RESUMO
Using Escherichia coli K-235 as a production strain in a fed-batch fermentation process with an optimized sorbitol/yeast extract medium, we were able to produce 640 U of CMP-Neu5Ac synthetase in 10 l scale (64 U/l) and 9200 U (total enzyme) in 200 l scale (390 U/kg wet weight). By simple one-step purification procedures, enzyme preparations were obtained that could be used efficiently for the synthesis of CMP-Neu5Ac from CTP and Neu5Ac with over 90% yield, from Neu5Ac, CMP, and ATP or phosphoenolpyruvate by in situ generation of CTP, and from CTP, pyruvate, and ManNAc or GlcNAc by in situ generation of Neu5Ac.
Assuntos
Ácido N-Acetilneuramínico do Monofosfato de Citidina/metabolismo , Escherichia coli/enzimologia , N-Acilneuraminato Citidililtransferase/metabolismo , Ácidos Siálicos/metabolismo , Proteínas de Bactérias/isolamento & purificação , Citidina Trifosfato/metabolismo , Fermentação , Ácido N-Acetilneuramínico , N-Acilneuraminato Citidililtransferase/isolamento & purificaçãoRESUMO
(R)-2-Hydroxy-4-phenylbutyric acid, an intermediate in the manufacture of inhibitors of angiotensin converting enzyme, can be produced continuously in an enzyme membrane reactor by enzymatic reduction of its corresponding alpha-keto acid. D-Lactate dehydrogenase (D-LDH) from Staphylococcus epidermidis was chosen as the most appropriate enzyme to carry out the NADH-dependent reduction. Formate dehydrogenase (FDH) was used for NADH regeneration. Detailed kinetic measurements and a mathematical model for the coupled enzyme reactions were applied to calculate the optimal conditions for continuous production of the alpha-hydroxy acid. A mass of 1 kg [corrected] (R)-2-hydroxy-4-phenylbutyric acid was synthesized in a 220 ml enzyme membrane reactor over a period of 4 weeks. A mean space-time-yield of 165 g l-1 d-1 was achieved at low enzyme consumptions of 150 U kg-1 alpha-hydroxy acid for FDH and D-LDH.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/síntese química , Butiratos/química , Lactato Desidrogenases , Fenilbutiratos , Estabilidade Enzimática , Cinética , L-Lactato Desidrogenase/metabolismo , Membranas/enzimologia , OxirreduçãoRESUMO
3-Amino-5-hydroxybenzoic acid was investigated for its ability to induce rifamycin biosynthesis in an appropriate mutant of Nocardia mediterranei and identified as a direct precursor of the seven-carbon amino starter-unit for the biosynthesis of ansamycins. A model for the biosynthesis of different types of ansamycins is presented and discussed.
Assuntos
Aminobenzoatos/metabolismo , Nocardia/metabolismo , Rifamicinas/biossíntese , Aminobenzoatos/síntese química , Benzoatos/metabolismo , Hidroxibenzoatos , Mutação , Nocardia/genéticaRESUMO
The mutant under study, designated A8, is derived from a Nocardia mediterranei strain, N813, which is a high rifamycin B producer. A8 is auxotrophic for aromatic amino acids and produces much less rifamycin B than the parent. A mixture of pentoses with D (--) ribulose as the main product is accumulated in the fermentation broth of this mutant. It was shown to be affected in its transketolase activity as no formation of D-sedoheptulose -7P from pentose-phosphates could be detected in vitro using crude extracts. The only pathway so far known which is derived from D-sedoheptulose-7P is the shikimate pathway leading to aromatic amino acids and vitamins. Biochemical and genetic investigations with mutant A8, which is defective in both the biosynthesis of rifamycins and the biosynthesis of shikimate pathway products, show that the seven-carbon amino unit of the rifamycin-chromophore must be derived from an intermediate of the shikimate pathway.
