Colorimetric Assay for Lysine Decarboxylase in Escherichia coli

A new assay is described for lysine decarboxylase. It is rapid and reproducible in assaying large numbers of samples, a situation in which earlier methods were less convenient. The new method is valuable in the study of peptide fractions and amino acid mixtures which stimulate induction of lysine decarboxylase. It may be useful for work on enzyme structure and modification, genetics, and kinetics.     

重组大肠杆菌发酵生产谷胱甘肽的氨基酸添加策略优化

对表达双功能谷胱甘肽合成酶的重组大肠杆菌发酵生产谷胱甘肽(Glutathione,GSH)进行氨基酸添加策略优化,结果表明:基本培养基中未添加氨基酸时GSH产量为0.81 g/L;诱导2 h后添加17 mmol/L半胱氨酸GSH产量为1.16 g/L,比不加氨基酸提高43%;添加17 mmol/L的3种前体氨基酸,GSH产量达到3.86 g/L,比只添加半胱氨酸提高2.33倍;进一步提高3种氨基酸添加量至25 mmol/L,GSH产量可达4.64 g/L,比不添加氨基酸提高4.73倍,总生产强度高达317.8 mg/(L·h),半胱氨酸转化为谷胱甘肽达到0.60 mol/mol;考察氨基酸添加模式发现一次性添加25 mmol/L氨基酸较恒速流加模式生产速率提高了29.8%。后续在50 L罐放大生产GSH,产量为4.31 g/L,总生产强度达到310.1 mg/(L·h),为工业化放大生产GSH奠定了基础。     

Lysine Suppressor in Escherichia coli

Head amber mutant of phage T4D was used to determine the efficiency of suppression and the amino acid inserted by Escherichia coli CA273 (sup-273 formerly Su(+) (beta)). The level of suppression determined was 8%, and the amino acid inserted was shown to be lysine.     

Uptake and Incorporation of Amino Acids and Peptides by Bacteroides amylophilus

Bacteroides amylophilus H-18 demonstrated a higher growth yield, a slightly higher growth rate, and a diminished lag period when Tryptose was added to the basal medium. This uptake of labeled amino acids was concentration-dependent, as the contribution of exogenous amino acid to the cell protein increased from 15.4 to 24.1% when the concentration of Casamino Acids in the medium was increased from 1.4 to 2.8 mg/ml. There was considerable redistribution of (14)C-label to other amino acids. Tryptic peptides of casein competed effectively with the amino acids for uptake. The (14)C-label from a protein was incorporated into B. amylophilus H-18 cells presumably after breakdown of the protein by the B. amylophilus H-18 protease.     

Amino sugar assimilation by Escherichia coli

The carbon skeleton of glucose is extensively randomized during conversion to cell wall glucosamine by Escherichia coli K-12. Exogenous glucosamine-1-(14)C is selectively oxidized, and isotope incorporation into cellular glucosamine is greatly diluted during assimilation. A mutant unable to grow with N-acetylglucosamine as a carbon and energy source was isolated from E. coli K-12. This mutant was found to be defective in glucosamine-6-phosphate deaminase. Glucosamine-1-(14)C and N-acetylglucosamine-1-(14)C were assimilated during the growth of mutant cultures without degradation or carbon randomization. Assimilated isotopic carbon resided entirely in cell wall glucosamine and muramic acid. Some isotope dilution occurred from biosynthesis, but at high concentrations (0.2 mm) of added N-acetylglucosamine nearly all cellular amino sugar was derived from the exogenous source. Growth of the mutant was inhibited with 1 mmN-acetylglucosamine.     

YjeH Is a Novel Exporter of l-Methionine and Branched-Chain Amino Acids in Escherichia coli

