Inclusion of nonproteinous amino acids in thermally prepared models for prebiotic protein

Sixteen nonproteinous amino acids (those not coded for in contemporary protein biosynthesis) were incorporated during the thermal formation of polyamino acids under postulated prebiotic conditions, although not all into a single polyamino acid. The copresence of proteinous or even α-amino acids was not required. (Norleucine color equivalents and elution times on a Beckman model 120C amino acid analyzer were determined for these nonproteinous amino acids). The results suggest that prebiotically available nonproteinous amino acids would have been constituents of prebiotic protein if the latter were formed thermally. Some differences in properties of the polyamino acids could be attributed to particular nonproteinous amino acid residues; however, the tested properties did not suggest a means for evolutionary selection against nonproteinous amino acids as a group. Selection against this class of amino acids in toto was likely a later, biotic, event.     

Obligatory amino acids in primitive proteins

Conformational similarity among amino acid residues, a property derived by analysing (φ, ψ)-probability distributions of 20 proteinous amino acids from 38 different globular proteins, is used to arrive at a set of six ‘obligatory’ amino acids of primitive proteins. The amino acids Ser, Val, Leu, Asp, Gly and Pro have been argued to be ‘obligatory’ and to represent, conformationally, the remaining amino acids. The reasons for consideration of these six residues as ‘obligatory’ are discussed. Methods to check the validity of our proposition are suggested.     

Abiotic synthesis of amino acids and imidazole by proton irradiation of simulated primitive earth atmospheres

Proton irradiation of simulated primitive earth atmosphere was performed, and amino acids and imidazole were analyzed. A mixture of carbon monoxide and nitrogen over water was irradiated by high energy protons (3 MeV, 0.6 µA) generated by a Van de Graaff accelerator for 2–5 h. Various kinds of proteinous and non-proteinous amino acids were detected in the irradiation products. Imidazole present in the irradiation products was also detected by high-performance liquid chromatography and mass spectrometry. The present results suggest that compounds of biological importance such as amino acids could be synthesized from primitive earth atmosphere by radiation of cosmic rays and/or solar flare particles.     

Organics in chimneys and water samples from deep-sea hydrothermal systems: implications for sub-vent biosphere.

Searching for life in extreme terrestrial environments can be a model of that for extraterrestrial life. Submarine hydrothermal system is one of promising sites for the frontier of life on the earth. Here seawater and vent chimnies were collected from deep-sea hydrothermal vents at Suiyo Seamount, Izu-bonin arc, Pacific Ocean as a part of Archaean Park Project. Pure seawater sample of 300 degrees C (purity>97%) could be collected. Dissolved and total hydrolyzable amino acids were determined by ion-exchange HPLC, and their enantiomeric ratio was measured by reversed-phase HPLC for the first time. Glycine and serine were two most abundant amino acids, followed by other proteinous amino acids such as alanine, glutamic acid and aspartic acid. Non-proteinous amino acids were detected as minor constituents. Most of the amino acids detected were of the L-form. Thus amino acids of abiotic origin were quite minor, and most of the amino acids detected were formed biologically. These results, together with analytical results of the vent chimney samples, suggest that there is active microbial activities near the hydrothermal systems.     

Evolutionary processes possibly limiting the kinds of amino acids in protein to twenty: a review.

Only 20 of more than 250 biosynthetic amino acids are common (coded) constituents of contemporary protein. In this paper, several stages of evolution, both prebiotic and biotic, are examined for means by which other (non-proteinous) amino acids may have been selected against. Simulated prebiotic experiments indicate that some non-proteinous amino acids were present prebiotically, that they could be incorporated during the formation of prebiotic protein, and that they would function in such protein. Biotic selection is thus indicated.Non-proteinous amino acids currently are available via biosynthetic pathways for potential incorporation into bioprotein. Codon-anticodon interaction, peptidyl transferases, and elongation and termination factors of protein synthesis do not show the specificity needed to preclude non-proteinous amino acids. Highly specific recognition among amino acids, tRNAs, and activating enzymes is concluded to be why the kinds of amino acids in contemporary protein are limited to twenty.Some of several theories concerning the origin, nature and evolution of the genetic code can readily accommodate non-proteinous amino acids. Some evidence suggests that such amino acids were eventually eliminated from protein because they were less suitable than related proteinous amino acids. However, deterministic or “direct interaction” theories currently lack sufficient experimental support to answer how non-proteinous amino acids were precluded; such theories, being testable, probably have the most potential for providing an answer.     

