L-缬氨酸的发酵工艺研究

目的:以黄色短杆菌XV0505为生产菌株,探讨发酵培养基和发酵控制条件对L-缬氨酸的产量和糖酸转化率的影响。方法:应用单因素实验确定发酵的工艺条件;利用纸层析-色斑洗脱比色法测定发酵液中L-缬氨酸的含量。结果:在最优发酵条件下,通过10L罐流加发酵72h,产酸量可达53.4g/L,糖酸转化率为26.7%,分别比补料分批发酵提高11.9%和3.5%。结论:环境因子和发酵控制工艺对发酵生产L-缬氨酸具有重要影响。     

低糖流加法生产L-缬氨酸发酵工艺条件的研究

研究了糖浓度对L -缬氨酸产生菌 (Brevibacteriumflavum)Apv- 2菌积累L -缬氨酸的影响 ,通过三十吨发酵罐发酵工艺条件的试验 ,在合适的外界条件下 ,确定了该菌种发酵L -缬氨酸的最佳初糖浓度和补糖浓度 ,在初糖质量浓度为 3.5 %～ 4 .5 %时 ,发酵至 16h开始连续流加葡萄糖 ,维持发酵培养基中残糖质量浓度为 1.2 %～ 1.5 % ,经过 4 8h左右发酵 ,L -缬氨酸发酵产酸率 3.3%～ 3.5 %。     

溶氧对L-缬氨酸发酵过程的影响

目的:以黄色短杆菌XV0505为供试菌,探索溶氧对L-缬氨酸发酵过程的影响及其控制策略。方法:利用5L发酵罐,考察了不同溶氧浓度对L-缬氨酸发酵的影响,并采用代谢流分析对其结果进行阐述,提出分阶段溶氧控制策略。结果:采用分阶段溶氧控制策略,即在0～24h溶氧浓度为20%,24～60h溶氧浓度为5%,发酵60h,L-缬氨酸可达到58.36g/L,比5%和20%溶氧浓度下分别提高了18.95%和13.56%。结论:溶氧浓度对L-缬氨酸发酵有重要影响。     

甘油转化生产1,3-丙二醇发酵液中甘油含量的测定

对文献介绍滴定法测定甘油的方法进行了改进,使之能够用于1,3－丙二醇发酵液中甘油含量测定。实验表明,化学滴定法测定结果具有较好的准确性和重复性,与酶法和变色酸比色法相比,测定结果接近,化学滴定法测定发酵液中甘油含量是一个较为经济简便的方法。     

L-缬氨酸合成的代谢流量分析

分别测定谷氨酸棒杆菌（Corynebacterium glutamicum）AS1-495及其3个逐个叠加不同遗传标记的突变株AA361、AAT231和AATV341在特定培养时段（26～28h）L缬氨酸等代谢物的胞外浓度,由此计算这一时段这些代谢物在发酵液中积累（或消耗）的速率,分别做出这4株菌在拟稳态下的代谢流量分布图,进而研究育种过程中不同遗传标记的叠加对代谢网络中L-缬氨酸合成流量分布的影响。结果表明遗传标记的引入使流量分配发生了重大变化,节点处的流量分配朝着有利于L缬氨酸合成的方向改变。6-磷酸葡萄糖节点处流入EMP途径和HMP途径的流量分配由17.0∶83.0变为24.3∶75.7;丙酮酸节点处流入L-缬氨酸合成途径和其他途径的流量分配由15.8∶842变为76.7∶23.3/L-缬氨酸合成的分支途径上的流量由最初的5.37增大为37.3,乳酸合成途径的流量从11.1最后降为1.16,L-缬氨酸产量由4g/L提高到24.5 g/L。代谢流量分布的变化趋势与L缬氨酸产量的变化趋势是互相吻合的。以2-噻唑丙氨酸抗性突变（2TAr）和L天冬氨酸氧肟酸盐超敏性突变（LAAHss）有效地进行代谢流遗传导向的事实,在代谢流量分析的层面上,证明结构类似物抗性突变和结构类似物超敏性突变是代谢流导向和设计育种的十分有效的手段,代谢流量分析会成为设计育种的校正方法。     

