RESUMEN
Isolated from pig liver as a crude, inhomogeneous enzyme fraction, pig liver esterase (PLE) was found to metabolize a wide range of substrates; often in a highly stereoselective manner. This crude esterase preparation, however, contains several iso-enzymes at proportions varying from batch to batch. Racemic methyl-(4E)-5-chloro-2-isopropyl-4-pentenoate is cleaved enantioselectively by crude PLE, but not by recombinantly expressed gamma-isoform of PLE. Concluding that another PLE iso-enzyme must carry the relevant activity, we cloned and sequenced cDNAs of several PLE isoforms and functionally expressed them in Pichia pastoris. One novel isoform termed alternative pig liver esterase (APLE) was found to hydrolyze methyl-(2R,4E)-5-chloro-2-isopropyl-4-pentenoate in a highly stereoselective manner (E>200). When heterologously expressed and directed for secretion in P. pastoris, APLE was found to be localized in the periplasm. The presence or absence of a putative C-terminal ER retention signal did neither influence functional expression nor cellular localization. The recombinant enzyme, purified by ion exchange chromatography, had a specific activity of 36U (mg protein)(-1) towards racemic methyl-(4E)-5-chloro-2-isopropyl-4-pentenoate.
Asunto(s)
Esterasas/metabolismo , Hígado/enzimología , Pichia/metabolismo , Sus scrofa/metabolismo , Secuencia de Aminoácidos , Animales , Catálisis , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Esterasas/química , Esterasas/aislamiento & purificación , Ácidos Grasos Monoinsaturados/metabolismo , Hidrólisis , Extractos Hepáticos , Espectrometría de Masas , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Periplasma/metabolismo , Transporte de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Alineación de Secuencia , Especificidad por SustratoRESUMEN
This article discusses the preparation, structure and objectives of the Bio-based Industries Joint Undertaking (BBI JU). BBI JU is a public-private partnership (PPP) between the European Commission (EC) and the Bio-based Industries Consortium (BIC), the industry-led private not-for-profit organisation representing the private sectors across the bio-based industries. The model of the public-private partnership has been successful as a new approach to supporting research and innovation and de-risking investment in Europe. The BBI JU became a reality in 2014 and represents the largest industrial and economic cooperation endeavour financially ever undertaken in Europe in the area of industrial biotechnologies. It is considered to be one of the most forward-looking initiatives under Horizon 2020 and demonstrates the circular economy in action. The BBI JU will be the catalyst for this strategy to mobilise actors across Europe including large industry, small and medium-sized enterprises (SMEs), all types of research organisations, networks and universities. It will support regions and in doing so, the European Union Member States and associated countries in the implementation of their bioeconomy strategies.
Asunto(s)
Biotecnología , Conservación de los Recursos Naturales , Desarrollo Económico , Europa (Continente) , Modelos Económicos , Asociación entre el Sector Público-PrivadoRESUMEN
(R)-2-chloromandelic acid represents a key pharmaceutical intermediate. Its production on large scale was hampered by low turnover rates and moderate enantiomeric excess (ee) using enzyme as well as metal catalysts. The cloning and heterologous overexpression of an (R)-hydroxynitrile lyase from Prunus amygdalus opened a way to large-scale production of this compound. Especially the rationally designed mutation of alanine to glycine at amino acid position 111 of the mature protein tremendously raised the yield for enantioselective conversion of 2-chlorobenzaldehyde to (R)-2-chloromandelonitrile, which can be hydrolysed to the corresponding alpha hydroxy acid. However, expression of this mutein was less efficient than for the unmodified enzyme. Subsequent LC/MS/MS-analysis of the protein sequence revealed that mutation A111G triggered the posttranslational deamidation of the neighbouring residue asparagine (N110) to aspartic acid. This finding on the one hand could explain the decreased secretion efficiency of the mutant as compared to the wildtype enzyme, but on the other hand raised the question which of the two residues was truly accountable for the enhanced conversion. The muteins N110D, A111G and N110DA111G were constructed and compared in terms of protein productivity and performance in chemical syntheses. The expression level of the double mutein was augmented significantly and the enantioselectivity remained high. Reduced protein expression of mutein PaHNL5-L1Q-A111G was remedied by mutational anticipation of posttranslational deamidation.
