellulolytic ethanol production.
Isolation of a thermophilic bacterium and analysis of its extracellular poly-meric substance
一株高温产粘菌株的筛选及其产胞外聚合物分析

Jing Liu,Jun Wang,Xinyu Bai,Ting M,Fenglai Liang,Rulin Liu,
刘静
,王君,白新宇,马挺,梁凤来,刘如林

微生物学报 , 2008,
Abstract: A thermophilic bacterial strain MS-1 was isolated from the water let from an oil well, and identified as Bacillus sp. by its 16S rDNA sequence and morphological characteristics. The bacterium was able to grow at 60 degrees C and produce extracellular polymeric substance (EPS). Analysis of the EPS showed that there was 48.3% - 54.5% polysaccharide, which was composed of mannose:glucose:galactose at a ratio of 2.04 : 1.00 : 0.89. The protein fraction was 37.2% - 42.4% in EPS, containing methionine, leucine, aspartic acid, alanine, histidine and serine.Transmission Electron Microscopy and Environmental Scanning Electron Microscopy were used to observe the extracellular polymeric substances. The strain is useful in microbial profile modification in high temperature oil fields.
Isolation, identification and metabolic characterization of an anaerobic thermophilic bacterium
一株嗜热厌氧杆菌的分离、鉴定及其代谢特征

LAN Gui-Hong,GE Ju,LIU Hai-Chang,TANG Quan-Wu,ZHANG Hui,QIAO Dai-Rong,CAO Yi,
兰贵红
,葛菊,刘海昌,唐全武,张辉,乔代容,曹毅

微生物学通报 , 2012,
Abstract: Objective] To isolate,protect thermophilic microbial resources from petroleum reservoirs and analyze these main metabolic characterization.Methods] The strain BF1 was isolated by Hungte anaerobic technique from Chenghai 1 Unit of Dagang oil field in China.Its taxonomic status determined by physiological,biochemical and 16S rRNA gene sequence analysis.Its effect of sulfur metabolism on the corrosion current was measured by electro-chemical analysis.Results] The strain BF1 was Gram-negative,strictly thermophilic anaero-bic,top-sporulating,non-motile,rods,0.42 μm×(1.6?5.4) μm,grew solitary,in pairs or in chains.Growth occurred at 45 °C?75 °C(optimum 60 °C),at pH 4.5?8.5(optimum 6.5).Spe-cific growth rate(μm) was 0.99 h?1 and doubling time was 42 min.Substrates included glu-cose,melizitose,raffinose,mannose,lactose,fructose and ribose.The main products of glu-cose fermentation were CO2,H2,acetate and ethanol.The strain could reduce thiosulfate and sulfite to sulfide,and its tolerance limits were 75 mmol/L and 50 mmol/L,respectively.The electrochemical impedance reduced from 2 099 Ω/cm2 to 776 Ω/cm2 and the corrosion current increased from 9.936e-006 A to 3.25e-005 A after thiosulfate(50 mmol/L) was reduced.The fatty acids were mainly composed of saturated long chain fatty acids,with C15:0 the most,accounting for 70.6%.The G+C content of DNA was 34.0 mol%.The 16S rRNA gene se-quence analysis indicated that the closest phylogenetic relatives were Thermoanaerobacter pseudethanolicus DSM2355T and T.brockii subsp.brockii DSM1457T,98.3% and 98.0%,re-spectively.However,the strain BF1 was different with T.pseudethanolicus DSM2355T and T.brockii subsp.brockii DSM1457T in doubling time,optimum temperature and substrates util-ized,and different with T.pseudethanolicus DSM 2355T in fatty acid profile.Conclusion] The strain BF1 may be a new species of Thermoanaerobacter genus,the exact taxonomic status of it requires DNA hybridization further.The corrosion current density improved by its metabolic element sulfur may cause corrosion of oil pipelines and equipments.
Isolation, Identification and Phylogenetic Analysis of A Thermophilic Cellulolytic Anaerobic Bacterium
嗜热厌氧纤维素降解细菌的分离、鉴定及其系统发育分析