L-缬氨酸生产菌的选育及其发酵培养基的模式识别优化

以黄色短杆菌TV10为出发菌株 ,经紫外线、硫酸二乙酯逐级诱变处理 ,在含磺胺胍 (SG)、α 氨基丁酸 (α AB)、2 噻唑丙氨酸 ( 2 TA)等氨基酸结构类似物平板上定向筛选 ,获得L 缬氨酸高产菌株TV2 30。应用模式识别方法 ,对L 缬氨酸发酵培养基进行优化。以培养基组成构筑模式空间 ,通过主成分分析 (PCA)揭示模式空间的可视优化区域 ,选择优化点并逆推回到高维空间得到最优培养基组成 ,结果该菌株可积累L 缬氨酸 2 6 .38g·L-1,比初始值提高 7.8%。     

L-缬氨酸发酵生产的育种思路及发酵条件优化策略

L-缬氨酸在医药及饲料领域中有着广泛的用途,根据L缬氨酸的生物合成途径及其代谢调节机制,利用代谢调控理论,重点阐述了L缬氨酸生产菌的育种思路及培养条件的优化,为缬氨酸发酵生产提供理论指导。     

β-发卡抗菌肽的全新设计及其生物学活性

通过缬氨酸和精氨酸的交替连接形成β-发卡结构的两条侧链,D-脯氨酸和甘氨酸形成β-转角单元以及侧链末端的两个半胱氨酸连接形成一个二硫键,来设计得到全新的由16残基构成的β-发卡抗菌肽VR。对设计得到的抗菌肽VR的生物学活性进行了检测,主要测定了新型β-发卡抗菌肽VR的最小杀菌浓度、对红细胞的溶血活性、杀菌动力学和盐敏感性。结果发现,VR和蜂毒素具有相似的杀菌活性,而溶血活性远低于蜂毒素,这表明VR比蜂毒素具有更高的细胞选择性。在NaCl的浓度低于100 mmol/L时,VR的杀菌活性没有受到影响;在NaCl的浓度为100 mmol/L时,VR具有50%的杀菌活性。综上可见,VR具有较优异的生物学活性,拥有成为抗生素替代物的发展潜力。     

L-缬氨酸产生菌的选育及其代谢流分析

以谷氨酸棒杆菌(Corynebacterium glutamicum)AS1.495(Leu^-)为出发菌株,通过多次亚硝基胍(NTG)诱变,给AS1．495(Leu^-)依次叠加L-AAH^as,2-TA^r,Vd^-的遗传标记,得到突变株AATV341(Leu^-,L-AAH^as,2-TA^r,Vd^-),可在8％的葡萄糖培养基积累L-缬氨酸24．5g／L,比出发菌株提高了5．13倍。同时运用代谢流量分析理论,测定出发菌株AS1．495及其突变株AATV341在L-缬氨酸合成阶段的代谢流量,并初步进行比较和分析,发现遗传标记的引入使流量分配发生了重大变化,流量分配朝着有利于L-缬氨酸合成的方向改变。     

粘虫谷氨酰胺转胺酶(MsTGase)活力测定条件的正交优化及其在幼虫体内的分布

【目的】本研究旨在优化Grossowicz氧肟酸比色法测定粘虫Mythimna separata谷氨酰胺转胺酶(Ms TGase)活力的组合条件,以Ms TGase酶活力为依据分析其在不同龄期幼虫体内的分布规律。【方法】取4龄粘虫幼虫,通过组织匀浆和沉析纯化制备Ms TGase,采用Grossowicz比色法测定Ms TGase酶活力,并对Grossowicz比色法的多重实验因素进行正交优化,进一步结合差速离心法分析不同龄期幼虫体内和亚细胞组分(细胞核和细胞碎片,线粒体,微粒体以及胞质溶胶)中Ms TGase酶活力。【结果】结果表明,酶浓度、底物浓度、反应体系pH值、反应温度及钙离子浓度等实验因素都对Ms TGase酶活力测定结果产生显著影响,其影响大小顺序为:酶浓度>温度> p H>底物浓度>Ca2+浓度。Ms TGase酶比活力测定的最优化条件:酶浓度20 mg/m L、底物浓度0. 04 mol/L、反应体系pH值6. 5、测定温度37℃,不添加钙离子。在1-5龄幼虫中以4龄幼虫的Ms TGase酶活力最高,其比活力也显著高于其他龄期的,且在1-5龄幼虫胞质溶胶中Ms TGase酶活力分别占各亚细胞组分酶活力总和的39%,25%,48%,60%和61%。【结论】所获得的最优化条件适用于粘虫Ms TGase酶活力测定。Ms TGase在粘虫体内呈显著的龄期表达特征和亚细胞分布规律。     

