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1.
Novel biosynthetic routes to non-proteinogenic amino acids as chiral pharmaceutical intermediates 总被引:1,自引:0,他引:1
David J. Ager Tao Li David P. Pantaleone Richard F. Senkpeil Paul P. Taylor Ian G. Fotheringham 《Journal of Molecular Catalysis .B, Enzymatic》2001,11(4-6):199-205
Transaminases catalyse the reversible transfer of amino and keto groups between an amino acid and keto acid substrate pair. Many bacterial transaminases accept a wide array of keto acids as amino acceptors and are useful as commercial biocatalysts in the preparation of amino acids. Since the reaction equilibrium typically lies close to unity, several approaches have been described to improve upon the 50% product yield, using additional enzymes. The present work describes an efficient means to significantly increase product yield in transamination using the aromatic transaminase of Escherichia coli encoded by the tyrB gene, with
-aspartate as the amino donor. This is achieved by the introduction of the alsS gene encoding the acetolactate synthase of Bacillus subtilis, which eliminates pyruvate and alanine produced as a by-product of aspartate transamination. The biosynthesis of the non-proteinogenic amino acid
-2-aminobutyrate is described using a recombinant strain of E. coli containing the cloned tyrB and alsS genes. The strain additionally carries the cloned ilvA gene of E. coli encoding threonine deaminase to produce the substrate 2-ketobutyrate from
-threonine. An alternate coupled process uses lysine -aminotransferase in concert with a transaminase using
-glutamate as the amino donor. 相似文献
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阿胶,新阿胶,黄明胶,马皮胶,杂皮胶的质量对比研究:Ⅰ.氨基酸分析 总被引:1,自引:0,他引:1
本文用835-50型氨基酸自动分析仪测定了阿胶、新阿胶、黄明胶、马皮胶和杂皮胶中游离氨基酸和总氨基酸的含量,并计算出游离氨基酸、总氨基酸按侧链基团分类和按医药用途分类的相对含量。结果表明,各种氨基酸含量一般以驴皮胶为较高,但与其他几种胶比较并无显著差异,似可以猪皮马皮、牛皮代替驴皮。这也说明,单从氨基酸的含量来鉴别胶的种类和质量似乎不可行。 相似文献
4.
A gas chromatographic method for the separation and quantitation of the 20 protein amino acids is described using N-methyl-N(tert.-butyldimethylsilyl)trifluoroacetamide, with 1% tert.-butyldimethylchlorosilane as catalyst, to prepare the tert.-butyldimethylsilyl amino acid derivatives. Alkylsilylation of amino acids proceeds at 140 degrees C in 20 min. The derivatives formed in the one-step reaction are used directly for gas-liquid chromatographic analysis, using a flame-ionization detector, without prior isolation or purification. Complete separation and quantitation of all protein amino acids are readily achieved using a 15-m DB-5 capillary column. Strict linearity extends from less than 15 to about 100 ng for all amino acids except Arg, which has a linear range from 50 to 300 ng. The limits of detection, however, range from one to several hundred nanograms. The method was used to analyze the free amino acid pool in carnation petals. 相似文献
5.
M Kempe B Ekberg K Mosbach 《International journal of peptide and protein research》1990,35(2):147-152
The enzymatic synthesis of dipeptide units of the D-D-configuration in aqueous media, catalysed by muramoyl-pentapeptide carboxypeptidase (E.C.3.4.17.8), is described. Ac-L-Lys(Ac)-D-Ala-D-Lac-OH and Ac-D-Ala-OMe were used as acyl-components. Neutral, basic, and hydrophobic amino acids acting as nucleophiles were incorporated. The enzyme is stereospecific in that only the D-enantiomers of amino acids or amino acid derivatives were incorporated. As nucleophiles, the unmodified amino acids resulted in higher product yields compared with using the corresponding amino acid derivatives. Product yields ranged from 40 to 87%. 相似文献
6.