Asunto(s)
Alanina/genética , Aldehído-Liasas/metabolismo , Ingeniería Genética , Glicina/genética , Procesamiento Proteico-Postraduccional , Prunus/enzimología , Aldehído-Liasas/genética , Sustitución de Aminoácidos , Benzaldehídos/química , Catálisis , Electroforesis en Gel de Poliacrilamida , Halógenos , Cinética , Proteínas Mutantes/biosíntesis , Proteínas Mutantes/metabolismo , Nitrilos , Fracciones Subcelulares/enzimologíaRESUMEN
Biocatalytic synthesis of enantiomerically pure compounds for pharmaceutical intermediates is gaining momentum. This is the result of advances in genomics, screening and evolution technologies leading to the increased availability of new and robust biocatalysts suited for industrial-scale application, and is stimulated by an increased demand for catalysts that are able to address the increased complexity of active pharmaceutical ingredients. The vast majority of biotransformation reactions for the manufacturing of optically active pharmaceutical intermediates are still based on enantioselective ketone reductions and enantiospecific hydrolyses. This review aims to point at alternative reaction types and integrated multi-enzymatic steps that are emerging in large-scale applications.
Asunto(s)
Biotecnología/métodos , Enzimas/química , Preparaciones Farmacéuticas/síntesis química , Bacterias/enzimología , Biotecnología/tendencias , Biotransformación , Catálisis , Enzimas/metabolismo , Fermentación , Hongos/enzimología , Estructura Molecular , EstereoisomerismoRESUMEN
The enantioselectivity of epoxide hydrolase from Agrobacterium radiobacter (EchA) was improved using error-prone PCR and DNA shuffling. An agar plate assay was used to screen the mutant libraries for activity. Screening for improved enantioselectivity was subsequently done by spectrophotometric progress curve analysis of the conversion of para-nitrophenyl glycidyl ether (pNPGE). Kinetic resolutions showed that eight mutants were obtained with up to 13-fold improved enantioselectivity toward pNPGE and at least three other epoxides. The large enhancements in enantioselectivity toward epichlorohydrin and 1,2-epoxyhexane indicated that pNPGE acts as an epoxyalkane mimic. Active site mutations were found in all shuffled mutants, which can be explained by an interaction of the affected amino acid with the epoxide oxygen or the hydrophobic moiety of the substrate. Several mutations in the shuffled mutants had additive effects.
Asunto(s)
Evolución Molecular Dirigida , Epóxido Hidrolasas/genética , Reacción en Cadena de la Polimerasa/métodos , Rhizobium/genética , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Cartilla de ADN , Epóxido Hidrolasas/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Rhizobium/enzimología , EstereoisomerismoRESUMEN
Biocatalysis has become an established technology for the industrial manufacture of fine chemicals. In recent years, a multitude of chemical companies have embraced biocatalysis for the manufacture of desired stereoisomers, and new or improved methods for the synthesis of enantiomerically pure alpha- and beta-amino acids, amines, amides, peptides, nitriles, alcohols, organic acids and epoxides have emerged. Furthermore, the selectivity and mild operational conditions of biocatalysts are increasingly applied in industry to modify complex target molecules. These recent innovations in the manufacture of industrial fine chemicals using biocatalysis are discussed from an industrial perspective.
