RESUMEN
During the aerobic batch cultivation of P. stipitis CBS 5776 with glucose, pyruvate decarboxylase was activated in a cell number-correlated manner. Activation started when a cell number between 7 x 10(7) and x 10(8) cells ml(-1) was reached and the enzyme activity increased during further cultivation. This induction might have been triggered either by an unknown quorum sensing system or by a shortage of cytoplasmic acetyl-CoA.
Asunto(s)
Oxígeno/farmacología , Pichia/enzimología , Piruvato Descarboxilasa/biosíntesis , Aerobiosis , Relación Dosis-Respuesta a Droga , Inducción Enzimática , Pichia/citología , Pichia/efectos de los fármacosRESUMEN
A newly developed Fmoc-Asp derivative, Fmoc-Asp beta-(2,3,4-trimethyl-pent-3-yl) ester, has been tried in the Fmoc-based SPPS of H-Val-Lys-Asp-Xaa-Tyr-Ile-OH, a well-established peptide model for studying base-catalysed aspartimide formation. When synthesizing the hexapeptide incorporating Gly, Arg(Pbf), Asn(Mtt), Asp(OtBu) or Cys(Acm) for Xaa, considerable amounts of aspartimide-related by-products were to be expected. The Asp(3) beta-carboxy protecting group and the duration of exposure to bases were varied. By-product formation could be reduced by incorporation of the new Asp derivative more efficiently than by introducing the less bulky Asp(OMpe). Significant improvements were observed in cases of prolonged contact with piperidine or DBU. Both beta-carboxy protecting groups were superior to the standard Asp(OtBu) which was also included in this study, but the additional stabilization gained by our new protecting group was valuable especially in syntheses of long peptides or difficult sequences.
Asunto(s)
Aminoácidos/química , Ácido Aspártico/análogos & derivados , Fluorenos/química , Oligopéptidos/síntesis química , Ácido Aspártico/química , Cromatografía Líquida de Alta PresiónRESUMEN
A novel surface-engineered strain of yeast Pichia pastoris was constructed that displays at its surface Kluyveromyces lactis Yellow Enzyme (KYE) fused to the C-terminal half of Saccharomyces cerevisiae alpha-agglutinin. The expression of the fusion protein was controlled by the AOX1-promoter. The new strain showed an increased sorption of the xenoestrogen Bisphenol A (BPA). It was shown that sorption of BPA depended on the presence of methanol in the growth medium and on the pH of the binding assays. The binding kinetics were typical for binding at a surface. The present results demonstrate that the alpha-agglutinin surface display system can be used in the yeast P. pastoris.
Asunto(s)
Kluyveromyces/enzimología , NADPH Deshidrogenasa/genética , NADPH Deshidrogenasa/metabolismo , Fenoles/metabolismo , Pichia/genética , Pichia/metabolismo , Adsorción , Oxidorreductasas de Alcohol/genética , Fusión Artificial Génica , Compuestos de Bencidrilo , Biodegradación Ambiental , Medios de Cultivo/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Genes Fúngicos , Concentración de Iones de Hidrógeno , Kluyveromyces/genética , Factor de Apareamiento , Proteínas de la Membrana/metabolismo , Metanol , Péptidos/genética , Pichia/enzimología , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
This paper presents a reevaluation of the synthesis and properties of Fmoc-His(3-Bum)-OH regarding its application in SPPS with minimal racemization of histidine residues during coupling and esterification reactions. By-product formation during the deprotection of the test peptides could be significantly reduced by scavenging the concomitantly formed HCHO, e.g. with methoxyamine.
Asunto(s)
Aminoácidos/química , Aminoácidos/síntesis química , Fluorenos/química , Fluorenos/síntesis química , Histidina/síntesis química , Biosíntesis de Péptidos , Cromatografía Líquida de Alta Presión , Fluorenos/farmacología , Histidina/análogos & derivados , Histidina/química , Histidina/farmacología , Espectroscopía de Resonancia Magnética , Modelos Químicos , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
The sequence dependence of base-catalysed aspartmide formation during Fmoc-based SPPS was systematically studied employing the peptide models H-Val-Lys-Asp-Xaa-Tyr-Ile-OH. The extent of formation of aspartimide and related by-products was determined by RP-HPLC. Considerable amounts of by-products were formed in the case of Xaa = Asp(OtBu), Arg(Pbf), Asn(Mtt), Cys(Acm) and unprotected Thr. Aspartimide formation could be diminished by incorporation of Asp(OMpe) or by employing milder methods for Fmoc cleavage, e.g. hexamethyleneimine/N-methylpyrrolidine/HOBt/NMP/DMSO 4:50:4:71:71 (v/v/w/v/v).
Asunto(s)
Aminoácidos/química , Ácido Aspártico/análogos & derivados , Ácido Aspártico/química , Fluorenos/química , Compuestos Bicíclicos Heterocíclicos con Puentes/química , Cromatografía Líquida de Alta Presión , Biosíntesis de Péptidos , Péptidos/química , Piperidinas/químicaRESUMEN
A variety of Asp beta-carboxy protecting groups, Hmb backbone protection and a range of Fmoc cleavage protocols have been employed in syntheses of the model hexapeptide H-VKDGYI-OH to investigate the aspartimide problem in more detail. The extent of formation of aspartimide and aspartimide-related by-products was determined by RP-HPLC. This study included three new Fmoc-Asp-OH derivatives: the beta-(4-pyridyl-diphenylmethyl) and beta-(9-phenyl-fluoren-9-yl) esters and also the orthoester Fmoc-beta-(4-methyl-2,6,7-trioxabicyclo[2.2.2]-oct-1-yl)-alanine. 3-Methylpent-3-yl protection of the Asp side chain resulted in significant improvements with respect to aspartimide formation. Complete suppression was achieved using the combination OtBu side chain protection and Hmb backbone protection for the preceding Gly residue.