营养及环境因素对黄色短杆菌发酵生产L-亮氨酸的影响

目的:研究葡萄糖、玉米浆、蛋氨酸等主要营养物质以及环境因素对发酵生产L-亮氨酸的影响。方法:应用单因素实验确定发酵条件,采用高效液相色谱法测定产量。结果:在最优发酵条件下,以种龄36h、接种量为10%,摇瓶产L-亮氨酸的量可达19.4g/L,采用10L罐分批补料发酵64h可积累29.47g/L的亮氨酸。结论:营养及环境因素对发酵生产L-亮氨酸具有重要影响。     

丙酮酸和乙酰-CoA协同促进L-亮氨酸的合成

【目的】通过理性改造柠檬酸合酶(citrate synthase,CS)、丙酮酸脱氢酶系E1p (pyruvate dehydrogenase complex,PDHC,编码基因aceE)和ATP-柠檬酸裂解酶(ATP-Citrate lyase,ACL),有效供应胞内丙酮酸和乙酰-CoA,以提高L-亮氨酸产量。【方法】以谷氨酸棒杆菌(Corynebacterium glutamicum)为底盘细胞,分析不同CS和PDHC酶活水平对L-亮氨酸合成的影响。随后,考查协同改造CS和PDHC或引入绿硫菌(Chlorobium tepidum)中ACL对L-亮氨酸合成的影响。【结果】低强度的CS酶活(即重组菌XL-3 P_(dapA-R2)gltA)有利于L-亮氨酸的合成,L-亮氨酸产量达到17.5±0.6 g/L。而改变PDHC酶活水平不利于L-亮氨酸的合成。此外,以启动子P_(dapA-R2)控制CS表达,而以启动子P_(gapA)控制PDHC表达时(即重组菌XL-4),可实现胞内丙酮酸和乙酰-CoA的有效供给,L-亮氨酸产量达到20.2±1.7 g/L,且显著降低副产物产量。若在重组菌XL-4中引入C.tepidum,ACL会显著抑制菌体生长而不利于L-亮氨酸合成,而引入到出发菌XL-3中因胞内丙酮酸和乙酰-CoA得到有效供给,目标重组菌XL-5L-亮氨酸产量达到18.5±1.2 g/L,比出发菌株XL-3增加了14.2%。【结论】重组菌XL-4中因协同控制CS和PDHC酶活,从而实现胞内丙酮酸和乙酰-CoA有效供给,促进L-亮氨酸的合成。该研究结果对后续利用代谢工程技术强化微生物合成L-亮氨酸等支链氨基酸具有重要的参考价值。     

亮氨酸和异亮氨酸研究题录选

1　以小什鱼为原料提取亮氨酸的试验 2　猪毛水解物中亮氨酸的离子交换柱分离 3　从猪血水解液中提取亮氨酸 4　以玉米麸质为原料提取L-亮氨酸 5　通过谷氨酸棒状杆菌由α酮异已酸生产L-亮氨酸的微生物生产法 6　L-异亮氨酸生产新技术 7　产L-异亮氨酸新菌株选育及其发酵产物提纯和鉴定 8　生产L-异亮氨酸的新方法 9　异亮氨酸产生菌推理选育思想 10 活细胞反应生产L-异亮氨酸工艺中附产物形成的抑制 11 异亮氨酸生产的新方法 12 L-异亮氨酸产生菌选育的研究 13 L-异亮氨酸质量标准 14 对四种异亮氨质量标准的评价 15 异亮氨酸细菌内毒素和热原检查的探讨 16 从面筋水解物中提取L—亮氨酸 17 从人发中连续提取亮氨酸和胱氨酸工艺初探 　　需要上述资料的读者,请与本刊编辑部联系,编辑部提供复印服务,复印费、邮寄费等共80 元。 　　联系电话：027-87664846 　　邮编：430072 　　地址：武汉市武汉大学《氨基酸和生物资源》编辑部     

