一株油藏嗜热厌氧杆菌的分离鉴定和代谢特征的分析

摘要：【目的】从高温油藏中发掘新的微生物种质资源。【方法】采用 Hungate 厌氧操作技术从大港油田采出水中分离到一株厌氧杆菌 HL-3。通过生理生化特征比较和16S rRNA序列比对,确定HL-3的分类地位。【结果】菌株HL-3为严格厌氧的革兰氏阴性杆菌。生长温度范围 40℃－75℃（最适温度 60℃）;pH 范围 5.0－8.0（最适 pH 6.5）;NaCl 浓度范围 0%－3.2%（最适NaCl浓度0.25%）。能够利用葡萄糖、核糖、甘露糖、木糖、纤维二糖等多种碳水化合物,发酵葡萄糖的产物是乙醇、乙酸、CO2及少量丙酸和丁醇。菌株HL-3的（G+C）mol%含量为 33.9%,与Thermoanaerobacter（嗜热厌氧杆菌属）中模式菌株T.uzonensis DSM18761T (EF530067)的16SrRNA 序列相似性为98.8%,与T.sulfurigignens DSM17917T (AF234164)的相似度次之为98.1％。菌株能够耐受浓度较高的亚硫酸根（0.1 mol/L）离子和浓度极高的硫代硫酸根（0.8 mol/L）。当硫代硫酸根浓度高于0.075 mol/L时,菌体内产生硫单质颗粒;同时,在培养血清瓶顶空中检测到硫化氢气体。菌株 HL-3与T.uzonensis DSM18761T对硫代硫酸根和亚硫酸根的耐受程度有很大不同。菌株HL-3对硫代硫酸根和亚硫酸根耐受程度及对硫代硫酸根的代谢机制与T.sulfurigignens DSM17917T(AF234164)极为相似,但二者代谢葡萄糖的产物却极不相同。【结论】所以菌株HL-3可能是Thermoanaerobacter属中的一个新种,其确切分类地位还有待用DNA分子杂交[1]的技术手段做进一步的鉴定。     

日本三宅岛火山土壤中硫代硫酸盐氧化菌的分离、生物学特性及鉴定

【目的】利用培养法从日本三宅岛火山土壤(堆积年限131年)中分离到一株能氧化分解硫代硫酸盐的细菌MU2A-22T。【方法】用培养法对该菌株MU2A-22T进行了生理生化性质以及分类学位置上的确定。【结果】菌株MU2A-22T为革兰氏阴性,短杆状或球状。理化性质表明该菌株能利用葡萄糖、L-阿拉伯糖、葡萄糖酸盐、己二酸酯、dL-苹果酸钠、硫代硫酸钠(最适浓度为2.5 mmol/L)为唯一碳源进行自养生长。最适生长温度为25°C 30°C,最适pH为6.0 8.0。菌株MU2A-22T的16S rRNA序列与菌株Paracoccus solventivorans 6637T亲缘关系最近,序列相似性为97%,编码核酮糖-1,5-二磷酸羧化酶/加氧酶(Rubisco)的基因也被确定。对Paracoccus属内几种近缘菌的脂肪酸分析,证明菌株MU2A-22T中含有Paracoccus属的特征氨基酸,其中含量大于10%的分别为C18:1(74.7%)和C18:0(12.1%)。DNA-DNA杂交实验表明,菌株MU2A-22T与Paracoccus solventivorans 6637TDNA的相似度为49.3%。MU2A-22T菌株G+C含量为66.5%66.7%。【结论】菌株MU2A-22T为Paracoccus属内的一新种菌(登录号GQ452286),命名为Paracoccus scorialis sp.nov.。     