Assuntos
Nocardia/metabolismo , Pentoses/metabolismo , Rifamicinas/biossíntese , Meios de Cultura , Fermentação , Mutação , Nocardia/genética , Nocardia/crescimento & desenvolvimentoRESUMO
The mutant under study, designated A10, is derived from a Nocardia mediterranei strain, N813, which is a high rifamycin B producer. A10 is auxotrophic for aromatic amino acids but unlike A8 (see preceding paper) produces the same amount of rifamycin B as the parent. Shikimic acid and 3-dehydroshikimic acid are accumulated in the fermentation broth of this mutant. It was shown to be blocked in one of the enzymes leading from shikimate to chorismate. No formation of shikimate-3-phosphate from shikimate and ATP could be detected in vitro using crude extracts of this mutant and of the parent. As mutant A10 is only defective in the biosynthesis of aromatic amino acids and not in the biosynthesis of rifamycins it would appear that the seven-carbon amino unit of the rifamycin-chromophore must be derived from an intermediate of the shikimate pathway not behind shikimate. By referring to the results of the preceding paper it can be seen that the origin of this moiety can definitely be localized between 3-deoxy-D-arabinoheptulosonic acid-7-phosphate and shikimate.
Assuntos
Nocardia/metabolismo , Rifamicinas/biossíntese , Ácido Chiquímico/metabolismo , Meios de Cultura , Fermentação , Mutação , Nocardia/genéticaRESUMO
A mutant strain derived from Nocardia mediterranei N813 was found to accumulate a number of novel ansamycins structurally related to the protostreptovaricins and to rifamycin W. One of the main components of this ansamycin complex, protorifamycin I (8-deoxyrifamycin W), was purified and identified by means of chemical and spectroscopic methods.
Assuntos
Rifamicinas/isolamento & purificação , Fenômenos Químicos , Química , Fermentação , Testes de Sensibilidade Microbiana , Mutação , Nocardia/genética , Nocardia/metabolismo , Rifamicinas/metabolismo , Rifamicinas/farmacologiaRESUMO
The transformation of rifamycin S into rifamycins B and L was reinvestigated in order to establish more detailed pathways. Our results exclude rifamycin O as a common progenitor in the biosyntheses of rifamycins B and L. Rifamycins B and L are formed from rifamycin S (SV) by different pathways using different C3-precursors for the biosynthesis of their glycolic acid moieties. A thiamine-dependent enzyme (decarboxylase) seems to be involved in the transformation reaction.
Assuntos
Rifamicinas/biossíntese , Rifamicinas/metabolismo , Acremonium/metabolismo , Antimetabólitos/farmacologia , Biotransformação , Glicolatos/metabolismo , Modelos Biológicos , Nocardia/metabolismoRESUMO
3'-Demethoxy-3'-hydroxystaurosporine, 1 (CGP 58,546), a novel staurosporine analogue, was isolated from a mutant of Streptomyces longisporoflavus R19 blocked in the last step of the biosynthetic pathway. CGP 58,546 was less potent than staurosporine, but it showed a more selective inhibition pattern against various subtypes of protein kinase C.
Assuntos
Alcaloides , Mutação , Streptomyces/metabolismo , Alcaloides/química , Alcaloides/farmacologia , Fenômenos Químicos , Físico-Química , Fermentação , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Mutagênese , Espectrometria de Massas de Bombardeamento Rápido de Átomos , Estaurosporina , Streptomyces/genética , Streptomyces/crescimento & desenvolvimentoRESUMO
A number of minor compounds were isolated from fermentations of the protorifamycin I producing strain Nocardia mediterranei F 1/24(1,2)) and identified by means of chemical and spectroscopic methods. Two types of structures were identified: Type 1: modified protorifamycins (derived from protorifamycin I) Type 2: defective rifamycins (8-deoxyrifamycins).