不同碳源生物转化合成L-亮氨酸的代谢计量分析

目的:建立黄色短杆菌利用不同碳源生物合成L-亮氨酸的代谢网络模型,并进行代谢网络计量分析.方法:通过对所构建的L-亮氨酸代谢网络模型进行途径分析,确定以果糖、葡萄糖、蔗糖或木糖为碳源时L-亮氨酸生物合成的基元模型、最大理论产率和不同模型的呼吸熵.结果:通过途径分析得到了L-亮氨酸生物合成的基元模型.以果糖、葡萄糖、蔗糖和木糖为碳源时L-亮氨酸的最大理论产率均为66.7%,其对应的最大呼吸熵分别为18、16、19、18.结论:L-亮氨酸理论得率与碳源种类无关;呼吸熵增加,能够有效提高L-亮氨酸合成代谢流,限制菌体量的过量生成.与其他碳源相比,蔗糖能够避免碳架溢流出现,合成L-亮氨酸能量代谢需求低;而葡萄糖能够较好地满足菌体生长和产酸的需求.     

通过谷氨酸棒状杆菌由α-酮异已酸生产L-亮氨酸的微生物生产法

本文研究用谷氨酸棒状杆菌(Co-rlnebacterium glutamicum)将α-酮异巳酸转化为L-亮氨酸的生产过程。在α-酮异已酸的浓度为20克/升的培养基中,经57小时的发酵大约有16克/升的L-亮氨酸被合成,其克分子产量为91%。若采用流加补料分批培养法时,在23小时内就可能从32克/升的α-酮异巳酸中产生出24克升的L-亮氨酸。有关的酶学研究表明,在这种谷氨酸生产菌中a酮异巳酸是经过转氨酶B的作用被转化为亮氨酸的。转氨作用所需的L谷氨酸则通过谷氨酸脱氨酶的作用再生。α-酮异巳酸加入培养基中后,转氨酶B的比活性提高三倍。     

L-亮氨酸的应用及其生产菌的育种思路

氨基酸是构成蛋白质的基本单位,是生物体内不可缺少的营养成份,L-亮氨酸是人体8种必需氨基酸之一,也是3种支链氨基酸之一,在医药、食品、饲料、化妆品等行业具有重要的用途,本文综述了L-亮氨酸的性质、生产方法及发酵法生产的育种思路。     

溶氧对L-苏氨酸发酵的影响

探索溶氧对L-苏氨酸发酵过程的影响及其控制方法。通过摇瓶装液量试验、不同溶氧控制方式考察发酵过程中溶氧对L-苏氨酸合成的影响。采用补料分批发酵工艺发酵L-苏氨酸,利用氨基酸分析仪测定发酵液中L-苏氨酸的产量,通过10L罐补料分批发酵36h,产酸可达118.9g/L,糖酸转化率为47.6%。可以得出溶氧对L-苏氨酸生物合成有重要影响,并建立了最佳溶氧控制条件。     

黄色短杆菌TK0303的L-亮氨酸生物合成途径分析

应用途径分析方法分析了在拟稳态时黄色短杆菌(Brevibacterium flavum)TK0303由葡萄糖发酵生产L-亮氨酸的代谢途径,确定了L-亮氨酸合成的最佳途径和最大理论产率。通过比较途径分析所获得的反应模型,确定了丙酮酸和乙酰辅酶A是L-亮氨酸合成途径的关键节点。在此基础上改变外界环境因子,强化L-亮氨酸生物合成途径中丙酮酸和乙酰辅酶A两个关键节点的代谢流,以期进一步提高L-亮氨酸产率。结果表明,经过谷氨酸以及醋酸铵的调节,代谢途径流量发生显著变化,L-亮氨酸产量有明显提高。     

