pta基因敲除对L-色氨酸发酵的影响

[目的]探究pta基因缺失对大肠杆菌发酵生产L-色氨酸的影响.[方法]运用Red重组技术敲除pta基因,构建pta缺失株E.coli TRTH△pta.利用30 L发酵罐进行分批补料发酵试验,考察重组菌E.coliTRTHApta发酵0生产L-色氨酸过程中生物量、L-色氨酸产量、有机酸含量、发酵液中NH4+浓度及变化....     

代谢副产物乙酸对L-色氨酸发酵的影响

分析了重组大肠杆菌(E.coli TRTH/pSV-709)发酵生产L-色氨酸的发酵过程,检测结果表明发酵液中有大量代谢副产物乙酸的积累。利用外源添加试验研究了乙酸对L-色氨酸发酵的影响,结果表明乙酸浓度高于2g/L时对L-色氨酸生产菌的生长和产酸均有抑制作用。分析了乙酸的产生机制,并采取了调节溶氧水平、确定合适初始葡萄糖浓度、限制葡萄糖流加及控制菌体比生长速率等措施来减少乙酸的生成。在优化条件下,乙酸含量与原工艺相比降低了51.35%,菌体生物量和L-色氨酸产量分别提高了51.07%和46.54%,实现了高密度发酵培养的目的。     

氮源NH4Cl浓度对粪产碱杆菌发酵生产热凝胶的影响

研究了利用粪产碱杆菌 (Alcaligenesfaecalis)发酵生产热凝胶的发酵条件 ,氮源是菌体生长的限制性底物 ,单纯地提高初始底物 (氮源 )浓度并不一定能促进细菌的生长和产物的合成。在分批发酵过程中 ,底物消耗导致培养环境pH的改变也是影响细菌进一步生长和产物合成的重要因素。通过增加培养基中初始氯化铵的浓度并同时控制发酵过程的pH条件 ,得到了较高的菌体浓度 ,热凝胶的合成水平也得到了显著提高。当培养基中NH₄Cl浓度提高到3.6g/L时 ,菌体浓度达到72g/L ,热凝胶合成的产量可达 30.5g L ,比原来NH₄Cl浓度为11g L时提高了51.7%。提高菌体浓度意味着需要提高溶氧水平来满足细菌的生长和代谢。初始氮源NH₄Cl浓度的增加虽然能使菌体浓度得到提高 ,但发酵过程对溶氧的需求也相应增加 ,需要提高搅拌转速和通风以增加供氧水平。但高搅拌速率产生的高剪切力对热凝胶的凝胶性能将产生破坏作用 ,因此在发酵过程中需要综合考虑细菌培养密度对合成热凝胶产量和质量的影响。     

外源H2O2对盐胁迫下小白菜种子萌发和幼苗生理特性的影响

以小白菜"甜脆青"为试材,研究不同浓度（5、10、25、50和100 mmol/L）过氧化氢（H₂O₂）浸种处理对100 mmol/L NaCl胁迫下小白菜（Brassica chinensis L.）种子萌发、幼苗生长及生理特性的影响。结果表明：100 mmol/L NaCl胁迫明显抑制小白菜种子的萌发状况和幼苗生长,发芽势、发芽指数、活力指数及幼苗根和芽长度和鲜重均明显降低,根和芽中CAT的活性及K⁺含量明显受到抑制,渗透调节物质、活性氧和MDA含量显著增加。不同浓度H₂O₂浸种处理提高了NaCl胁迫下小白菜种子发芽势、发芽指数和活力指数,促进小白菜根和芽的生长,增强了NaCl胁迫下根和芽中SOD、CAT和APX的活性及K⁺含量,降低O₂^-·产生速率及H₂O₂和MDA含量,进一步促进脯氨酸和可溶性糖含量的增加,降低体内Na⁺含量。其中以10 mmol/L H₂O₂处理缓解盐胁迫效果最好,明显缓解NaCl胁迫对小白菜种子萌发和幼苗生长的抑制。     