目的:建立检测人胎盘组织液氨基氮含量的方法,为产品质量控制和生产工艺稳定性提供判断手段。方法:加入福尔马林溶液后,以麝香草酚蓝为指示剂,用NaOH滴定液滴定。结果:当福尔马林溶液为中性、NaOH滴定液浓度为0.04mol/L时,甲醛滴定氨基氮的方法可靠。结论:本方法回收率高,操作简便,可以作为检测人胎盘组织液氨基氮含量的方法。 相似文献
7.
To study the role of 5-methylcytidine in the aminoacylation of mammalian tRNA, bulk tRNA specifically deficient in 5-methylcytidine was isolated from the livers of mice treated with 5-azacytidine (18 mg/kg) for 4 days. For comparison, more extensively altered tRNA was isolated from the livers of mice treated with DL-ethionine (100 mg/kg) plus adenine (48 mg/kg) for 3 days. The amino acid acceptor capacity of these tRNAs was determined by measuring the incorporation of one of eight different 14C-labeled amino acids or a mixture of 14C-labeled amino acids in homologous assays using a crude synthetase preparation isolated from untreated mice. The 5-methylcytidine-deficient tRNA incorporated each amino acid to the same extent as fully methylated tRNA. The tRNA from DL-ethionine-treated livers showed an overall decreased amino-acylation capacity for all amino acids tested. The 5-methylcytidine-deficient tRNA from DL-ethionine-treated mice were further characterized as substrates in homologous rate assays designed to determine the Km and V of the aminoacylation reaction using four individual 14C-labeled amino acids and a mixture of 14C-labeled amino acids. The Km and V of the reactions for all amino acids tested using 5-methylcytidine-deficient tRNA as substrate were essentially the same as for fully methylated tRNA. However, the Km and V were increased when liver tRNA from mice treated with DL-ethionine plus adenine was used as substrate in the rate reaction with [14C]lysine as label. Our results suggest that although extensively altered tRNA is a poorer substrate than control tRNA in both extent and rate of aminoacylation, 5-methylcytidine in mammalian tRNA is not involved in the recognition of the tRNA by the synthetase as measured by aminoacylation activity. 相似文献
8.
High-sensitivity amino acid analysis: methodology for the determination of amino acid compositions with less than 100 picomoles of peptides 总被引:14,自引:0,他引:14
Highly sensitive methodology is described for the determination of amino acid compositions of picomole quantities of peptides using an automated fluorescence amino acid analyzer with o-phthalaldehyde as the detection agent. All commonly occurring amino acids, including cystine, proline, and tryptophan, can be quantitated. High sensitivity is primarily achieved by using simple procedures which effectively and reliably reduce the level of interfering contamination present in buffers and reagents or introduced during sample handling. Accurate and reproducible amino acid compositions are obtained with 50 pmol or less peptide. 相似文献
9.
A simple, rapid method for measuring individual amino acids in physiological fluids using high-performance liquid chromatography [HPLC]/fluorescence detection has been characterized. Solutions containing free amino acids are first derivatized with orthophthalaldehyde, and then chromatographed on C18 reversed-phase columns. Sixteen amino acids are readily separated and quantitated, with a run time of about 60 min, and a sensitivity of at least 10 pmoles. Applying this technique to solutions of amino acid standards, and methanol supernatants of a large pool of blood, we find within-assay and between-assay coefficients of variation generally to be as good as those obtained using a standard amino acid analyzer. Serum amino acid measurements have also been obtained under a number of experimental conditions in rats, and yielded values comparable to those obtained using the amino acid analyzer. Samples of cerebrospinal fluid have also been analyzed via this new procedure. This technique offers numerous advantages over the oftentimes laborious, expensive, and time-consuming procedures required by conventional amino acid analyzers, and thus should find widespread application. 相似文献
10.
Detecting thermophilic proteins is an important task for designing stable protein engineering in interested temperatures.