支链氨基酸的代谢

<正> 支链氨基酸包括缬氨酸、亮氨酸和异亮氨酸。因为在它们的碳链中都有分支,故总称这三种氨基酸为支链氨基酸或分支氨基酸。它们都属于体内不能合成而必需从食物中获得的必需氮基酸。在支链氨基酸中最早发现的氨基酸是亮氨酸。但一直到30年代后     

活性炭-离子交换树脂联合柱层析分离猪血粉水解液中L-苯丙氨酸等多种氨基酸的新工艺

<正> 本文报道了活性炭——离子交换树脂联合柱层析分离猪血粉水解液中L-苯丙氨酸等多种氨基酸的新工艺。本工艺将猪血粉水解液通过活性炭柱吸附,依次用IN氨水,适当浓度乙醇解吸得到L-苯丙氨酸,L-酪氨酸;活性炭柱流出液再通过离子交换柱交换吸附,依次用0.05、0.1、2N氨水解吸得到L-组氨酸、L-亮氨酸、L-精氨酸、L-赖氨酸。上述六种氨基酸对猪血粉的百分收率分别为2.5、0.8、5.4、4.1、3.4、2.9。     

Amino acid and dipeptide absorption in rats fed chemically defined diets of differing fiber composition

In rats chronically fed a fiber-free diet or one of the three nutritionally equivalent fiber-containing diets, in vivo jejunal absorption of L-leucine from a free amino acid mixture of L-leucine and glycine as well as equimolar solutions of the dipeptides, L-leucyl-glycine and glycyl-L-leucine, were compared. In addition, total and brush border amino peptidase activities for the two dipeptide substrates were examined in small bowel segments. With two of three fiber diet groups, absorption of L-leucine from the free amino acid solution was reduced without a detectable change in peptide transport or aminopeptidase activities. This investigation provides evidence that peptide transport mechanisms are relatively spared with long-term feeding of fiber-containing diets similar to observations recorded in disease states associated with protein-energy malnutrition.     

Interactions between the branched-chain amino acids in the growth of Spirodela polyrhiza

The joint action of L-valine and L-isoleucine, L-leucine and L-isoleucine, and L-valine and L-leucine on the growth of Spirodela polyrhiza was established. The effect of one branched-chain amino acid on growth inhibition by another one was compared with the non-specific antagonisms which glycine and L-alanine exert on growth inhibition by singly supplied branched-chain amino acids. In this way specific and non-specific interactions could be distinguished. It appeared that: (1) L-isoleucine was a specific antagonist of L-valine; (2) L-leucine was a specific antagonist of L-isoleucine; (3) L-valine and L-leucine were synergistic growth inhibitors. Further, it was found that: (4) growth inhibition by L-leucine was specifically antagonized by simultaneously supplied L-valine and L-isoleucine; (5) an excess of L-isoleucine strongly inhibited the conversion of exogenous valine into leucine; (6) accumulation of valine was typical of isoleucine-induced growth inhibition. The results are consistent with the view that growth inhibition by L-valine and L-leucine is due to the blocking of acetohydroxy acid synthetase, the first common enzyme in the valine-isoleucine biosynthetic pathway. Growth inhibition by L-isoleucine, however, seems to result from inhibition of leucine synthesis at a step after 2-oxoisovaleric acid. Some aspects of the regulation of branched-chain amino acid biosynthesis in higher plants are discussed.     

Does leucine- and norleucine-induced insulin release depend on amino acid aminotransferase activity?

In the absence of another exogenous nutrient, L-leucine but not L-norleucine stimulates insulin release from rat pancreatic islets, although the corresponding keto acids, 2-ketoisocaproate and 2-ketocaproate, are equally potent secretagogues. Such a situation cannot be ascribed to the preferential transamination of L-leucine as compared to L-norleucine in islet homogenates. Indeed, in the presence of a suitable activator of glutamate dehydrogenase, L-leucine and L-norleucine stimulate secretion to the same extent. It is concluded that the rate of transamination of these amino acids in intact islet cells depends on the availability of a 2-keto acid partner rather than on the assayed amino acid aminotransferase activity.     