NH4+离子和Cl-离子影响谷氨酰胺发酵的研究

在微酸性条件下,当有高浓度铵盐存在时,谷氨酸发酵将转向谷氨酰胺发酵。本文阐述了用硫酸铵和氯化铵作氮源时的不同发酵结果,以及用氯化铵时NH₄⁺离子和Cl^-离子对发酵的影响。     

氮素形态对杉木幼苗侧根生长和叶片光合特性的影响

以3月龄的杉木实生苗为试验材料,分析了不同氮素形态——硝态氮(NO₃^- N)、铵态氮(NH4⁺ N)和硝酸铵(NH₄NO₃)(氮素浓度均为3 mmol·L^-1)对杉木幼苗侧根生长、叶片光合气体交换参数和叶绿素荧光参数的影响,以揭示杉木幼苗对不同形态氮的偏好性,以及不同形态氮肥下杉木幼苗侧根生长和光合生理的响应特征,为杉木苗期氮肥管理提供理论依据。结果显示：(1)不同氮素形态对杉木幼苗地上部和侧根生物量具有显著影响,其中NH₄⁺ N处理下幼苗地上部和侧根生物量最大,NO₃^- N处理次之,而NH₄NO₃处理最小。(2)NH₄⁺ N和NO₃^- N处理下杉木幼苗总根长、根系总表面积和根系总体积均显著高于NH₄NO₃处理(P＜0.05),且NH₄⁺ N处理又显著高于NO₃^- N处理,但不同氮形态处理间侧根数量差异不显著。(3)NH₄⁺ N处理下杉木幼苗叶片净光合速率、气孔导度和蒸腾速率明显高于NO₃^- N和NH₄NO₃处理,但NO₃^- N和NH₄NO₃处理之间无明显差异。(4)NH₄⁺ N处理下杉木叶片初始荧光强度低于NO₃^- N处理,而最大荧光强度、可变荧光强度和PSⅡ潜在活性却高于全硝氮和硝铵氮处理。上述结果表明,NH₄⁺ N处理不仅有利于杉木幼苗侧根生长发育,且其叶片具有较强的光合能力,较高的PSⅡ中心稳定性、光化学活性以及电子传递效率,从而更有利于植株生长。因此,从根系生长和光合特性来看,杉木幼苗对铵态氮具有偏好性。     

气提式内循环硝化反应器运行性能的研究

气提式内循环反应器具有很好的生物硝化性能,能承受高进水氨浓度（78.49mmol/L）,具有高容积转化效率（163.18 mmol/L·d）,运行性能稳定（氨去除率保持在94.42%以上）。在气提式内循环反应器的运行过程中,可产生硝化颗粒污泥。颗粒污泥开始出现的时间约为45d,颗粒污泥的粒径平均值0.83 mm,沉降速度55.53m/h,氨氧化活性0.95mmol (NH⁺₄-N)/g(VS)·d。硝化颗粒污泥也具有厌氧氨氧化活性,氨氧化速率0.23mmol (NH⁺₄-N)/g(VS)·d,亚硝酸还原速率0.24mmol (NO^-₂-N)/g(VS)·d。     

温度对大肠杆菌L-色氨酸发酵过程的影响

目的：研究变温控制对大肠杆菌TRTH L-色氨酸补料分批发酵过程中生物量、色氨酸产量、比生长速率及质粒稳定性的影响。方法：利用5L自控发酵罐对L-色氨酸补料分批发酵过程进行温度控制,对不同温度下相关参数进行分析比较,确定优化的温度控制方案。结果：以30-36％顺序升温的工艺进行发酵得到理想结果,与单一温度控制策略相比,L-色氨酸产量提高了15．4％;色氨酸的比合成速率提高了21．6％;质粒稳定性增加,未出现质粒丢失现象,质粒拷贝数保持在恒定水平。结论：温度对大肠杆菌L-色氨酸发酵有重要影响。     