Amino acid efflux transport systems have important physiological functions and play vital roles in the fermentative production of amino acids. However, no methionine exporter has yet been identified in Escherichia coli. In this study, we identified a novel amino acid exporter, YjeH, in E. coli. The yjeH overexpression strain exhibited high tolerance to the structural analogues of l-methionine and branched-chain amino acids, decreased intracellular amino acid levels, and enhanced export rates in the presence of a Met-Met, Leu-Leu, Ile-Ile, or Val-Val dipeptide, suggesting that YjeH functions as an exporter of l-methionine and the three branched-chain amino acids. The export of the four amino acids in the yjeH overexpression strain was competitively inhibited in relation to each other. The expression of yjeH was strongly induced by increasing cytoplasmic concentrations of substrate amino acids. Green fluorescent protein (GFP)-tagged YjeH was visualized by total internal reflection fluorescence microscopy to confirm the plasma membrane localization of YjeH. Phylogenetic analysis of transporters indicated that YjeH belongs to the amino acid efflux family of the amino acid/polyamine/organocation (APC) superfamily. Structural modeling revealed that YjeH has the typical “5 + 5” transmembrane α-helical segment (TMS) inverted-repeat fold of APC superfamily transporters, and its binding sites are strictly conserved. The enhanced capacity of l-methionine export by the overexpression of yjeH in an l-methionine-producing strain resulted in a 70% improvement in titer. This study supplements the transporter classification and provides a substantial basis for the application of the methionine exporter in metabolic engineering.     

Escherichia coli K-12 Mutants Altered in the Transport of Branched-Chain Amino Acids

Two mutants of Escherichia coli K-12 are described which are resistant to the inhibition that valine exerts on the growth of E. coli. These mutants have lesions at two different loci on the chromosome. One of them, brnP, is linked to leu (87% cotransduction) and is located between leu and azi represented on the map at 1 min; the other, brnQ, is linked to phoA (96% cotransduction), probably between proC and phoA and represented at 10 min. These mutants are resistant to valine inhibition but are sensitive to dipeptides containing valine. Since it is known that dipeptides are taken up by E. coli through a transport system(s) different from those used by amino acids, this sensitivity to the peptides suggests an alteration in the active transport of valine. The mutants are resistant to valine only if leucine is present in the growth medium; the uptake of valine is less in both mutants than it is in wild-type E. coli, and it is reduced even further if leucine is present. Under these conditions the total uptake of valine is almost completely abolished in the brnQ mutant. The brnP mutant takes up about 60% as much valine as does the wild type, but no exogenous valine is incorporated into proteins. The apparent K(m) and V(max) of isoleucine, leucine, and valine for the transport system are reported; the brnP mutant, when compared to the wild type, has a sevenfold higher K(m) for isoleucine and a 17-fold lower K(m) for leucine; the V(max) for the three amino acids is reduced in the brnQ mutant, up to 20-fold for valine. The transport of arginine, aspartic acid, glycine, histidine, and threonine is not altered in the brnQ mutant under conditions in which that of the branched amino acids is. Evidence is reported that O-methyl-threonine enters E. coli through the transport system for branched amino acids, and that thiaisoleucine does not.     

DNA-Unwinding Dynamics of Escherichia coli UvrD Lacking the C-Terminal 40 Amino Acids

The E. coli UvrD protein is a nonhexameric DNA helicase that belongs to superfamily I and plays a crucial role in both nucleotide excision repair and methyl-directed mismatch repair. Previous data suggested that wild-type UvrD has optimal activity in its oligomeric form. However, crystal structures of the UvrD-DNA complex were only resolved for monomeric UvrD, using a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C). However, biochemical findings performed using UvrDΔ40C indicated that this mutant failed to dimerize, although its DNA-unwinding activity was comparable to that of wild-type UvrD. Although the C-terminus plays essential roles in nucleic acid binding for many proteins with helicase and dimerization activities, the exact function of the C-terminus is poorly understood. Thus, to understand the function of the C-terminal amino acids of UvrD, we performed single-molecule direct visualization. Photobleaching of dye-labeled UvrDΔ40C molecules revealed that two or three UvrDΔ40C molecules could bind simultaneously to an 18-bp double-stranded DNA with a 20-nucleotide, 3′ single-stranded DNA tail in the absence of ATP. Simultaneous visualization of association/dissociation of the mutant with/from DNA and the DNA-unwinding dynamics of the mutant in the presence of ATP demonstrated that, as with wild-type UvrD, two or three UvrDΔ40C molecules were primarily responsible for DNA unwinding. The determined association/dissociation rate constants for the second bound monomer were ∼2.5-fold larger than that of wild-type UvrD. The involvement of multiple UvrDΔ40C molecules in DNA unwinding was also observed under a physiological salt concentration (200 mM NaCl). These results suggest that multiple UvrDΔ40C molecules, which may form an oligomer, play an active role in DNA unwinding in vivo and that deleting the C-terminal 40 residues altered the interaction of the second UvrD monomer with DNA without affecting the interaction with the first bound UvrD monomer.     