Candida L-norleucine,leucine:2-oxoglutarate aminotransferase. Purification and properties

A new enzyme which catalyzes the transamination of L-norleucine (2-aminohexanoic acid) and L-leucine with 2-oxoglutarate was purified to homogeneity from cells of Candida guilliermondii var. membranaefaciens. The relative molecular mass determined by gel filtration was estimated to be close to 100,000. The transaminase behaved as a dimer which consists of two subunits identical in molecular mass (Mr 51,000). The enzyme has a maximum activity in the pH range of 8.0-8.5 and at 55 degrees C. 2-Oxoglutarate, and to a lesser extent pyridoxal 5'-phosphate, were effective protecting agents against increasing temperature. The enzyme exhibits absorption maximum at 330 nm and 410 nm. L-Norleucine, and L-leucine to a lesser extent, are the best amino donors with 2-oxoglutarate as amino acceptor. The Km values for L-norleucine, L-leucine and 2-oxoglutarate determined from the Lineweaver-Burk plot were 1.8 mM, 6.6 mM and 2.0 mM respectively. A ping-pong bi-bi mechanism of inhibition with alternative substrates is found when the enzyme is in the presence of both L-norleucine and L-leucine. The inhibitory effect of various amino acid analogs on the transamination reaction between L-norleucine and 2-oxoglutarate was studied and Ki values were determined.     

Uptake of L-proline by Histoplasma capsulatum.

The uptake and incorporation of L-proline by yeast cells of the dimorphic zoopathogen Histoplasma capsulatum were studied. The amino acid was assimilated in at least two ways: by an active transport system with a Km of 1.7 X 10(-5) M and by simple diffusion. The active transport system was sterospecific and severely restricted to neutral aliphatic side-chain amino acids. Certain analogues inhibited L-proline uptake and prevented incorporation of the amino acid into cellular constituents. The inhibition of L-proline uptake by L-leucine was competitive. Since L-leucine and L-proline are seemingly transported by a system with similar characteristics, must be concluded, as originally postulated, that the buckled ring of L-proline, in solution, acts as an aliphatic side chain and that this cyclic amino acid is transported by a system more or less specific for amino acids with neutral aliphatic side chains.     

Amino acid transport by membrane vesicles of virally transformed and nontransformed cells: effects of sodium gradient and cell density.]

Mixed membrane vesicle populations composed of plasma membrane and endoplasmic reticulum were prepared from Balb/c 3T3 and simian virus 40-transformed Balb/c 3T3 mouse fibroblasts. The initial rates of uptake of L-leucine and alpha-aminoisobutyric acid by these vesicles were stimulated by a NaCl gradient (external greater than internal). Cation specificity for stimulation of L-leucine uptake was Na+ greater than Li+ greater than K+. NaSCN was as effective as NaCl. Stimulation of uptake of both amino acids by a NaCl gradient was twice as great in vesicles from transformed as compared to non-transformed cells. The NaCl gradient produced transient accumulation of both L-leucine and alpha-aminoisobutyric acid to twice the equilibrium level in vesicles from transformed cells. No such "overshoot" was observed in vesicles from nontransformed cells. In vesicles from the contact-inhibitable Balb/c 3T3 cells, transport of alpha-aminoisobutyric acid, but non L-leucine, exhibited a density-dependent decrease in Na+ gradient induced stimulation, from 248% for sub-confluent to 109% with confluent cells. No density-related changes in uptake were noted with vesicles from the transformed cells. These studies suggest that variation in amino acid uptake associated with viral transformation may be related, at least in part, to alterations in Na+ permeability of the surface membrane.     