不同浓度Fe2+与Zn2+对羊草幼苗生长和生理特性的影响

以羊草幼苗为研究对象,通过调整全营养培养基(CK,0.05 mmol/L Fe²⁺、0.015 mmol/L Zn²⁺)中铁或者锌含量设置0、10倍、20倍Fe²⁺(Zn²⁺)浓度处理Fe₀(Zn₀)、Fe₁₀(Zn₁₀)、Fe₂₀(Zn₂₀),以及在高铁培养基中单独添加0.15 mmol/L Zn²⁺或同时添加10 mmol/L Ca²⁺、5 mmol/L Mg²⁺、20 mmol/L K⁺处理,测定培养6 d后幼苗生长指标和矿质元素含量、以及高铁(Fe₂₀)处理下幼苗根中抗氧化指标和相关基因表达量,探究不同浓度Fe²⁺、Zn²⁺对羊草幼苗生长、矿质元素吸收积累及抗氧化指标、基因表达的影响。结果表明：(1)缺锌(Zn₀)显著抑制羊草幼苗鲜重的增加和Zn元素的积累,但促进Fe、Mg元素的积累;高浓度锌(Zn₁₀、Zn₂₀)显著促进幼苗叶片生长和Zn元素的积累;缺铁(Fe₀)显著抑制幼苗的根长、鲜重和Fe元素的积累,促进Mg、Zn元素的积累;高浓度铁(Fe₁₀、Fe₂₀)显著抑制羊草幼苗根叶生长、根毛发育和Ca、Zn、Mg、K元素的积累。(2)增加Zn²⁺和Ca²⁺、Mg²⁺、K⁺浓度无法恢复高铁胁迫对幼苗生长的抑制作用。(3)高浓度铁(Fe₂₀)处理羊草幼苗48 h后,根部过氧化物酶、超氧化物歧化酶、过氧化氢酶、抗坏血酸过氧化物酶、谷胱甘肽还原酶活性和丙二醛、抗坏血酸、还原型谷胱甘肽含量显著升高;烟酰胺合成酶基因、过氧化物酶基因表达量显著下调,植物类萌发素蛋白基因表达量显著上调。研究发现,羊草幼苗生长发育和矿质元素积累对环境中Zn²⁺浓度变化不敏感,却受到环境中高浓度Fe²⁺的显著抑制,并造成严重的氧化胁迫伤害,这种伤害无法在添加Zn²⁺或同时添加Ca²⁺、Mg²⁺、K⁺的条件下恢复。     

碳源和氮源对水母雪莲悬浮培养细胞生长和黄酮合成的影响

在MS培养基上进行水母雪莲(Saussurea medusa Maxim)细胞悬浮培养时研究了碳源和氮源的影响。结果表明碳源以蔗糖最适合,蔗糖浓度则以40g/L较好,细胞生长量干重为18.12g/L,总黄酮合成量达到1423.25mg/L。在培养过程中,水母雪莲细胞能够快速将蔗糖水解为葡萄糖和果糖,并首先利用葡萄糖。氮源总浓度(包括NH⁺₄和NO^-₃)为60～120mmol/L,NH⁺₄/NO^-₃比例为20/40有利于雪莲细胞生长和黄酮合成。用HPLC检测显示4′,5,7-三羟基3′,6-二甲氧基黄酮(Jaceosidin)和4′,5,7-三羟基-6-甲氧基黄酮(Hispidulin)2种黄酮的含量分别达到细胞干重的1.46%和0.010%     