一株高效降解木糖的耐酸、嗜热厌氧杆菌的生理特性及产物分析

【目的】分离高效降解木糖的嗜热厌氧杆菌菌株,用于发酵生产生物燃料乙醇,为后继的构建基因工程菌株及联合生物工艺提供材料。【方法】运用亨盖特厌氧操作技术从胜利油田油层采出液两年的富集样中分离到一株嗜热厌氧杆菌xyl-d。采用形态学观察、生理生化指标鉴定及基于16S rRNA的系统发育学分析确定其分类地位。【结果】菌株xyl-d为革兰氏阴性厌氧杆菌,菌体大小为(1.35-5.08)μm×(0.27-0.40)μm,单生、成对或成簇生长,芽胞圆形,端生。温度生长范围30-85℃(最适温度65℃);pH范围3.0-10.0(最适pH 7.5);NaCl浓度范围0%-4%(最适NaCl浓度2.0%)。发酵D-木糖的产物是乙醇、乙酸、CO2及少量的异丁醇、丙酸。菌株xyl-d的(G+C)mol%含量为45.6%,与热厌氧杆菌属模式菌株威吉利热厌氧杆菌(Thermoanaerobacter wiegelii)DSM10319T及嗜热乙醇杆菌(Thermoanaerobacter ethanolicus)DSM2246T的16S rRNA序列相似性均为99.3%。菌株利用D-木糖产乙醇的最佳初始pH为8.5;少量酵母粉能刺激生长并显著提高发酵D-木糖的产醇率,使乙醇成为主要的发酵产物;培养基中乙醇浓度达到7%(V/V)时菌体生长受到抑制,最佳生长条件下D-木糖的降解率可达91.37%,最佳产醇条件下发酵1摩尔D-木糖可产生1.29摩尔的乙醇。【结论】菌株xyl-d是从特殊生境(油藏)中分离到的一株高效降解D-木糖的耐酸、嗜热的厌氧杆菌,其为半纤维素降解产乙醇的联合生物工艺提供了菌源。     

来源于中国南海的微生物新种乙醇速生杆菌(Celeribacter ethanolicus)多相分类研究（英文）

【目的】研究来源于南海海水的一株速生杆菌属菌株NH195T的多相分类。【方法】采用表型、基因型和化学分类方法,并综合系统发育关系结果,分析菌株NH195T的分类学地位。【结果】菌株NH195T是一株革兰氏阴性、好氧、杆状、无运动性细菌;能积累Poly-β-hydroxy butyrate(PHB);能在0.5%-10.0%(质量体积比)NaCl浓度,pH 5.0-9.0和20-40°C条件下生长,最适NaCl生长浓度为1.0%-3.0%;氧化酶、触酶和脲酶反应结果阳性。菌株NH195T主要呼吸醌为Q-10,主要脂肪酸为C18:1ω7c、C18:1ω6c和11 methyl C_(18:1)ω7c,主要极性脂为磷脂酰胆碱、磷脂酰甘油、双磷脂酰甘油、一个未知的氨基脂和两个未知脂。基因组G+C含量为61.3 mol%。基于16S rRNA基因的系统发育结果显示,菌株NH195T隶属于速生杆菌属;其与速生杆菌属标准菌株的16S rRNA基因相似性范围为94.4%-97.7%。菌株NH195T与速生杆菌属标准菌株C.halophilus ZXM137T和C.indicus P73T的平均核苷酸一致性(ANI)分别为78.6%和78.0%;基于基因组数据计算所得的DNA杂交同源率分别为26.1%和23.0%。【结论】基于表型和基因型结果,菌株NH195T代表了速生杆菌属一个新物种,命名为Celeribacter ethanolicus,标准菌株为NH195T(CGMCC 1.15406T=JCM 31095T)。     

一株源自渤海海域高温酸败油井采出水的硫酸盐还原菌筛选与活性抑制

【背景】海域油藏环境中硫酸盐还原菌(sulfate-reducing prokaryotes,SRP)的生长和代谢能够产生大量H_2S,引发酸败(souring)及微生物腐蚀等多方面的问题。但有关油藏生境中SRP的微生物多样性与生理活性及其危害控制的认识仍十分有限。【目的】深入了解我国渤海海域某高温酸败油藏中SRP的生理特点,探究SRP危害的控制策略。【方法】采用Hungate厌氧纯培养技术从渤海海域某高温酸败油井采出水中分离筛选SRP,并考察优势菌株的生理特点,综合16S rRNA基因序列比对分析菌株系统发育地位,着重评价不同杀菌剂及使用剂量对优势菌株产H_2S活性的抑制效力。【结果】酸败采出井的采出水中优势SRP菌株WJ_1的细胞呈长杆状,菌体长度为2.0-5.5μm,有运动性。WJ_1与解糖泽恩根氏菌(Soehngenia saccharolytica)模式菌株BOR-Y~T的16S rRNA基因序列一致性达99%。WJ_1最适生长温度约为37°C,能够耐受60°C高温;最适pH值约为8.0 (范围5.0-10.0),最适盐度约为0%-3%,盐度高于5%不能生长。WJ_1可利用丙酸钠、乳酸钠、乙酸盐等多种碳源,能以硫酸盐、亚硫酸盐、硫代硫酸盐为唯一电子受体产生H2S,不能利用单质硫。次氯酸钠(600 mg/L)、苄基三甲基氯化铵(600 mg/L)对WJ_1产H_2S活性无明显抑制效果。戊二醛(30 mg/L)、溴硝醇(10 mg/L)或四羟甲基硫酸磷(120 mg/L)可抑制WJ_1产H_2S活性达30 d以上。【结论】渤海海域某高温酸败油井采出水中优势SRP菌株WJ_1与S. saccharolytica BOR-Y~T的16S rRNA基因序列一致性最高,但两者生理特性差异明显;溴硝醇、戊二醛、四羟甲基硫酸磷是控制该酸败油井的潜在有效杀菌剂。     