Asunto(s)
Aminoácidos/biosíntesis , Catálisis , Industria Química/métodos , Industria Química/tendencias , Enzimas/química , Enzimas/metabolismo , Patentes como Asunto/estadística & datos numéricos , Alcoholes/metabolismo , Aminas/metabolismo , Biotransformación , Carbohidratos/biosíntesis , Industria Química/estadística & datos numéricos , Hidrolasas/metabolismo , Microbiología Industrial/métodos , Microbiología Industrial/estadística & datos numéricos , Microbiología Industrial/tendencias , Oxidorreductasas/biosíntesisRESUMEN
The impact of directed evolution and site-specific mutagenesis on the industrial utility of enzymatic catalysis through the modification of enzyme structure and function is clearly an important area of research in bioprocess engineering. High-throughput screening for novel or improved enzyme activities, both by more efficiently exploring nature's diversity and by creating new diversity in the test tube, allows new bioprocesses to be developed. Similarly, innovations in enzyme technology that address novel ways to apply enzymes in bioprocesses also have an impact on bioprocess engineering. Several recent developments have been made in this latter aspect of bioprocess engineering.
Asunto(s)
Biotecnología/métodos , Enzimas/química , Enzimas/metabolismo , Modelos Químicos , Biotecnología/tendencias , Catálisis , Evolución Molecular Dirigida , Estabilidad de Enzimas , Enzimas/genética , Iones/química , Lipasa/metabolismo , Mutagénesis , Mutagénesis Sitio-Dirigida , Sensibilidad y Especificidad , Estereoisomerismo , Especificidad por Sustrato , Agua/metabolismoRESUMEN
Epoxide hydrolases play an important role in the biodegradation of organic compounds and are potentially useful in enantioselective biocatalysis. An analysis of various genomic databases revealed that about 20% of sequenced organisms contain one or more putative epoxide hydrolase genes. They were found in all domains of life, and many fungi and actinobacteria contain several putative epoxide hydrolase-encoding genes. Multiple sequence alignments of epoxide hydrolases with other known and putative alpha/beta-hydrolase fold enzymes that possess a nucleophilic aspartate revealed that these enzymes can be classified into eight phylogenetic groups that all contain putative epoxide hydrolases. To determine their catalytic activities, 10 putative bacterial epoxide hydrolase genes and 2 known bacterial epoxide hydrolase genes were cloned and overexpressed in Escherichia coli. The production of active enzyme was strongly improved by fusion to the maltose binding protein (MalE), which prevented inclusion body formation and facilitated protein purification. Eight of the 12 fusion proteins were active toward one or more of the 21 epoxides that were tested, and they converted both terminal and nonterminal epoxides. Four of the new epoxide hydrolases showed an uncommon enantiopreference for meso-epoxides and/or terminal aromatic epoxides, which made them suitable for the production of enantiopure (S,S)-diols and (R)-epoxides. The results show that the expression of epoxide hydrolase genes that are detected by analyses of genomic databases is a useful strategy for obtaining new biocatalysts.
Asunto(s)
Bases de Datos Genéticas , Epóxido Hidrolasas , Genoma , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Catálisis , Epóxido Hidrolasas/química , Epóxido Hidrolasas/clasificación , Epóxido Hidrolasas/genética , Epóxido Hidrolasas/metabolismo , Compuestos Epoxi/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Células Eucariotas , Genoma Arqueal , Genoma Bacteriano , Genoma Fúngico , Proteínas de Unión a Maltosa , Modelos Moleculares , Filogenia , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Relación Estructura-ActividadRESUMEN
Aldolases are emerging as powerful and cost efficient tools for the industrial synthesis of chiral molecules. They catalyze enantioselective carbon-carbon bond formations, generating up to two chiral centers under mild reaction conditions. Despite their versatility, narrow substrate ranges and enzyme inactivation under synthesis conditions represented major obstacles for large-scale applications of aldolases. In this study we applied directed evolution to optimize Escherichia coli 2-deoxy-D-ribose 5-phosphate aldolase (DERA) as biocatalyst for the industrial synthesis of (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside. This versatile chiral precursor for vastatin drugs like Lipitor (atorvastatin) is synthesized by DERA in a tandem-aldol reaction from chloroacetaldehyde and two acetaldehyde equivalents. However, E. coli DERA shows low affinity to chloroacetaldehyde and is rapidly inactivated at aldehyde concentrations useful for biocatalysis. Using high-throughput screenings for chloroacetaldehyde resistance and for higher productivity, several improved variants have been identified. By combination of the most beneficial mutations we obtained a tenfold improved variant compared to wild-type DERA with regard to (3R,5S)-6-chloro-2,4,6-trideoxyhexapyranoside synthesis, under industrially relevant conditions.