Mechanism of energy coupling to entry and exit of neutral and branched chain amino acids in membrane vesicles of Streptococcus cremoris

The energetics of neutral and branched chain amino acid transport by membrane vesicles from Streptococcus cremoris have been studied with a novel model system in which beef heart mitochondrial cytochrome c oxidase functions as a proton-motive force (delta p) generating system. In the presence of reduced cytochrome c, a large delta p was generated with a maximum value at pH 6.0. Apparent H+/amino acid stoichiometries (napp) have been determined at external pH values between 5.5 and 8.0 from the steady state levels of accumulation and the delta p. For L-leucine napp (0.8) was nearly independent of the pH. For L-alanine and L-serine napp decreased from 0.9-1.0 at pH 5.5 to 0-0.2 at pH 8.0. The napp for the different amino acids decreased with increasing external amino acid concentration. At pH 6.0, first order rate constants for amino acid exit (kex) under steady state conditions for L-leucine, L-alanine, and L-serine were 1.1-1.3, 0.084, and 0.053 min-1, respectively. From the pH dependence of kex it is concluded that amino acid exit in steady state is the sum of two processes, pH-dependent carrier-mediated amino acid exit and pH-independent passive diffusion (external leak). The first order rate constant for passive diffusion increased with increasing hydrophobicity of the side chain of the amino acids. As a result of these processes the kinetic steady state attained is less than the amino acid accumulation ratio predicted by thermodynamic equilibrium. The napp determined from the steady state accumulation represents, therefore, a lower limit. It is concluded that the mechanistic stoichiometry (n) for L-leucine, L-alanine, and L-serine transport most likely equals 1.     

E.S.R. of spin-trapped radicals in gamma-irradiated polycrystalline amino acids. Chromatographic separation of radicals

The free radicals produced by gamma-radiolysis of polycrystalline amino acids (L-valine, L-leucine, L-isoleucine and L-proline) at room temperature in the absence of air were investigated by spin trapping with 2-methyl-2-nitrosopropane (MNP). The spin adducts produced by dissolving the irradiated solids in aqueous MNP solutions were separated by high-performance liquid chromatography and then identified by e.s.r. Deamination (ring-opening reaction for L-proline) was observed for all amino acid. For L-valine and L-leucine, H-abstraction from the tertiary carbon in the side chains occurred. For isoleucine, H-abstractions from the alpha-carbon of the amino acid and from a non-terminal carbon in the side chain were found.     

Uptake of L-leucine and L-phenylalanine across the basolateral cell surface in isolated oxyntic glands.

The time course, kinetic, specificity and sodium-dependence of L-leucine and L-phenylalanine uptake by rabbit isolated oxyntic glands were studied in order to identify the systems involved in the transport of branched-chain and aromatic neutral amino acids through the basolateral cell membrane. The uptake was measured directly in the disrupted cells after incubation of the glands with the 3H-labelled amino acid both in a sodium-containing and a sodium-free medium. The uptake of L-leucine was largely carrier-mediated whilst L-phenylalanine was taken up by either carrier-mediated and nonsaturable processes. Both amino acids were taken up by a Na(+)-independent process. The kinetic parameters of L-leucine and L-phenylalanine carrier-mediated influx were, respectively: Kt = 2.71 mM and Jmax = 1390 nmol mg-1 s-1, Kt = 1.03 mM and Jmax = 176 nmol mg-1 s-1. From cross-inhibition studies it can be inferred that L-leucine is primarily transported by a Na(+)-independent system which shows specificity for bulky side chains dipolar amino acids. The system displays similar affinities for L-phenylalanine (Ki = 2.81 mM) and L-isoleucine (Ki = 2.62 mM). Similar results were obtained from self-inhibition experiments: the Ki of the carrier-mediated uptake of L-leucine and L-phenylalanine were 2.12 and 2.40 mM (from a Hanes plot) or 3.2 and 0.8 mM (from a Dixon plot), respectively. It is concluded that a sodium-independent transport system, like Christensen's 'L' type, is shared by branched-chain and aromatic dipolar amino acids, which only shows slight differences in their affinities for the carrier.