In this work, we develop a simple but efficient method to classify thermophilic proteins from mesophilic ones using the amino
acid and dipeptide compositions. Since most of the amino acid and dipeptide compositions are redundant, we propose a new forward
floating selection technique to select only a useful subset of these compositions as features for support vector machine-based
classification. We test the proposed method on a benchmark data set of 915 thermophilic and 793 mesophilic proteins. The results
show that our method using 28 amino acid and dipeptide compositions achieves an accuracy rate of 93.3% evaluated by the jackknife
cross-validation test, which is higher not only than the existing methods but also than using all amino acid and dipeptide
compositions. 相似文献
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13.
Cloning and characterization of the cDNAs for human and rabbit interleukin-1 precursor. 总被引:27,自引:3,他引:27
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Y Furutani M Notake M Yamayoshi J Yamagishi H Nomura M Ohue R Furuta T Fukui M Yamada S Nakamura 《Nucleic acids research》1985,13(16):5869-5882
DNA sequence complementary to the mRNA for rabbit interleukin-1 precursor (preIL-1) has been cloned from the cDNA library constructed using partially purified poly(A)+RNA from induced rabbit alveolar macrophages by mRNA hybridization-translation assay. By using this cDNA as a probe, human IL-1 cDNA was isolated from the cDNA library prepared using poly(A)+RNA from induced HL-60 cells, a human monocyte-like cell line. The amino acid sequences of the human and rabbit preIL-1 deduced from the cDNA sequences reveal their primary structures which consists of 271 and 267 amino acid residues, respectively. The amino acid sequence is 64% conserved between human and rabbit. The difference in number of amino acid residues results from the carboxy-terminal extention of 4 amino acid residues in human preIL-1. Expression of the cloned human cDNA in E. coli yielded biologically active IL-1. 相似文献
14.
Y Shirasu H Yoshida T Mikayama S Matsuki J Tanaka H Ikenaga 《Journal of biochemistry》1986,99(6):1707-1712
We have cloned a DNA that is complementary to the messenger RNA that encodes porcine pancreatic elastase 1 from pancreas using rat pancreatic elastase 1 cDNA as a probe. This complementary DNA contains the entire protein coding region of 798 nucleotides which encodes an elastase of 266 amino acids, and 22 and 136 nucleotides of the 5' and 3'-untranslated sequences. When this deduced amino acid sequence was compared with known amino acid sequences, a carboxy-terminal 240 amino acids long peptide was found to be identical with a mature form of porcine pancreatic elastase 1, except for two amino acids. The porcine enzyme contains the same number of amino acid residues as the rat enzyme, and their amino acid sequences are 85% homologous. Taking the above findings together, we conclude that the cloned cDNA encodes a mature enzyme of 240 amino acids including a leader and activation peptide of 26 amino acids. We expressed the cloned porcine pancreatic elastase 1 cDNA in E. coli as a lac-fused protein. The resulting fused protein showed enzymatic activity and immunoreactivity toward anti-elastase serum. 相似文献
15.
Changes in Free Amino Acid Production and Intracellular Amino Acid Pools of Bacillus licheniformis as a Function of Culture Age and Growth Media 总被引:11,自引:8,他引:3
Changes in the endogenous intracellular amino acid pool and total free amino acid production in Bacillus licheniformis grown in minimal media were investigated. The total intracellular pool increased during exponential growth and then decreased rapidly after the end of growth. Most of the amino acids were present at low concentrations, but glutamate and alanine comprised 60 to 90% of the total intracellular free amino acid at most times during the growth cycle. It was concluded that, in addition to providing monomers for protein synthesis, the intracellular amino acid pool may be maintained for the storage of energy-providing metabolic intermediates and possibly as a balance to the ionic strength of the medium. The total free amino acid production by the cell was found to be dependent upon the composition of the salts medium as well as the culture age under conditions in which the carbon and nitrogen sources were the same. A 10-fold increase in extracellular amino acid was observed as the cells changed from vegetative to sporulation metabolism, mostly due to the extrusion of intracellular amino acid. The impact of this increase upon amino acid uptake and pulse-labeling studies using unwashed cells is discussed. 相似文献
16.
Jun.-Prof. Dr. Christoph Kaleta Sascha Schäuble Ursula Rinas Stefan Schuster 《Biotechnology journal》2013,8(9):1105-1114
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. 相似文献
17.