丙氨酸转氨酶修饰对大肠杆菌L-色氨酸合成的影响

探究大肠杆菌细胞内负责L-丙氨酸合成的转氨酶对菌株代谢及L-色氨酸合成的影响。运用Red重组技术分别对编码L-丙氨酸转氨酶的基因alaA、alaC和avtA进行敲除。通过摇瓶和50 L罐中探究其对L-色氨酸积累、L-丙氨酸代谢及菌体生长变化情况。结果显示,除3种L-丙氨酸转氨酶全部缺失的工程菌L-丙氨酸合成受阻、菌体生长受到较强抑制外,其它各任意一种或两种丙氨酸转氨酶缺失菌株的生长并未有较大差异,但色氨酸的合成变化显著。其中alaA和alaC双基因缺失的E.coli FS-T4工程菌,摇瓶发酵L-色氨酸产量达6.08 g/L,L-丙氨酸含量仅0.16 g/L,较出发菌株分别提高了26.7%和降低了91.0%。在50 L罐中E.coli FS-T4工程菌L-色氨酸产量最高可达41.9 g/L,糖酸转化率达20.5%,分别较出发菌株提高了13.8%和5.1%。转氨酶AlaA和AlaC的同时缺失,既可以满足细胞整体氨基酸池的需要,而且有利于减少杂酸的积累,使得更多的碳源流向L-色氨酸的合成。     

Enzymatic production of L-tryptophan from DL-serine and indole by a coupled reaction of tryptophan synthase and amino acid racemase

Enzymatic production of L-tryptophan from DL-serine and indole by a coupled reaction of tryptophan synthase and amino acid racemase was studied. The tryptophan synthase (EC 4.2.1.20) of Escherichia coli catalyzed beta-substitution reaction of L-serine into L-tryptophan and the amino acid racemase (EC 5.1.1.10) of Pseudomonas putida catalyzed the racemization of D-serine simultaneously in one reactor. Under optimal conditions established for L-tryptophan production, a large-scale production of L-tryptophan was carried out in a 200-liter reactor using intact cells of E. coli and P. putida. After 24 h of incubation with intermittent indole feeding, 110 g liter-1 of L-tryptophan was formed in molar yields of 91 and 100% for added DL-serine and indole, respectively. Continuous production of L-tryptophan was also carried out using immobilized cells of E. coli and P. putida. The maximum concentration of L-tryptophan formed was 5.2 g liter-1 (99% molar yield for indole), and the concentration decreased to 4.2 g liter-1 after continuous operation for 20 days.     

L-Tryptophan prevents Escherichia coli biofilm formation and triggers biofilm degradation

The effect of deletion of trp operon and tna operon on the Escherichia coli biofilm formation was investigated in order to elucidate the role of L-tryptophan metabolism in biofilm formation. trp operon deletion mutants ΔtrpC, ΔtrpD and ΔtrpE deficient in L-tryptophan biosynthesis showed higher biofilm formation. In addition, ΔtnaC with increased L-tryptophan degradation activity showed higher biofilm formation. On the contrary, ΔtnaA deletion mutant which lost L-tryptophan degradation activity showed low biofilm formation. From these results, it was suggested that decrease of intracellular L-tryptophan level induced biofilm formation and increase of L-tryptophan repressed biofilm formation. So the effect of the addition of L-tryptophan to the medium on the E. coli biofilm formation was investigated. L-Tryptophan addition at starting culture decreased biofilm formation and furthermore L-tryptophan addition after 16 h culture induced the degradation of preformed biofilm. From the above results, it was suggested that maintenance of high intracellular L-tryptophan concentration prevents E. coli biofilm formation and elevation of intracellular L-tryptophan concentration triggers degradation of matured biofilm.     

重组大肠杆菌生产L-色氨酸发酵条件优化

为了提高重组大肠杆菌FB讲／pSV－04发酵生产L－色氨酸的产量,减少代谢副产物乙酸的生成,考察了比生长速率和无机盐对重组大肠杆菌发酵生产L．色氨酸的影响。在确定了合适的比生长速率和无机盐浓度之后,乙酸积累很少,L-色氨酸的产量为53．4g／L,比优化前提高了141．6％。经30L发酵罐初步放大,L－色氨酸的产量达53．6g／L,发酵结果稳定,具有工业化应用前景。     