Enzymatic Production of Amino Acids

Abstract

Insects have extremely sensitive systems of olfaction. These systems have been explored as potential sensors for odourants associated with forensics, medicine, security, and agriculture application. Most sensors based on insect olfaction utilize associative learning to “program” the insects to exhibit some form of behavioural response to a target odourant. To move to the next stage of development with whole-insect programmable sensors, an examination of how odourants are captured, processed and used to create behaviour is necessary. This review article examines how the neurophysiological, molecular, genetic and behavioural system of olfaction works and how an understanding of these systems should lead the way to future developments in whole-insect programmable sensors.     

Mutant of Escherichia coli K-12 Defective in the Transport of Basic Amino Acids

Escherichia coli K-12 possesses two active transport systems for arginine, two for ornithine, and two for lysine. In each case there is a low- and a high-affinity transport system. They have been characterized kinetically and by response to competitive inhibition by arginine, lysine, ornithine and other structurally related amino acids. Competitors inhibit the high-affinity systems of the three amino acids, whereas the low-affinity systems are not inhibited. On the basis of kinetic evidence and competition studies, it is concluded that there is a common high-affinity transport system for arginine, ornithine, and lysine, and three low-affinity specific ones. Repression studies have shown that arginine and ornithine repress each other's specific transport systems in addition to the repression of their own specific systems, whereas lysine represses its own specific transport system. The common transport system was found to be repressible only by lysine. A mutant was studied in which the uptake of arginine, ornithine, and lysine is reduced. The mutation was found to affect both the common and the specific transport systems.     

In Silico Study of Different Signal Peptides to Express Recombinant Glutamate Decarboxylase in the Outer Membrane of Escherichia coli

International Journal of Peptide Research and Therapeutics - Gamma amino butyric acid (GABA) is used as drugs, food ingredients, and dietary supplements. l-glutamate is converted to GABA by the...     

Genetic Analysis of Glutamate Transport and Glutamate Decarboxylase in Escherichia coli

The location of the Escherichia coli K-12 genes determining or regulating glutamate transport, and the location of the gene determining glutamate decarboxylase synthesis, were established by conjugation. The ability to grow on glutamate as the sole source of carbon and energy was used to select for glutamate transport recombinants. Two genes determining the ability to grow on glutamate as the sole source of carbon and energy were mapped. One (gltC) is located near mtl (mannitol), and the other (gltH) appears to be located between the gal (galactose) and trp (tryptophan) loci. The glutamate decarboxylase gene (gad) is strongly linked to gltC. The gltC(+) recombinants grow on glutamate much faster and accumulate this amino acid to a greater extent than do the gltH(+) recombinants. The gltH(+) gene functioned only in one female strain (P678), whereas the gltC gene functioned in all the female strains tested (P678, C600, W1).     

Lysine Decarboxylase Activity in Broth and Agar Media

Four lysine decarboxylase media were studied by testing them with 305 Enterobacteriaceae and 42 nonfermenting bacilli. A comparison was made between lysine decarboxylase broth medium (Moeller base) and Johnson's semisolid agar without lactose and Bachrach's broth medium and lysine-agar slants which contain lactose. The nonlactose media, lysine decarboxylase broth and the semisolid medium of Johnson, were the best media for use with all of the bacteria studied. The exclusion of lactose from lysine decarboxylase medium seems desirable to extend the usefulness of this medium among members of the Enterobacteriaceae. When the results with lysine decarboxylase broth and Johnson's semisolid medium without lactose were compared, a 6% difference existed between the results obtained with lysine decarboxylase broth and Johnson's semisolid agar. When the results with Bachrach's broth and lysine-agar slants with lactose were compared, a 1% difference existed between Bachrach's broth and the agar slant method. At times, reading and interpretation were difficult because of intermediate degrees of color change. The inability of Pseudomonas aeruginosa or Herellea to utilize glucose under the anaerobic condition of the medium makes the lysine decarboxylase test an undesirable procedure for these organisms. Of the four test media used, the lysine-lactose-agar slants seemed to be the least desirable because of the more frequent occurrence of indistinct color reactions and shifts in color.     

Rapid Glutamate Decarboxylase Assay for Detection of Escherichia coli

Volume 59, no. 12, p. 4347, column 1, line 3 from bottom: "500 x g" should read "2,500 x g." [This corrects the article on p. 4347 in vol. 59.].     