Transaminases catalyze amino transfer reactions from amino donors such as amino acids or amines to keto acids or ketones to give chiral amino acid or amines in optically pure form. α-Amino acid dehydrogenases catalyze the asymmetric reductive amination of α-keto acids using ammonia as amino donor to furnish L -amino acids. The distinct features and synthetic application of these two enzymes are reviewed in an effort to illustrate their promising and challenging aspects in serving as approaches to the direct asymmetric synthesis of optically pure amines from the corresponding keto compounds, a formidable problem in organic chemistry. 相似文献
18.
Hara KY Kim S Yoshida H Kiriyama K Kondo T Okai N Ogino C Fukuda H Kondo A 《Applied microbiology and biotechnology》2012,93(4):1495-1502
Glutathione is a valuable tri-peptide that is widely used in the pharmaceutical, food, and cosmetic industries. Glutathione
is produced industrially by fermentation using Saccharomyces cerevisiae, and supplementation of fermentation with several amino acids can increase intracellular GSH content. More recently, however,
focus has been given to protein as a resource for biofuel and fine chemical production. We demonstrate that expression of
a protease on the cell surface of S. cerevisiae enables the direct use of keratin and soy protein as a source of amino acids and that these substrates enhanced intracellular
GSH content. Furthermore, fermentation using soy protein also enhanced cell concentration. GSH fermentation from keratin and
to a greater extent from soy protein using protease-displaying yeast yielded greater GSH productivity compared to GSH fermentation
with amino acid supplementation. This protease-displaying yeast is potentially applicable to a variety of processes for the
bio-production of value-added chemicals from proteinaceous biomass resources. 相似文献
19.
H J Horstmann 《Analytical biochemistry》1979,96(1):130-138
A method for the quantitation of protein in biological material is described which gives the same response for all proteins irrespective of their amino acid composition. The method is based on the ninhydrin reaction of amino acids released after total acid hydrolysis of 5- to 20-μl solutions containing 1 to 100 μg of protein. The ammonia is released from the hydrolysate by diffusion and the amino acids are quantitated without fractionation using the continuous-flow system of an amino acid analyzer. Calibration is obtained with solutions of known amino acid content. The protein of a sample is calculated by multiplying the nanomoles of total amino acids found by a conversion factor F. F is the weight in micrograms of 1 nmol of the specific mixture of amino acid residues that the protein of the sample is composed of F has to be determined once for all further quantitations of the same material by quantitative amino acid analysis following standard procedures. By this method as little as 30 ng of protein per aliquot of hydrolysate analyzed can be determined. 相似文献
20.
S. Bouifraden C. Drouot M. El Hadrami F. Guenoun L. Lecointe N. Mai M. Paris C. Pothion M. Sadoune B. Sauvagnat M. Amblard J. L. Aubagnac M. Calmes P. Chevallet J. Daunis C. Enjalbal J. A. Fehrentz F. Lamaty J. P. Lavergne R. Lazaro V. Rolland M. L. Roumestant Ph. Viallefont Y. Vidal Dr. J. Martinez 《Amino acids》1999,16(3-4):345-379
Some of the chemistry of amino acids going on in our laboratory (Laboratoire des Amino acides Peptides et Protéines) is described as well as some mass spectrometry methodology for their characterization particularly on solid supports. Several aspects are presented including: (i) the stereoselective synthesis of natural and unnatural amino acids using 2-hydroxypinan-3-one as chiral auxiliary; (ii) the stereoselective synthesis of natural and unnatural amino acids by deracemization of alpha-amino acids via their ketene derivatives; (iii) the synthesis of alpha-aryl-alpha-amino acids via reaction of organometallics with a glycine cation; (iv) the diastereoselective synthesis of glycosyl-alpha-amino acids; (v) the synthesis of beta-amino acids using alpha-aminopyrrolidinopiperazinediones as chiral templates; (vi) the reactivity of urethane-N-protected N-carboxyanhydrides. To characterize natural and non natural amino acids through their immonium ions by mass spectrometry, some methodology is also described. 相似文献