一株食醋污染菌CICC 10774 的鉴定及其生长代谢特性

【目的】对食醋污染菌CICC 10774进行多相分类学鉴定,并对其生长特征和代谢产物进行研究。【方法】通过16S r RNA基因序列系统发育学分析,结合形态特征和生理生化特性确定该菌株的分类学地位。通过分光光度法测定菌株生长的最适温度和最适p H值,确定其最佳培养条件。采用HPLC测定该菌株代谢产物。【结果】多相鉴定分析表明菌株CICC 10774为耐酸乳杆菌(Lactobacillus acetotolerans),革兰氏染色呈阳性,兼性厌氧生长,具有明胶酶活性,能够利用葡萄糖、果糖、甘露糖、N-乙酰葡萄糖胺、熊果苷、七叶灵、水杨苷、纤维二糖、海藻糖和龙胆二糖作为碳源底物生长。生长代谢特性研究表明,CICC 10774的最适生长温度为37°C,最适p H值为5.0,代谢产物主要为醋酸和乳酸,是一株典型的耐酸菌。【结论】对引起食醋污染的耐酸乳杆菌CICC 10774生长代谢特征进行了研究,为食醋生产企业生产过程的微生物控制提供理论基础。     

6种区分乳酸乳球菌乳酸亚种和乳酸乳球菌乳脂亚种的分子生物学方法比较

【目的】比较并评价6种分子生物学技术对乳酸乳球菌乳酸亚种(Lactococcus lactis subsp.lactis)和乳酸乳球菌乳脂亚种(Lactococcus lactis subsp.cremoris)的区分效果。【方法】采用16S rRNA基因序列分析技术,16S-23S rRNA间区序列多态性分析技术,变性梯度凝胶电泳技术(DGGE),随机扩增多态性分析技术(RAPD),重复基因外回文序列分析技术(rep-PCR)和限制性酶切片段多态性分析技术(RFLP)对4株Lactococcus lactis subsp.lactis和Lactococcus lactis subsp.cremoris参考菌株进行了区分,并对这6种方法的区分效果进行了比较评价。【结果】16S rRNA基因序列分析技术,16S-23S rRNA间区序列多态性分析技术无法区分Lactococcus lactis subsp.lactis和Lactococcus lactis subsp.cremoris,而其余4种技术可以实现区分。【结论】变性梯度凝胶电泳(DGGE),随机扩增多态性分析技术(RAPD)耗时短,操作简单,试验结果准确稳定,更适合Lactococcus lactis subsp.lactis和Lactococcus lactis subsp.cremoris的快速准确区分。     

粪臭素高效降解菌YKSW-6的分离、鉴定及降解特性

【背景】粪臭素是畜牧堆肥中有机污染物的主要成分,造成养殖场及周边环境恶化,粪臭素污染问题亟待解决,利用微生物降解粪臭素是一种环保节能的有效方法。【目的】分离鉴定粪臭素高效降解菌株,研究其降解特性,为粪臭素降解提供高效的菌种资源,为该菌株应用于臭味污染环境的净化提供基础。【方法】以粪臭素为唯一碳源的无机盐培养基作为培养基质,从猪粪堆肥样品中分离筛选粪臭素高效降解菌株,通过形态特征和16S rRNA基因序列分析进行分离菌株的初步鉴定,分析其生长规律及粪臭素降解特性,并利用气相色谱质谱联用(GC-MS)对菌株代谢粪臭素的产物进行分析。【结果】从样品中分离获得一株能以粪臭素为唯一碳源的细菌YKSW-6菌株,形态学和16S rRNA基因序列分析初步鉴定该菌株为戈登氏红球菌(Rhodococcus gordoniae)。接种量为10%时,该菌培养14 h对100 mg/L的粪臭素降解率达到100%。其能够利用D-山梨醇、溴-丁二酸等18种碳源,对亚碲酸钾、溴酸钾等13种化学敏感物具有抗性。菌株YKSW-6在5%接种量、温度30-42℃和pH值为6.0-9.0时对100 mg/L的粪臭素降解效率均能达到100%,菌株生长和降解粪臭素的最佳条件为：pH 7.2,温度37℃,转速180 r/min。GC-MS结果表明,粪臭素在菌株的作用下C2先被氧化,转变为3-甲基羟基吲哚,随后进一步被氧化为N-(2-乙酰基苯基)甲酰胺。同时中间产物还有苯乙醛和苯乙酸。【结论】红球菌YKSW-6为目前已报道的降解粪臭素能力较强的菌株,丰富了粪臭素降解菌种的资源库,为实际环境微生物修复应用提供了理论参考。     