ppc基因敲除对大肠杆菌L-色氨酸发酵的影响

目的：减少大肠杆菌L-色氧酸前体物质磷酸烯醇式丙酮酸向草酰乙酸的代谢流,提高其L-色氨酸的产量。方法：以大肠杆菌TRTH0709为出发菌株,利用Red重组敲除技术敲除磷酸烯醇式丙酮酸羧化酶（Ppc）编码基因PPc,并经测序和酶活性检测确证;对出发菌株和基因敲除菌株进行L-色氖酸发酵,对比分析发酵结果。结果：测序和酶活性检测结果表明ppc基因被成功敲除。发酵结果表明,与出发菌株相比,基因敲除菌株TRTH0709△ppc生长速度减慢,最终生物量减少32％,L-色氯酸产量降低27％,但糖酸转化率提高6％;向发酵培养基中添加1％琥珀酸后,TRTH0709△ppc的生长速率和产酸量有所提高,但仍与出发菌株有一定差距。结论：虽然ppc基因敲除对菌体生长和产酸量影响较大,但能有效提高其糖酸转化率;选育Ppc弱化的突变株以达到减弱代谢流且不影响菌体生长,以及增加,L-色氨酸积累的目的,将是本研究今后的主要方向。     

利用大肠杆菌细胞工厂生产吲哚-3-乙酸的研究

目的:利用重组大肠杆菌全细胞转化色氨酸生产IAA。方法:在大肠杆菌胞内构建两条全新的IAA合成途径,即吲哚-3-乙酰胺(indole-3-acetamide,IAM)途径和色胺(tryptamine,TRP)途径。结果:IAM途径涉及两个酶,分别是色氨酸-2-单加氧酶(IAAM)和酰胺酶(AMI1),构建好的重组大肠杆菌TPA-4以2g/L的色氨酸为底物,可以产生0.803g/L的IAA;敲除控制色氨酸合成副产物吲哚的tnaA基因后,菌株MPA-3的IAA产量达到1.43g/L,提高了78%。第二条TRP途径合成IAA涉及三个酶:左旋色氨酸脱羧酶(TDC),二胺氧化酶(AOC1)和吲哚-3-乙醛脱氢酶(IAD1)。包含这条途径的重组大肠杆菌TPTA-2以2g/L的色氨酸为底物能够合成13.0mg/L的IAA。在菌株MPTA-3中,最终产生了21.0mg/L的IAA,产量增加了61.5%。结论:首次通过IAM途径和TRP途径利用重组大肠杆菌全细胞催化生产IAA,其中IAM途径的IAA产量较高,有较高的工业化应用前景。     

Cloning of the Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase gene in Escherichia coli. Nucleotide sequence determination and properties of the plasmid-encoded enzyme

The Bacillus subtilis gene encoding glutamine phosphoribosylpyrophosphate amidotransferase (amidophosphoribosyltransferase) was cloned in pBR322. This gene is designated purF by analogy with the corresponding gene in Escherichia coli. B. subtilis purF was expressed in E. coli from a plasmid promoter. The plasmid-encoded enzyme was functional in vivo and complemented an E. coli purF mutant strain. The nucleotide sequence of a 1651-base pair B. subtilis DNA fragment was determined, thus localizing the 1428-base pair structural gene. A primary translation product of 476 amino acid residues was deduced from the DNA sequence. Comparison with the previously determined NH2-terminal amino acid sequence indicates that 11 residues are proteolytically removed from the NH2 terminus, leaving a protein chain of 465 residues having an NH2-terminal active site cysteine residue. Plasmid-encoded B. subtilis amidophosphoribosyltransferase was purified from E. coli cells and compared to the enzymes from B. subtilis and E. coli. The plasmid-encoded enzyme was similar in properties to amidophosphoribosyltransferase obtained from B. subtilis. Enzyme specific activity, immunological reactivity, in vitro lability to O2, Fe-S content, and NH2-terminal processing were virtually identical with amidophosphoribosyltransferase purified from B. subtilis. Thus E. coli correctly processed the NH2 terminus and assembled [4Fe-4S] centers in B. subtilis amidophosphoribosyltransferase although it does not perform these maturation steps on its own enzyme. Amino acid sequence comparison indicates that the B. subtilis and E. coli enzymes are homologous. Catalytic and regulatory domains were tentatively identified based on comparison with E. coli amidophosphoribosyltransferase and other phosphoribosyltransferase (Argos, P., Hanei, M., Wilson, J., and Kelley, W. (1983) J. Biol. Chem. 258, 6450-6457).     