L-缬氨酸高产菌株的选育研究

以产L-缬氨酸的谷氨酸棒状杆菌(Corynebacterium glutamicum)为原始菌株,利用注入低能氮离子束进行一系列诱变,获得一株稳定的高产L-缬氨酸突变菌株。摇瓶培养96h后发酵能力可达38.0g·L-1,较出发菌株提高18.01%。通过对摇瓶中葡萄糖、玉米浆浓度及培养条件进行优化,发酵能力达到40.6g·L-1,50L发酵罐的发酵能力可达70g·L-1左右。     

Amino Acid Activation in Subcellular Fractions of Tetrahymena pyriformis*

SYNOPSIS. The presence of amino acid activating enzymes was demonstrated in the ciliated protozoan Tetrahymena pyriformis. By employing a sensitive hydroxamate assay procedure, the activation of L-valine was assayed in various subcellular fractions of the ciliate, and some characteristics of the enzyme activity in the most active fraction were determined. Most of the activity resided in pH 5 fractions isolated from high speed supernatants of ciliates disrupted by various physical and chemical methods. No activity could be demonstrated in isolated cilia, in pellicles with attached kinetosomes, in microsomes or in macronuclei, providing these organelles were thoroughly washed. A washed mitochondrial preparation isolated by the Mager and Lipmann procedure activated L-valine; mitochondria isolated by the procedure of Hogg and Kornberg did not. The pH 5 fraction isolated from the 102,000 X g supernatant of digitonin-lysed ciliates was stable for several weeks when stored in 0.1 M Tris buffer, pH 7.6 at – 25 C. The activity of this fraction with respect to L-valine activation was dependent on the presence of ATP¹ and magnesium in the reaction mixture. The optimal concentrations of these components and of L-valine and hydroxylamine were determined, and the linearity of activity with time and enzyme concentration was demonstrated. Valine activation was not modified by dialysis of the pH 5 fraction, or treatment with RNase, or the addition of boiled pH 5 fraction.     

L-valine transport by the actinomycete Actinomyces species 26-115, the producer of actinomycin C]

Transport of L-valine by Actinomyces species 26-115, an organism producing actinomycin C depended on L-valine concentration in the medium and temperature and required a source of intrinsic energy. Km for L-valine transport was 3.5.10(-6)--6.0.10(-6) M. It somewhat differed from experiment to experiment. The above system transported also other neutral amino acids. L-isoleucine was a competing inhibitor of L-valine transport. The transport of L-valine was stereospecific. The activity of the transport system was regulated by the intracellular content of L-valine. Probably because of this the amino acid transport depended on the culture age, so far as the level of free valine in the mycelium at various stages of development was different.     

NMR analyses of the conformations of L-isoleucine and L-valine bound to Escherichia coli isoleucyl-tRNA synthetase

The 400-MHz 1H NMR spectra of L-isoleucine and L-valine were measured in the presence of Escherichia coli isoleucyl-tRNA synthetase (IleRS). Because of chemical exchange of L-isoleucine or L-valine between the free state and the IleRS-bound state, a transferred nuclear Overhauser effect (TRNOE) was observed among proton resonances of L-isoleucine or L-valine. However, in the presence of isoleucyl adenylate tightly bound to the amino acid activation site of IleRS, no TRNOE for L-isoleucine or L-valine was observed. This indicates that the observed TRNOE is due to the interaction of L-isoleucine or L-valine with the amino acid activation site of IleRS. The conformations of these amino acids in the amino acid activation site of IleRS were determined by the analyses of time dependences of TRNOEs and TRNOE action spectra. The IleRS-bound L-isoleucine takes the gauche+ form about the C alpha-C beta bond and the trans form about the C beta-C gamma 1 bond. The IleRS-bound L-valine takes the gauche- form about the C alpha-C beta bond. Thus, the conformation of IleRS-bound L-valine is the same as that of IleRS-bound L-isoleucine except for the delta-methyl group. The side chain of L-isoleucine or L-valine lies in an aliphatic hydrophobic pocket of the active site of IleRS. Such hydrophobic interaction with IleRS is more significant for L-isoleucine than for L-valine. The TRNOE analysis is useful for studying the amino acid discrimination mechanism of aminoacyl-tRNA synthetases.     