桑氏链霉菌KJ40全基因组测序及分析

【背景】桑氏链霉菌(Streptomyces sampsonii)KJ40是一株具有防病、促生多重功能的放线菌,有作为生物农药的潜力。目前还没有相关研究报道S.sampsonii全基因组序列,这限制了其功能基因、代谢产物合成途径及比较基因组学等研究。【目的】解析S.sampsonii KJ40的基因组序列信息,以深入研究该菌株防病促生机制及挖掘次级代谢产物基因资源。【方法】利用Illumina HiSeq高通量测序平台对KJ40菌株进行全基因组测序,使用相关软件对测序数据进行基因组组装、基因预测和功能注释、预测次级代谢产物合成基因簇、共线性分析等。【结果】基因组最后得到9个Scaffolds和578个Contigs,总长度为7 261 502 bp,G+C%含量平均为73.41%,预测到6 605个基因、1 260个串联重复序列、804个小卫星序列、67个微卫星序列、90个tRNA、9个rRNA和19个sRNA。其中,2 429、3 765、2 890、6 063和1 911个基因分别能够在COG、GO、KEGG、NR和Swiss-Prot数据库提取到注释信息。同时,还预测得到21个次级代谢产物合成基因簇。基因组测序数据提交至NCBI获得Gen Bank登录号:LORI00000000。S.sampsonii KJ40与Streptomyces coelicolor A3(2)、Streptomyces griseus subsp.griseus NBRC 13350三株链霉菌基因组存在翻转、易位等基因组重排,3个基因组共有1 711个蛋白聚类簇。【结论】研究为从基因组层面上解析KJ40菌株具有良好促生防病效果的内在原因提供基础数据,为深入了解链霉菌次级代谢合成途径提供参考信息,对S.sampsonii后续相关研究具有重要意义。     

一株油藏嗜热厌氧杆菌的分离、鉴定及代谢产物特征

[目的]了解油藏环境中细菌的生理生化特性及代谢产物.[方法]采用Hungate厌氧操作技术从胜利油田罗801区块油层采出水中分离到一株厌氧杆菌SC-2.采用生理生化鉴定结合16S rDNA序列的系统发育学分析确定该菌株的系统发育地位,用气相色谱分析其代谢产物.[结果]菌株SC-2为严格厌氧的革兰氏阴性杆菌,菌体大小为0.38 um×1.7um-3.9um,单生、成对或成串生长,产端生芽孢.温度生长范围40℃-75℃(最适温度70℃);pH范围5.5-9.5(最适pH 6.5);NaCl浓度范围0%～5%(最适NaCl浓度0%).能够利用葡萄糖、麦芽糖、甘露糖、木糖等多种碳水化合物,发酵葡萄糖的产物是乙醇、乙酸、丙酸、H2、CO2及少量的乳酸.菌株SC-2的(G C)mol%含量为30.8%,与Thermoanaerobacter mathranii subsp.mathranii的16S rDNA序列相似性为99.85%.菌株利用葡萄糖产乙酸、乙醇的最佳初始pH为8.0;酵母粉能刺激生长并显著提高发酵葡萄糖的产酸、产醇率;培养基中添加4%(V/V)的乙醇能明显抑制菌体生长.[结论]菌株SC-2是从特殊生境(油层采出水)中分离到的一株嗜热、耐盐的厌氧菌,其发酵葡萄糖产生的代谢产物有利于改善油藏中的微环境.菌株SC-2与T.mathranii subsp.mathranii 11426T的最适pH和最大耐受NaCl浓度有所不同,且二者的(G C)mol%含量差异较大.     