不同产地天麻氨基酸的含量测定

氨基酸用途广泛,人们对氨基酸质量和品种的要求也越来越高.我国氨基酸从无到有,到现在的大市场份额,经历了五十多年的发展历程,其中包括谷氨酸、赖氨酸、苏氨酸和蛋氨酸在内的大品种氨基酸已经发展成为全球的主要供应品种,部分小品种氨基酸已形成自己的优势.绿色制造和现代生物技术的应用将成为氨基酸行业发展的主流.本文重点论述了我国氨基酸品种的现状、水平以及未来发展方向.     

Stimulation of cytochrome synthesis in Escherichia coli by cyclic AMP

A cyclic AMP-requiring mutant of Escherichia coli K12 which grows slowly on glucose was found to contain reduced levels of cytochrome b₁ and cytochrome oxidase o. The addition of exogenous cyclic AMP stimulated the synthesis of these cytochrome components and restored growth on glucose to the normal rate observed with the parental strain. Cytochrome synthesis in the parental strain was also stimulated by exogenous cyclic AMP. These studies have provided evidence that cyclic AMP participates in regulating cytochrome synthesis in E. coli, and, coupled with other observations, have suggested a role for this cyclic nucleotide in controlling the construction and operation of the organism's membrane system.     

几种氨基酸在庆大霉素生产中作用的研究

庆大霉素是氨基糖苷类广谱抗生素,其不仅用于临床治病,而且广泛应用于畜牧业。由于其发酵周期长,产素率低,生产成本高,因此改进生产方法势在必行。本文报道了在绛红色小单孢菌产生庆大霉素的培养过程中,添加一定量的甘氨酸、蛋氨酸、赖氨酸、酪氨酸能够有效地提高微生物细胞的代谢能力,缩短发酵培养周期,提高产素率。本方法是在小试（摇瓶）成功基础上,用5L玻璃发酵罐运转一个多月,取一个月的平均罐批数据表明：新方法较原工艺发酵周期缩短30% ~45%,罐批产量增加14% 左右,产素率提高30 % ~95%（因菌种生产能力不同而异）,产品质量符合中国药典2000版（CP2000）、英国药典2000版（BP2000）、美国药典26版（USP26）,生产成本大幅度降低,具有很强的市场竞争力。     

Production L-tryptophan by Escherichia coli cells

Whole cells of Escherichia coli B 10 having high tryptophan synthetase activity were used directly as an enzyme source to produce L-tryptophan from indole and L- or D,L-serine. This strain is tryptophan auxotrophic, which is tryptophanase negative and, in addition, L- and D-serine deaminase negative under production conditions. To avoid inhibition of tryptophan synthetase by a high concentration of indole, nonaqueous organic solvents, Amberlite XAD-2 adsorbent, and nonionic detergents were used as reservoirs of indole in the reaction mixture for the production of L-tryptophan. As a result, different effects were observed on the production of L-tryptophan. Particularly, among the nonionic detergents, Triton X-100 was very efficient. Using Triton X-100 for production of L-tryptophan from indole and L- or D,L-serine by whole cells of Escherichia coli B 10, 14.14 g/100 mL and 14.2 g/100 mL of L-tryptophan were produced at 37 degrees C for 60 h.     

大肠杆菌的直流电场刺激过程

以钛网电极和铂网电极对培养瓶中大肠杆菌生长过程进行加电刺激,研究其在直流电场作用下的生长情况,并结合循环伏安扫描、恒电流、十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)及测定菌体ATP酶活力等技术对大肠杆菌的直流电场刺激过程进行研究。结果表明,在0-0.2275mA/cm2范围内,随着电流密度的增加,直流电场对大肠杆菌生长量的增长促进作用逐渐增加,而0.0455mA/cm2的电场则是获得最大活菌量的最适电流密度;通过对析氢活性不同的铂网电极与钛网电极通加相同电流密度的电场,发现铂电极培养体系菌体生长优于钛电极培养体系菌体的生长。经验证发现引起这种变化的原因主要是水的阴极电解产物吸附氢和氢气比例的不同引起的;同时发现在0.091mA/cm2电流密度下,直流电场能有效提高ATP酶的活力,在8h时通电菌样酶活为不通电菌样酶活的3.2倍;通过对0.0455mA/cm2直流电场刺激后的菌体蛋白进行SDS-PAGE分析发现加电菌体在分子量25kD与35kD左右多肽表达量明显高于不加电菌体的多肽表达量,而在分子量为66.2kD左右时多肽表达量低于不加电菌体多肽表达量。