Asunto(s)
Aldehído-Liasas/química , Aldehído-Liasas/genética , Industria Química/métodos , Evolución Molecular Dirigida/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/químicaRESUMEN
D-phenylglycine (D-Phg) is an important side chain building block for semi-synthetic penicillins and cephalosporins such as ampicillin and cephalexin. To produce d-Phg ultimately from glucose, metabolic engineering was applied. Starting from phenylpyruvate, which is the direct precursor of L-phenylalanine, an artificial D-Phg biosynthesis pathway was created. This three-step route is composed of the enzymes hydroxymandelate synthase (HmaS), hydroxymandelate oxidase (Hmo), and the stereoinverting hydroxyphenylglycine aminotransferase (HpgAT). Together they catalyse the conversion of phenylpyruvate via mandelate and phenylglyoxylate to D-Phg. The corresponding genes were obtained from Amycolatopsis orientalis, Streptomyces coelicolor, and Pseudomonas putida. Combined expression of these activities in E. coli strains optimized for the production of L-phenylalanine resulted in the first completely fermentative production of D-Phg.
Asunto(s)
Escherichia coli/genética , Escherichia coli/metabolismo , Glucosa/metabolismo , Glicina/análogos & derivados , Ingeniería de Proteínas/métodos , Mejoramiento Genético/métodos , Glicina/genética , Glicina/metabolismo , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
An industrially attractive L-specific amidase was purified to homogeneity from Ochrobactrum anthropi NCIMB 40321 wild-type cells. The purified amidase displayed maximum initial activity between pH 6 and 8.5 and was fully stable for at least 1 h up to 60 degrees C. The purified enzyme was strongly inhibited by the metal-chelating compounds EDTA and 1,10-phenanthroline. The activity of the EDTA-treated enzyme could be restored by the addition of Zn2+ (to 80%), Mn2+ (to 400%), and Mg2+ (to 560%). Serine and cysteine protease inhibitors did not influence the purified amidase. This enzyme displayed activity toward a broad range of substrates consisting of alpha-hydrogen- and (bulky) alpha,alpha-disubstituted alpha-amino acid amides, alpha-hydroxy acid amides, and alpha-N-hydroxyamino acid amides. In all cases, only the L-enantiomer was hydrolyzed, resulting in E values of more than 150. Simple aliphatic amides, beta-amino and beta-hydroxy acid amides, and dipeptides were not converted. The gene encoding this L-amidase was cloned via reverse genetics. It encodes a polypeptide of 314 amino acids with a calculated molecular weight of 33,870. Since the native enzyme has a molecular mass of about 66 kDa, it most likely has a homodimeric structure. The deduced amino acid sequence showed homology to a few other stereoselective amidases and the acetamidase/formamidase family of proteins (Pfam FmdA_AmdA). Subcloning of the gene in expression vector pTrc99A enabled efficient heterologous expression in Escherichia coli. Altogether, this amidase has a unique set of properties for application in the fine-chemicals industry.
Asunto(s)
Amidohidrolasas/genética , Amidohidrolasas/metabolismo , Ochrobactrum anthropi/enzimología , Amidohidrolasas/antagonistas & inhibidores , Secuencia de Aminoácidos , Secuencia de Bases , Secuencia de Consenso , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Ochrobactrum anthropi/crecimiento & desarrollo , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Mapeo Restrictivo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Estereoisomerismo , Especificidad por Sustrato , TermodinámicaRESUMEN
Biocatalysis has emerged as an important tool in the industrial synthesis of bulk chemicals, pharmaceutical and agrochemical intermediates, active pharmaceuticals, and food ingredients. However, the number and diversity of the applications are modest, perhaps in part because of perceived or real limitations of biocatalysts, such as limited enzyme availability, substrate scope, and operational stability. Recent scientific breakthroughs in genomics, directed enzyme evolution, and the exploitation of biodiversity should help to overcome these limitations. As a result, we expect many new industrial applications of biocatalysis to be realized, from single-step enzymatic conversions to customized multistep microbial synthesis by means of metabolic pathway engineering.