Protein evolution: intrinsic preferences in peptide bond formation: a computational and experimental analysis

Two possibilities exist for the evolution of individual enzymes/proteins from a milieu of amino acids, one based on preference and selectivity and the other on the basis of random events. Logic is overwhelmingly in favour of the former. By protein data base analysis and experiments, we have provided data to show the manifestation of two types of preferences, namely, the choice of the neighbour and its acceptance from the amino end (left) or the carboxyl end (right). The study tends to show that if the 20 proteinous amino acids were made to combine in water, the resulting profile would be nonrandom. Such selectivity could be a factor in protein evolution. Dedicated to the memory of Darshan Ranganathan.     

Sequence alignment approach to pick up conformationally similar protein fragments.

Crystal structure data of globular proteins were used to prepare (phi, psi) probability maps of 20 proteinous amino acids. These maps were compared grid-wise with each other and a conformational similarity index was calculated for each pair of amino acids. A weight matrix, called Conformational Similarity Weight (CSW) matrix, was prepared using the conformational similarity index. This weight matrix was used to align sequences of 21 pairs of proteins whose crystal structures are known. The aligned regions with more than seven contiguous amino acids were further analysed by plotting average weight (W) values of overlapping hepatapeptides in these regions and carrying out curve fitting by Fourier series having TEN harmonics. The protein fragments corresponding to the half-linewidth of peaks were predicted as fragments having similar conformation in the protein pair under consideration. Such an approach allows us to pick up conformationally similar protein fragments with more than 67% accuracy.     

Synthesis of peptides from amino acids and ATP with lysine-rich proteinoid

Summary Lysine-rich proteinoids in aqueous solution catalyze the formation of peptides from free amino acids and ATP. This catalytic activity is not found in acidic proteinoids, even though the latter contain some basic amino acid. The pH optimum for the synthesis is about 11, but is appreciable below 8 and above 13. Temperature data indicate an optimum at 20°C or above, with little increase in rate to 60°C. Pyrophosphate can be used instead of ATP, with lesser yields resulting. The ATP-aided syntheses of peptides in aqueous solution occur with several types of proteinous amino acid.Proofs should be sent to S.W. Fox, Institute for Molecular and Cellular Evolution, University of Miami, 521 Anastasia Avenue, Coral Gables, FL 33134     

Nitrogen Constituents in Manganese-Deficient Lemon Leaves

Total (Kjeldahl) nitrogen content of manganese-deficient lemon leaves was found to be about 40% higher (on a dry weight basis) than in the analog control leaves. The nitrate-N content rose in the deficient leaves from 18.2 mg/100 g to 95.6 mg/100 g dry weight. Among the N constituents studied, the free amino acids increased by about 42%, the protein amino acids by about 22%, and the total protein by more than 22%, all on a fresh weight basis. Acrylamide gel electrophoresis and ammonium sulfate fractionation showed qualitative differences in the various proteins. The increase of the protein fraction in the -Mn leaves may be attributed in part to the decrease of dry matter in these leaves (25.8% as compared to 30.7% in the control leaves) and in part to accumulation of apparent proteinous material in manganese-deficient tissues.     

A model for the origin of stable protocells in a primitive alkaline ocean

When a mixture of the eighteen proteinous amino acids are suitably heated in the dry state with seawater salts, a copolyamino acid results. One fraction of this polymer is found, through isoelectric focusing, to consist of a mixture of acidic and basic proteinoids, each of sharply limited heterogeneity. When one fraction of the seawater proteinoid is dissolved in hot water, and the solution is cooled, proteinoid microspheres result. These have properties in common with simpler types, but are also stable at pH values to 9, in common with microspheres prepared by mixing acidic and basic proteinoids. These processes thus constitute a simple model for the origin of a protocell stable in a primitive alkaline ocean.     

Biodegradability and biodegradation of poly(lactide)

Poly(lactide) (PLA) has been developed and made commercially available in recent years. One of the major tasks to be taken before the widespread application of PLA is the fundamental understanding of its biodegradation mechanisms. This paper provides a short overview on the biodegradability and biodegradation of PLA. Emphasis is focused mainly on microbial and enzymatic degradation. Most of the PLA-degrading microorganisms phylogenetically belong to the family of Pseudonocardiaceae and related genera such as Amycolatopsis, Lentzea, Kibdelosporangium, Streptoalloteichus, and Saccharothrix. Several proteinous materials such as silk fibroin, elastin, gelatin, and some peptides and amino acids were found to stimulate the production of enzymes from PLA-degrading microorganisms. In addition to proteinase K from Tritirachium album, subtilisin, a microbial serine protease and some mammalian serine proteases such as α-chymotrypsin, trypsin, and elastase could also degrade PLA.     