Assuntos
Antibacterianos/biossíntese , Nocardia/metabolismo , Antibacterianos/isolamento & purificação , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Fenômenos Químicos , Química , Fermentação , Lactamas Macrocíclicas , Espectroscopia de Ressonância MagnéticaRESUMO
Proansamycin B-M1 and protorifamycin I-M1 were isolated as minor compounds from fermentations of the protorifamycin I producing strain Nocardia mediterranei F 1/24, identified by means of chemical and spectroscopic methods and shown to be degradation products of the hypothetical proansamycin B postulated in part I of this series of papers and of protorifamycin I, respectively.
Assuntos
Antibacterianos/isolamento & purificação , Antibacterianos/análise , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Biotransformação , Candida albicans/efeitos dos fármacos , Fermentação , Lactamas Macrocíclicas , Nocardia/metabolismoRESUMO
A number of rifamycin non-producing UV-mutants derived from Nocardia mediterranei strains N813 (rifamycin B producer) and A10 (aro--mutant excreting shikimate derived from strain N813) were found to accumulate an identical complex of aromatic components instead of rifamycin B. The main component of this aromatic complex, product P8/1-OG, was isolated from six of these P--mutant strains and identified spectroscopically as a very early precursor in the biosynthesis of rifamycins.
Assuntos
Antibacterianos/biossíntese , Nocardia/metabolismo , Fenômenos Químicos , Físico-Química , Lactamas Macrocíclicas , Mutação , Nocardia/genética , Rifamicinas/biossínteseRESUMO
D-Ins(1,3,4,5)P4 and unnatural L-Ins(1,3,4,5)P4 were prepared in gram-quantities from D- and L-2,6-di-O-benzyl-myo-inositol by a chemical phosphorylation and deprotection step in high yield and purity without extensive purification. The optically pure benzyl derivatives were obtained by enzyme-catalyzed resolution of racemic 2,6-di-O-benzyl-myo-inositol under acyl-transfer conditions in vinyl acetate as the acyl donor. The lipase of Candida antarctica only acetylated regio- and enantio-selectively the L-enantiomer, providing exclusively L-5-acetyl-2,6-di-O-benzyl-myo-inositol, whereas the D-enantiomer remained unchanged.
RESUMO
Recombinant Lactobacillus leichmannii ribonucleosidetriphosphate reductase (RTPR, E.C.1.17.4.2) constitutively expressed by E. coli HB101 pSQUIRE has been purified from sonicated cell material in a one-step procedure by PEG 4000 (16% (w/w))/phosphate (7% (w/w)) liquid-liquid extraction. A high yield of 75.1% RTPR in the top phase and a partitioning of 8.5:1 between total RTPR activity in top and bottom phase were obtained in this preparative system. The RTPR-containing top phase was used to reduce ATP in the 2'-position on a gram scale with high final conversion and yield proving the ribonucleotide reductase approach feasible for the preparative synthesis of 2'-deoxyribonucleotides. High concentrations of sodium acetate in the reaction served to substitute for allosteric effectors of RTPR. 1,4-Dithio-DL-threitol was used as an artificial reducing agent for RTPR.
Assuntos
Trifosfato de Adenosina/química , Ribonucleotídeo Redutases/metabolismo , Trifosfato de Adenosina/metabolismo , Sulfato de Amônio , Escherichia coli/enzimologia , Indicadores e Reagentes , Cinética , Lactobacillus/enzimologia , Oxirredução , Polietilenoglicóis , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleotídeo Redutases/isolamento & purificaçãoRESUMO
Optically pure D-tert.-leucine was obtained by the enzymatic hydrolysis of (+/-)-N-acetyl-tert. leucine chloroethyl ester after about 50% conversion, this being catalyzed by a protease from Bacillus licheniformis (Alcalase), and subsequent acidic saponification of the recovered ester. Among the methyl, ethyl, octyl, chloroethyl and trichloroethyl esters, the chloroethyl ester exhibited the highest rate of hydrolysis.