Asunto(s)
Biotecnología , Catálisis , Industria Química , Enzimas/química , Enzimas/metabolismo , Carbono/química , Química Orgánica , Biología Computacional , Evolución Molecular Dirigida , Ecosistema , Estabilidad de Enzimas , Ingeniería Genética , Cinética , Fenómenos Químicos Orgánicos , Oxidación-Reducción , Ingeniería de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Tecnología FarmacéuticaRESUMEN
Comparative screening of gene expression libraries employing the potent industrial host Pichia pastoris for improving recombinant eukaryotic enzymes by protein engineering was an unsolved task. We simplified the protocol for protein expression by P. pastoris and scaled it down to 0.5-ml cultures. Optimising standard growth conditions and procedures, programmed cell death and necrosis of P. pastoris in microscale cultures were diminished. Uniform cell growth in 96-deep-well plates now allows for high-throughput protein expression and screening for improved enzyme variants. Furthermore, the change from one host for protein engineering to another host for enzyme production becomes dispensable, and this accelerates the protein breeding cycles and makes predictions for large-scale production more accurate.
Asunto(s)
Apoptosis/fisiología , Pichia/metabolismo , Ingeniería de Proteínas/métodos , ADN de Hongos/química , ADN de Hongos/genética , Biblioteca de Genes , Glucosa/metabolismo , Etiquetado Corte-Fin in Situ , Mutagénesis Insercional , Pichia/enzimología , Pichia/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Transformación Genética/fisiologíaRESUMEN
Recombinant Escherichia coli JM101(pSPZ10) cells produce the styrene monooxygenase of Pseudomonas sp. strain VLB120, which catalyzes the oxidation of styrene to (S)-styrene oxide at an enantiomeric excess larger than 99%. This biocatalyst was used to produce 388 g of styrene oxide in a two-liquid phase 30-L fed-batch bioconversion. The average overall volumetric activity was 170 U per liter over a period of more than 10 h, equivalent to mass transfer rates of 10.2 mmoles per liter per hour at a phase ratio of 0.5. At this transfer rate, the biotransformation system appeared to be substrate mass-transfer limited. The reactor had an estimated power input in the order of 5 W. L(-1), which is close to values typically obtained with commercially operating units. The product could be easily purified by fractional distillation to a purity in excess of 97%. The process illustrates the feasibility of recombinant whole cell biotransformations in two-liquid phase systems with toxic substrates and products.
Asunto(s)
Compuestos Epoxi/síntesis química , Escherichia coli/genética , Escherichia coli/metabolismo , Oxigenasas/metabolismo , Estireno/metabolismo , Línea Celular , Compuestos Epoxi/metabolismo , Regulación Bacteriana de la Expresión Génica , Oxigenasas/genética , Proyectos Piloto , Pseudomonas/enzimología , Pseudomonas/genética , Recombinación Genética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , EstereoisomerismoRESUMEN
Shikimic acid is a high valued compound used as a key starting material for the synthesis of the neuramidase inhibitor GS4104, which was developed under the name Tamiflu for treatment of antiviral infections. An excellent alternative to the isolation of shikimic acid from fruits of the Illicium plant is the fermentative production by metabolic engineered microorganisms. Fermentative production of shikimic acid was most successfully carried out by rational designed Escherichia coli strains by blocking the aromatic amino acid pathway after the production of shikimic acid. An alternative is to produce shikimic acid as a result of dephosphorylation of shikimate-3-phosphate. Engineering the uptake of carbon, the regulatory circuits, central metabolism and the common aromatic pathway including shikimic acid import that have all been targeted to effect higher productivities and lower by-product formation are discussed.