Screening,Purification and Properties of a Specific Antiphage Substance,AFS, against Filamentous Phage of Escherichia coli

We have made screening for antiphage antibiotics using an assay system of a male-specific filamentous phage, fl, and Escherichia coli as its host, and found a proteinous active substance named AFS (anti-filamentous phage substance) which was produced by an Actinomyces belonging to the group of Streptomyces lavendulae. It was purified from the filtered broth of the strain by ammonium sulfate precipitation and chromatographies of SP-Sephadex, CM-Sephadex and Sephadex G–50. The purified preparation was judged to be homogeneous by several column chromatographies and electrophoresis. Its molcular weight was about 5100 and isoelectric point was pH 9.9. Fourty two residues of amino acids, rich in the hydrophobic ones, were contained in one mole of AFS, but no carbohydrate was detected.     

Metabolic costs of amino acid and protein production in Escherichia coli

Escherichia coli is the most popular microorganism for the production of recombinant proteins and is gaining increasing importance for the production of low-molecular weight compounds such as amino acids. The metabolic cost associated with the production of amino acids and (recombinant) proteins from glucose, glycerol and acetate was determined using three different computational techniques to identify those amino acids that put the highest burden on the biosynthetic machinery of E. coli. Comparing the costs of individual amino acids, we find that methionine is the most expensive amino acid in terms of consumed mol of ATP per molecule produced, while leucine is the most expensive amino acid when taking into account the cellular abundances of amino acids. Moreover, we show that the biosynthesis of a large number of amino acids from glucose and particularly from glycerol provides a surplus of energy, which can be used to balance the high energetic cost of amino acid polymerization.     

氨基酸生产的代谢工程研究进展与发展趋势

氨基酸发酵是我国发酵工业的支柱产业,近年来,随着代谢工程的快速发展,氨基酸的代谢工程育种蓬勃发展。传统的正向代谢工程、基于组学分析与计算机模拟的反向代谢工程以及借鉴自然进化的进化代谢工程,都有越来越多的应用。在氨基酸的工业生产中涌现出了一系列具有高效生产、抗逆性强等优良性状的菌株。日益剧烈的市场竞争对菌株的选育提出了新的要求,如开发高附加值氨基酸品种、菌株代谢的动态调控、适应新工艺的要求等。文中介绍了氨基酸生产相关的代谢工程研究进展以及未来的发展趋势。     

Insecticidal activity of proteinous venom from tentacle of jellyfish Rhopilema esculentum Kishinouye

Insecticidal activity of proteinous venom from tentacle of jellyfish Rhopilema esculentum Kishinouye was determined against three pest species, Stephanitis pyri Fabriciusa, Aphis medicaginis Koch, and Myzus persicae Sulzer. R. esculentum full proteinous venom had different insecticidal activity against S. pyri Fabriciusa, A. medicaginis Koch, and M. persicae Sulzer. The 48 h LC50 values were 123.1, 581.6, and 716.3 microg/mL, respectively. Of the three pests, R. esculentum full proteinous venom had the most potent toxicity against S. pyri Fabriciusa, and the corrected mortality recorded at 48 h was 97.86%. So, S. pyri Fabriciusa could be a potential target pest of R. esculentum full proteinous venom.     

Production of amino acids by free-living heterotrophic nitrogen-fixing bacteria

Summary Interest of microbial production of amino acids has been increased greatly since development of biotechnological methods. These methods represent a perspective way applied in a future large-scale manufacture of inexpensive amino acids. In this context, the isolation of producing organisms that may be exploited in the desing of alternative methods for the production of amino acids could be of primary importance.In this review we will describe the liberation of amino acids (methionine, lysine, arginine, tryptophane and glutamic acid) byAzotobacter andAzospirillum during growth in culture media with different carbon sources under diazotrophic and adiazotrophic conditions. These organisms may be useful in developing new methods for the industrial production of amino acids.     