Han Ruyang Min Hang Che Meici Zhao Yuhua,
韩如旸
,闵航,陈美慈,赵宇华

微生物学报 , 2002,
Abstract: Four strains of thermophilic cellulolytic anaeobic bacteria were isolated from fresh feces, heat compost, cellulolytic mixed culture with a method based on adherence of cellulolytic bacteria to cellulose. The cells of isolates were straight or slightly curved rods that were 0.4 micron-0.6 micron x 3 microns-15 microns, Gram negative, strictly anaerobic, sulfate reduction negative, spore-forming bacteria. Most of the cells had oval terminal spores, while subterminal spores, middle spores, two or more spores also could be observed and spore formation could occurred in any position. The isolates degraded cellulose filter paper, cellulose powder Whatman CF II, microcrystalline cellulose, cellulose powder MN300 and unpretreated maize stem core, sugarcane residue and rice straw. The pH and temperature ranges for growth on cellulose were 6.2-8.9 and 45 degrees C-65 degrees C respectively with the optima, 7.0-7.5 and 55 degrees C-60 degrees C, respectively. The major fermentation products from cellulose were acetic acid, ethanol, CO2, H2. The isolates could ferment cellobiose, glucose, fructose, maltose, and sorbital. The phylogenetic analysis based on 16S rDNA suggested strain EVA1 was the closest relative of Clostridium thermocellum with 99.8% sequence similarity.
Isolation, identification and screening of potential xylanolytic enzyme from litter degrading fungi
M Palaniswamy, BV Pradeep, R Sathya, J Angayarkanni
African Journal of Biotechnology , 2008,
Abstract: Consortia of litter degrading fungal species were developed from different baiting substrates collected in and around Western ghat forest ecosystem, Coimbatore, Tamilnadu, India. Fifty-three litter degrading fungal species were isolated by nylon litterbag technique. The production of endo-b-1,4-xylanase (1,4-b-D-xylan xylanohydrolase, E.C. 3.2.1.8), b-D-xylosidase (1,4-b-xylan xylanohydrolase, E.C. 3.2.1.37) and protease was studied using oat spelt xylan as carbon source. Results showed that all fifty-three fungal species isolated from various litter samples produced fairly good xylanolytic enzyme activity. The xylanase and b-D-xylosidase activity ranges from 4.41 to 132.20 U and 48.72 to 1510.32 U, respectively. Growth was determined in terms of mycelial dry weight, which ranged between 0.209 and 1.047 mg/ml. The protease enzyme activity was from 19.7 to 60.8 U. This is the first report concerning xylanolytic enzyme production by the litter degrading fungi, isolated from litter samples.
Ribotyping Identification of Thermophilic Bacterium from Papandayan Crater  [PDF]
Akhmaloka,A. Suharto,S. Nurbaiti,I N. Tika
ITB Journal of Engineering Science , 2006,
Abstract: A few thermophilic bacteria were isolated from a hot spring located in Papandayan Crater, Garut. One of the organisms showed a well growth at temperature of up to 80oC. Chromosomal DNA from the organism was isolated and used to amplify 16S rRNA gene fragment. The gene was amplified by a set of universal primers (27F and 1492R) resulting in a 1.5 kb DNA fragment. The gene was cloned and sequenced. The phylogenetic tree, homological analysis, and detailed comparison of the sequences showed that 16S rRNA gene sequence of the Papandayan isolate is unique compared to other known strains, however the sequence had closest similarities with Bacillus caldolyticus and Bacillus caldotenax.
Extremely thermophilic microorganisms and their polymer-hidrolytic enzymes
Andrade, Carolina M.M.C.;Pereira Jr., Nei;Antranikian, Garo;
Revista de Microbiologia , 1999, DOI: 10.1590/S0001-37141999000400001
Abstract: thermophilic and hyperthermophilic microorganisms are found as normal inhabitants of continental and submarine volcanic areas, geothermally heated sea-sediments and hydrothermal vents and thus are considered extremophiles. several present or potential applications of extremophilic enzymes are reviewed, especially polymer-hydrolysing enzymes, such as amylolytic and hemicellulolytic enzymes. the purpose of this review is to present the range of morphological and metabolic features among those microorganisms growing from 70oc to 100°c and to indicate potential opportunities for useful applications derived from these features.
Extremely thermophilic microorganisms and their polymer-hidrolytic enzymes  [cached]
Andrade Carolina M.M.C.,Pereira Jr. Nei,Antranikian Garo
Revista de Microbiologia , 1999,
Abstract: Thermophilic and hyperthermophilic microorganisms are found as normal inhabitants of continental and submarine volcanic areas, geothermally heated sea-sediments and hydrothermal vents and thus are considered extremophiles. Several present or potential applications of extremophilic enzymes are reviewed, especially polymer-hydrolysing enzymes, such as amylolytic and hemicellulolytic enzymes. The purpose of this review is to present the range of morphological and metabolic features among those microorganisms growing from 70oC to 100°C and to indicate potential opportunities for useful applications derived from these features.
Production and properties of Xylanase from thermophilic Bacillus sp.
Cordeiro, Carlos Alberto Martins;Martins, Meire Lelis Leal;Luciano, Angélica Bárbara;Silva, Roberta Freitas da;
Brazilian Archives of Biology and Technology , 2002, DOI: 10.1590/S1516-89132002000600002
Abstract: an aerobic, thermophilic, xylanolytic bacterium was isolated from local soil. the results of 16s rrna sequence comparisons indicated that the isolate was closely related to bacillus caldoxylolyticus and bacillus sp strain ak1. these organisms exhibited 94% levels of ribossomal dna sequence homology. studies on the xylanase characterisation from liquid cultures grown on beechwood xylan revealed that the enzyme retained 100% of activity for 2 hours at temperatures ranging from 30 to 50o c, while at 60, 70 and 100o c, 10%, 11% and 29% of the original activities were lost, respectively. the optimum ph of the enzyme was found to be between 6.5 and 7.0. after incubation of crude enzyme solution for 24 hours at 25o c and at ph 5.5 to 8.0, a decrease of about 12% of its original activity was observed.
Production and properties of Xylanase from thermophilic Bacillus sp.  [cached]
Cordeiro Carlos Alberto Martins,Martins Meire Lelis Leal,Luciano Angélica Bárbara,Silva Roberta Freitas da
Brazilian Archives of Biology and Technology , 2002,
Abstract: An aerobic, thermophilic, xylanolytic bacterium was isolated from local soil. The results of 16S rRNA sequence comparisons indicated that the isolate was closely related to Bacillus caldoxylolyticus and Bacillus sp strain AK1. These organisms exhibited 94% levels of ribossomal DNA sequence homology. Studies on the xylanase characterisation from liquid cultures grown on beechwood xylan revealed that the enzyme retained 100% of activity for 2 hours at temperatures ranging from 30 to 50o C, while at 60, 70 and 100o C, 10%, 11% and 29% of the original activities were lost, respectively. The optimum pH of the enzyme was found to be between 6.5 and 7.0. After incubation of crude enzyme solution for 24 hours at 25o C and at pH 5.5 to 8.0, a decrease of about 12% of its original activity was observed.
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