Utilization of serine, leucine, isoleucine, and valine by Thermoanaerobacter brockii in the presence of thiosulfate or Methanobacterium sp. as electron acceptors

Thermoanaerobacter brockii fermented serine to acetate and ethanol. It oxidized leucine to isovalerate, isoleucine to 2-methylbutyrate, and valine to isobutyrate only in the presence of thiosulfate, or when co-cultured with Methanobacterium sp. This oxidative deamination was rendered thermodynamically possible by the ability ofT. brockii to reduce thiosulfate to sulfide or the transfer of reducing equivalents to the hydrogenotrophic methanogen. The results suggest that T. brockii may be of ecological significance in thermal environments in the turnover of amino acids, especially with thiosulfate or H(2)-utilizing methanogens are present.     

Isolates of Thermoanaerobacter thermohydrosulfuricus from decaying wood compost display genetic and phenotypic microdiversity

In this study, 12 strains of Thermoanaerobacter were isolated from a single decaying wood compost sample and subjected to genetic and phenotypic profiling. The 16S rRNA encoding gene sequences suggested that the isolates were most similar to strains of either Thermoanaerobacter pseudethanolicus or Thermoanaerobacter thermohydrosulfuricus. Examination of the lesser conserved chaperonin-60 (cpn60) universal target showed that some isolates shared the highest sequence identity with T.?thermohydrosulfuricus; however, others to Thermoanaerobacter wiegelii and Thermoanaerobacter sp. Rt8.G4 (formerly Thermoanaerobacter brockii Rt8.G4). BOX-PCR fingerprinting profiles identified differences in the banding patterns not only between the isolates and the reference strains, but also among the isolates themselves. To evaluate the extent these genetic differences were manifested phenotypically, the utilization patterns of 30 carbon substrates were examined and the niche overlap indices (NOI) calculated. Despite showing a high NOI (>?0.9), significant differences existed in the substrate utilization capabilities of the isolates suggesting that either a high degree of niche specialization or mechanisms allowing for non-competitive co-existence, were present within this ecological context. Growth studies showed that the isolates were physiologically distinct in both growth rate and the fermentation product ratios. Our data indicate that phenotypic diversity exists within genetically microdiverse Thermoanaerobacter isolates from a common environment.     

Hydrogen oxidation abilities in the presence of thiosulfate as electron acceptor within the genusThermoanaerobacter

Thermoanaerobacter (T.) brockii, T. ethanolicus, andT. thermohydrosulfuricus were tested for their capacities to oxidize H₂ in the presence of thiosulfate.T. brockii oxidized H₂ actively, whileT. ethanolicus andT. thermohydrosulfuricus oxidized it poorly. At the end of the exponential growth, H₂ was oxidized byT. brockii in the presence of an energy source and thiosulfate. This oxidative process improved the growth ofT. brockii. Thermoanaerobacter species could be divided into two groups with regard to their H₂ metabolism in the presence of thiosulfate. Thiosulfate reduction by species of the genusThermoanaerobacter is of significance in mineralizing organic matter in thermophilic environments.     

Enzymatic properties of recombinant kojibiose phosphorylase from Caldicellulosiruptor saccharolyticus ATCC43494

One kojibiose phoshorylase (KP) homolog gene was cloned from Caldicellulosiruptor saccharolyticus ATCC43494. Recombinant KP from C. saccharolyticus (Cs-KP) expressed in Escherichia coli showed highest activity at pH 6.0 at 85 °C, and was stable from pH 3.5 to 10.0 and up to 85 °C for phosphorolysis. Cs-KP showed higher productivity of kojioligosaccharides of DP ≧ 4 than KP from Thermoanaerobacter brockii ATCC35047.     

Peptide and amino acid oxidation in the presence of thiosulfate by members of the genus Thermoanaerobacter

Thermoanaerobacter brockii, T. ethanolicus, T. thermohydrosulfuricus, T. finnii, and Thermoanaerobacter strain SEBR 5268 (an isolate from an oil-producing well) were studied for their ability to oxidize proteinaceous compounds that included gelatin, peptides, and casamino acids. All bacteria tested used peptides and amino acids, but only slightly. However, in the presence of thiosulfate all the Thermoanaerobacter species showed a substantial improvement in growth and/or the production of acetate, isovalerate, isobutyrate, and sulfide. Propionate was a minor product of peptide or amino acid oxidation. The reduction of thiosulfate during growth on peptides by members of the Thermoanaerobacter species is a trait that closely resembles that of archaeal hyperthermophiles during growth on peptides and amino acids with elemental sulfur as electron acceptor.     