Elevated glutathione production by adding precursor amino acids coupled with ATP in high cell density cultivation of Candida utilis

Aims: Three precursor amino acids and adenosine triphosphate ( ATP) are necessary for fermentative production of glutathione. In this study, our aims were to develop a strategy to enhance glutathione production by adding three precursor amino acids coupled with ATP in high cell density (HCD) cultivation of Candida utilis. Methods and Results: A high-glutathione yeast strain, C. utilis WSH 02-08, was used in this study. Whole fermentative process for glutathione production was divided into two phases of cell growth and glutathione synthesis. Cells concentration was increased by HCD cultivation. Meanwhile, intracellular glutathione content was enhanced by the addition of three precursor amino acids. Concentrations of three precursor amino acids added at stationary phase were optimized by response surface methodology. Moreover, the addition of ATP 15 h after the addition of the three amino acids can further enhance glutathione production. Based on aforementioned phenomenon, a strategy of adding three precursor amino acids coupled with ATP was developed to enhance glutathione production. Conclusion: Without the addition of three precursor amino acids and the ATP, a total glutathione of 1123 mg l⁻¹ was achieved after 60-h cultivation. In comparison, addition of three precursor amino acid counterparts resulted in a total glutathione of 1841 mg l⁻¹. Moreover, by adding amino acids combined with ATP, a total glutathione of 2043 mg l⁻¹ was achieved after 72-h cultivation, increased by 81·9% and 11%, respectively, as compared with the control and the one without ATP addition. Significance and Impact of the Study: This is the first report on investigating changes of the intracellular three precursor amino acids and ATP, and γ-glutamylcysteine synthase activity in HCD cultivation of C. utilis for glutathione production. A strategy of combining addition of three precursor amino acids with ATP was developed to enhance glutathione production in C. utilis.     

Correlation between the consumption of amino acids and the production of the antibiotic gallidermin by Staphylococcus gallinarum

The correlation between the consumption of amino acids and the production of the polypeptide antibiotic gallidermin by Staphylococcus gallinarum Tü 3928 was investigated by on-line determination of amino acids and pulse experiments. A prolonged production phase together with an increase in gallidermin formation of about 25% was obtained during pulse and fed-batch experiments with the amino acids glutamic acid, glycine, serine and threonine.     

Production and Characterization of N- and C-terminally Truncated Mtx2: a Mosquitocidal Toxin from Bacillus sphaericus

Mosquitocidal toxin 2 (Mtx2) is a mosquito-larvicidal protein produced during vegetative stage of Bacillus sphaericus. The toxin consists of 292 amino acids and has a molecular weight of 31.8 kDa. To determine the active core region of the toxin, amino acids at N- and C-termini were sequentially removed. Deletion up to 23 amino acids from the N-terminus (Met1-Tyr23) did not significantly affect protein production and the toxin activity, whereas removal of 26 amino acids from the N-terminus (Met1-Lys26) completely abolished toxicity even though the protein production remained unchanged. Deletion of only 5 amino acids from the C-terminal end yielded the protein that could not be solubilized and rendered the toxin inactive. The results demonstrated that the C-terminal end of Mtx2 is required for proper folding and toxicity. Amino acids at the N-terminus up to Tyr23 did not play a significant role in protein production and toxicity whereas amino acids between Thr24 and Lys26 are required for full toxicity.     

冬虫夏草菌菌丝体中的氨基酸及其衍生物的代谢组学分析

为探究冬虫夏草菌（Ophiocordyceps sinensis）菌丝体氨基酸及其衍生物的差异性,采用超高效液相色谱串联质谱（Ultra performance liquid Chromatography, tandem mass spectrometry, UPLC-MS/MS）技术对3株冬虫夏草菌（玉树菌株XSH、达日菌株DR、贵德菌株LJ）菌丝体的氨基酸及其衍生物进行靶向定量检测,利用主成分分析（Principal component analysis, PCA）和正交偏最小二乘判别分析（Orthogonal signal correction and partial least squares-discriminant analysis, OPLS-DA）考察样品分类情况、筛选差异代谢物。结果表明,3株菌株菌丝体中均检测到72种氨基酸及其衍生物,发现除组氨酸外可合成蛋白质的19种氨基酸,菌株XSH的总氨基酸及必需氨基酸绝对含量均高于其他菌株。菌株XSH相比菌株DR和菌株LJ分别含有29种和28种差异氨基酸及其衍生物,菌株DR相比菌株LJ含有10种差异氨基酸及其衍生物,此外,3株菌株菌丝体含有3个共有的差异氨基酸及其衍生物,分别为高精氨酸、N-乙酰-L-谷氨酰胺、肌氨酸。京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes,KEGG）通路富集分析显示,差异氨基酸及其衍生物在精氨酸和脯氨酸代谢通路中更活跃,不同产区冬虫夏草菌株的氨基酸含量可能受精氨酸和脯氨酸代谢通路的影响。基于研究结果发现不同产区的冬虫夏草菌株的氨基酸及其衍生物存在较大的差异。