发酵液中L-色氨酸分离纯化工艺研究

通过静态吸附实验,考察了温度、pH值对001×7阳离子交换树脂平衡吸附量的影响,并测定了吸附动力学曲线。通过动态实验,测定了动态吸附曲线和洗脱曲线。最后确定了001×7阳离子交换树脂分离纯化L-色氨酸的最佳工艺条件:用001×7阳离子交换树脂吸附L-色氨酸,以浓度为2 mol.L-1氨水进行洗脱,收集的流份经D315阴离子交换树脂脱色,浓缩结晶后得L-色氨酸成品,总提取率为73.0%。     

Intracellular Trp repressor levels in Escherichia coli.

A radioimmunoassay for the Trp repressor protein of Escherichia coli was developed with antisera raised against purified Trp repressor protein. This assay was used to directly measure the intracellular Trp repressor content in several E. coli K-12 and B/r strains. Repressor levels varied from 2.5- to 3-fold in response to L-tryptophan concentration in the growth medium (15 to 44 ng of repressor per mg of protein). Neither cell growth rate nor culture age had a significant effect on repressor concentrations within the cell. Addition of L-tryptophan to the growth medium resulted in lowered intracellular levels of Trp repressor. The absolute amounts of native Trp repressor molecules per cell varied between 120 and 375 dimers in the presence and absence of L-tryptophan in the culture medium, respectively. Assuming an intracellular volume of 7.3 microliters/10(10) E. coli cells, the Trp repressor concentration varied from 270 to 850 nM in response to extracellular tryptophan levels. These findings represent the first direct measurements of Trp repressor levels in E. coli and confirm the autoregulatory nature of the trpR gene.     

The tryptophan synthase bienzyme complex transfers indole between the alpha- and beta-sites via a 25-30 A long tunnel

The bacterial tryptophan synthase bienzyme complexes (with subunit composition alpha 2 beta 2) catalyze the last two steps in the biosynthesis of L-tryptophan. For L-tryptophan synthesis, indole, the common metabolite, must be transferred by some mechanism from the alpha-catalytic site to the beta-catalytic site. The X-ray structure of the Salmonella typhimurium tryptophan synthase shows the catalytic sites of each alpha-beta subunit pair are connected by a 25-30 A long tunnel [Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., & Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871]. Since the S. typhimurium and Escherichia coli enzymes have nearly identical sequences, the E. coli enzyme must have a similar tunnel. Herein, rapid kinetic studies in combination with chemical probes that signal the bond formation step between indole (or nucleophilic indole analogues) and the alpha-aminoacrylate Schiff base intermediate, E(A-A), bound to the beta-site are used to investigate tunnel function in the E. coli enzyme. If the tunnel is the physical conduit for the transfer of indole from the alpha-site to the beta-site, then ligands that block the tunnel should also inhibit the rate at which indole and indole analogues from external solution react with E(A-A). We have found that when D,L-alpha-glycerol 3-phosphate (GP) is bound to the alpha-site, the rate of reaction of indole and nucleophilic indole analogues with E(A-A) is strongly inhibited. These compounds appear to gain access to the beta-site via the alpha-site and the tunnel, and this access is blocked by the binding of GP to the alpha-site. However, when small nucleophiles such as hydroxylamine, hydrazine, or N-methylhydroxylamine are substituted for indole, the rate of quinonoid formation is only slightly affected by the binding of GP. Furthermore, the reactions of L-serine and L-tryptophan with alpha 2 beta 2 show only small rate effects due to the binding of GP. From these experiments, we draw the following conclusions: (1) L-Serine and L-tryptophan gain access to the beta-site of alpha 2 beta 2 directly from solution. (2) The small effects of GP on the rates of the L-serine and L-tryptophan reactions are due to GP-mediated allosteric interactions between the alpha- and beta-sites.(ABSTRACT TRUNCATED AT 400 WORDS)