L-valine production with pyruvate dehydrogenase complex-deficient Corynebacterium glutamicum

Corynebacterium glutamicum was engineered for the production of L-valine from glucose by deletion of the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex and additional overexpression of the ilvBNCE genes encoding the L-valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and transaminase B. In the absence of cellular growth, C. glutamicum DeltaaceE showed a relatively high intracellular concentration of pyruvate (25.9 mM) and produced significant amounts of pyruvate, L-alanine, and L-valine from glucose as the sole carbon source. Lactate or acetate was not formed. Plasmid-bound overexpression of ilvBNCE in C. glutamicum DeltaaceE resulted in an approximately 10-fold-lower intracellular pyruvate concentration (2.3 mM) and a shift of the extracellular product pattern from pyruvate and L-alanine towards L-valine. In fed-batch fermentations at high cell densities and an excess of glucose, C. glutamicum DeltaaceE(pJC4ilvBNCE) produced up to 210 mM L-valine with a volumetric productivity of 10.0 mM h(-1) (1.17 g l(-1) h(-1)) and a maximum yield of about 0.6 mol per mol (0.4 g per g) of glucose.     

Linking central metabolism with increased pathway flux: L-valine accumulation by Corynebacterium glutamicum

Mutants of Corynebacterium glutamicum were made and enzymatically characterized to clone ilvD and ilvE, which encode dihydroxy acid dehydratase and transaminase B, respectively. These genes of the branched-chain amino acid synthesis were overexpressed together with ilvBN (which encodes acetohydroxy acid synthase) and ilvC (which encodes isomeroreductase) in the wild type, which does not excrete L-valine, to result in an accumulation of this amino acid to a concentration of 42 mM. Since L-valine originates from two pyruvate molecules, this illustrates the comparatively easy accessibility of the central metabolite pyruvate. The same genes, ilvBNCD, overexpressed in an ilvA deletion mutant which is unable to synthesize L-isoleucine increased the concentration of this amino acid to 58 mM. A further dramatic increase was obtained when panBC was deleted, making the resulting mutant auxotrophic for D-pantothenate. When the resulting strain, C. glutamicum 13032DeltailvADeltapanBC with ilvBNCD overexpressed, was grown under limiting conditions it accumulated 91 mM L-valine. This is attributed to a reduced coenzyme A availability and therefore reduced flux of pyruvate via pyruvate dehydrogenase enabling its increased drain-off via the L-valine biosynthesis pathway.     

微量法与试管比色法检测ALT的比较

经实验建立了一种用于检测血浆中丙氨酸氨基转移酶(ALT)含量的微量测定法,并与比色法（传统赖氏法)进行了比较。用两种方法检测定值血清、室内质控及样品并比较标准曲线后,结果无显著性差异,同时微量法重复性较好,结果表明微量法测定ALT酶活力可以替代比色法测定血浆中ALT含量,适合大批量血浆ALT含量的快速检测。     

Order of binding of substrate to valyl-tRNA synthetase from Bacillus stearothermophilus in amino acid activation reaction

Amino acid activation reaction with valyl-tRNA synthetase (EC 6.1.1.9) from Bacillus stearothermophilus was studied kinetically by measuring ATP-PPi exchange to find the order of the binding of substrate to the enzyme. The effects of the concentration of the substrates (L-valine and ATP) and two dead-end inhibitors (L-valinol and adenosine) on the reaction rate were analyzed. The results indicate that L-valine and ATP are bound to the enzyme in a random sequence. This conclusion is consistent with the one previously suggested by static binding experiments.