Cloning, sequencing, and characterization of the bifunctional xylosidase-arabinosidase from the anaerobic thermophile thermoanaerobacter ethanolicus

The gene for the bifunctional xylosidase-arabinosidase (xarB) from the thermophilic anaerobe Thermoanaerobacter ethanolicus JW200 was cloned, sequenced, and expressed in Escherichia coli (Genebank Accession No. AF135015). Analysis of the recombinant enzyme revealed activity against multiple substrates with the highest affinity towards p-nitrophenyl beta-D-xylopyranoside (pNPX) and highest activity against p-nitrophenyl alpha-L-arabinopyranoside (pNPAP), respectively. Thus, we classify this enzyme as a bifunctional xylosidase-arabinosidase. Even though both sequences are 96% identical on the amino acid level, excluding the amino-terminal end, a frame-shift mutation in the 5' region of the gene in T. brockii ATCC 33075 and a deletion in a downstream open reading frame in T. ethanolicus seem to have occurred through evolutionary divergence of these two species. This represents an interesting phenomenon of molecular evolution of bacterial species, as PCR analysis of the region around the deletion indicates that the deletion is not present in T. brockii ssp. finnii and T. brockii ssp. brockii type strain HTD4.     

Characterization of Halanaerocella petrolearia gen. nov., sp. nov., a new anaerobic moderately halophilic fermentative bacterium isolated from a deep subsurface hypersaline oil reservoir

An anaerobic, halophilic, and fermentative bacterium, strain S200(T), was isolated from a core sample of a deep hypersaline oil reservoir. Cells were rod-shaped, non-motile, and stained Gram-positive. It grew at NaCl concentrations ranging from 6 to 26% (w/v), with optimal growth at 15% (w/v) NaCl, and at temperatures between 25 and 47°C with an optimum at 40-45°C. The optimum pH was 7.3 (range 6.2-8.8; no growth at pH 5.8 and pH 9). The doubling time in optimized growth conditions was 3.5 h. Strain S200(T) used exclusively carbohydrates as carbon and energy sources. The end products of glucose degradation were lactate, formate, ethanol, acetate, H(2), and CO(2). The predominant cellular fatty acids were non-branched fatty acids C(16:1), C(16:0), and C(14:0). The G + C mole% of the DNA was 32.7%. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that strain S200(T) formed a distinct lineage within the family Halobacteroidaceae, order Halanaerobiales, and was most closely related to Halanaerobaculum tunisiense DSM 19997(T) and Halobacteroides halobius DSM 5150(T), with sequence similarity of 92.3 and 91.9%, respectively. On the basis of its physiological and genotypic properties, strain S200(T) is proposed to be assigned to a novel species of a novel genus, for which the name Halanaerocella petrolearia is proposed. The type strain of Halanaerocella petrolearia is strain S200(T) (=DSM 22693(T) = JCM 16358(T)).     

利用冰核蛋白N末端结构域在大肠杆菌表面展示粘质沙雷氏菌脂肪酶

【目的】利用细胞表面工程技术将活性脂肪酶展示于大肠杆菌细胞表面并对展示脂肪酶的酶学性质进行研究。【方法】将丁香假单胞菌冰核蛋白N末端结构域序列与粘质沙雷氏菌脂肪酶编码基因融合,构建成脂肪酶表面展示载体,并转化大肠杆菌BL21(DE3)。【结果】重组菌以终浓度0.05 mmol/L异丙基硫代-D-半乳糖苷(IPTG)、25°C条件下诱导培养,16 h后表面展示脂肪酶活力达到最大值1 852 U/g细胞干重。表面展示酶的最适pH为9.0,最适反应温度为40°C,表面展示酶热稳定性较游离酶有较大提高,在40°C孵育1 h后仍能保持90%以上的酶活力。【结论】以上结果表明细菌表面展示技术为脂肪酶固定提供了一个很有前景的替代方法。     

Gene encoding a trehalose phosphorylase from Thermoanaerobacter brockii ATCC 35047

A gene encoding a trehalose phosphorylase was cloned from Thermoanaerobacter brockii ATCC 35047. The gene encodes a polypeptide of 774 amino acid residues. The deduced amino acid sequence was homologous to bacterial maltose phosphorylases and a trehalose 6-phosphate phosphorylase catalyzing anomer-inverting reactions. On the other hand, no homology was found between the T. brockii enzyme and an anomer-retaining trehalose phosphorylase from Grifola frondosa.