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- 1. MSG production Fermentors at Kyowa Akko Corp. The volume of each is 63,420 gallons and the height is about 100 ft tall. Hofu, Japan PRODUCCION DE AMINOACIDOS
- 2. PRINCIPALES USOS 60 % industria alimentaria 30 % suplementacion de dietas animales 10 %. medicina, cosmetica e industria quimica SABORIZANTES CALIDAD DE PRODUCTO SUPLEMENTACION DE GRANOS Y PIENSOS NUEVAS APLICACIONES: Fibras de polialanina FABRICACION DE POLIMEROS Resinas de isociananto de Lys Cueros sinteticos con polimetilglutamato COSMETICOS champues, bronceadores, dentifricos, surfactantes FARMACOS Tirosina como precursor de L- Dopa, bloqueantes de receptores,etc
- 3. VOLUMENES DE PRODUCCION aminoacido 1981 1985 1992 1996 (ton/año) (ton/año) (ton/año) L-Arg 400 1000 L-Asp 250 4000 6000 7,000 L-Cys 200 1000 1,500 L-Glu 80000 340000 600000 1,000,000 Gly 3500 6000 22,000 L-Lys 20000 70000 200000 250,000 DL-Met 120000 250000 350,000 L-Phe 150 3000 6000 8,000 L-Thr 160 200 4,000 Produccion total en el año 2001: 1.8 millones de toneladas Degussa AG ( Germany) Kyowa (Japon) y Ajinomoto (Japon) producen ¾ de la produccion mundial
- 4. METODOS DE PRODUCCION 1- EXTRACCION A PARTIR DE HIDROLIZADOS DE PROTEINA: Se aplica solamente para Cys, Tyr y Leu 2- SINTESIS QUIMICA. Se obtienen mezclas racemicas que luego se resuelven con enzimas de origen microbiano. 3- METODOS MICROBIOLOGICOS a) Fermentacion directa a partir de una fuente de carbono Se aplica a la produccion de ac. Glutamico, Lys, Thr, Trp y la mayoria de los AA empleando mutantes tradicionales o cepas recombinantes
- 5. Resolucion de mezclas racemicas Para Met, Ala y Gly
- 6. ADH o AA DH + NAD(P)H Alfa- cetoacidos + amonio Aminoacido Aspartasa (DE E. COLI) + NAD(P)H Fumarato + amonio Aspartico Fenilalanina deshidrogenasa + NAD(P)H Fenilpiruvato + NH4 Fenilalanina ( Brevibacterium flavum) Conversion de Alfa cetoacidos
- 7. pH 8.5 Obtencion de Hidantoinas y Uso posterior de Hidantoinasa y Carbamoylasa de especies de Arthrobacter
- 8. CEPAS UTILIZADAS EN LA PRODUCCION MICROBIOLOGICA DE AMINOACIDOS •-Cepas salvajes o aisladas de la naturalezaCepas salvajes o aisladas de la naturaleza •-Mutantes auxotroficasMutantes auxotroficas •--Mutantes regulatoriasMutantes regulatorias •--Mutantes combinadasMutantes combinadas •- Cepas obtenidas por manipulacion genetica de mutantes,- Cepas obtenidas por manipulacion genetica de mutantes, amplificando operones que codifican para la ruta metabolica oamplificando operones que codifican para la ruta metabolica o ampliando el rango de sustratos utilizables, etcampliando el rango de sustratos utilizables, etc • -USANDO PLASMIDOS VECTORES -Usando fagos - Usando fusion de protoplastos para combinar cepas con caracteristicas deseables
- 9. PRODUCCION DE AMINOACIDOS CON CEPAS SALVAJESPRODUCCION DE AMINOACIDOS CON CEPAS SALVAJES En 1908 se aislo el Acido Glutamico de un alimento autoctono oriental por lo que se empezo a producir comercialmente a partir de la hidrólisis de proteinas. En 1957 se aisla una cepa microbiana capaz de producir y secretar al medio aprox. 1 g/ L de Ac. Glutamico. Desde entonces se aislaron muchas cepas capaces de acumular mas de 30 g/ L del aminoacido. Generos productores: Corynebacterium, Brevibacterium y Microbacterium. Caracteristicas morfologicas y bioquimicas: Gram positivos, no esporulados, inmoviles, forma de coco o cocobacilo. Pueden emplear glucosa, acetato o etanol como fuentes de carbono. No crecen en metanol pero algunas cepas lo hacen en alcanos.
- 10. Vias glicoliticas relacionadas con la produccion de AA La via de las pentosas es menos activa La glucosa se metaboliza preferentemente por la via de Embden Meyerhoff dando intermediarios C2 y C3. malato carboxilasa piruvato-carboxilasa via del glioxalato biotina glutamato NADPH +NH3 -Para que el α-cetoglutarato se acumule es necesario bloquear el pasaje de α-cetoglutarato a succinato. De esta forma se acumula isocitrato que se deriva a α-cetoglutarato. Los C4 necesarios (malato, oxalacetato ) se obtienen por las 3 vias anapleroticas.
- 11. BIOSINTESIS Y REGULACION DE LA PRODUCCION YBIOSINTESIS Y REGULACION DE LA PRODUCCION Y SECRECION DE ACIDO GLUTAMICO ENSECRECION DE ACIDO GLUTAMICO EN CORYNEBACTERIUMCORYNEBACTERIUM -Las cepas productoras de acido glutamico se caracterizan por : • Su requerimiento de biotina (auxotrofas) • Por poseer una muy baja actividad de la enzima α- ceto glutarato deshidrogenasa. • Algun mecanismo de transporte para la exportacion del AA
- 12. Contribucion de las vias anapleroticas en Corynebacterium
- 13. ASPARTATO TREONINA, METIONINA, LISINA, ETC GLUCOSA PEP OXALACETATO CITRATO αCETO- GLUTAMICO GLUTARATO Para limitar el control feed-back puede aumentarse la secrecion de glutamico al medio modificando la permeabilidad celular. El aumento de permeabilidad se logra por formacion defectuosa de la pared celular mediante el crecimiento a alta temperatura, la inclusión de detergentes, de acidos grasos no saturados o inhibidores de la síntesis de pared celular como penicilina : •PAPEL DE LA BIOTINAPAPEL DE LA BIOTINA Como todos los aminoacidos, el acido glutamico regula su propia biosintesis por control feed-back. La auxotrofia a biotina y el crecimiento en medios deficientes en este cofactor provocan una inhibición de la enzima Acetil- Co A carboxilasa que convierte AcetilCoA + CO2 en malonil CoA, es decir, el primer paso para la síntesis de acidos grasos. Como consecuencia de esta deficiencia los auxotrofos a biotina tienen una marcada alteración en sus membranas y con ello se estimula significativamente la producción y excrecion de glutámico (Hoischen and Kramer, 1989)
- 14. 1- LIMITACION DE LA CANTIDAD DE BIOTINA EN EL MEDIO (PARA CEPAS AUXOTROFAS A BIOTINA) PUES EN CONDICIONES DE EXCESO DE BIOTINA NO SE ACUMULA ACIDO GLUTAMICO, LA FUENTE DE CARBONO SE OXIDA TOTALMENTE A C02 (EL MICROORGANISMO SOLO CRECE). 2- LIMITANDO EL CULTIVO EN ACIDO OLEICO (PARA CEPAS AUXOTROFAS AL ACIDO OLEICO) 3- ADICION DE ACIDOS GRASOS SATURADOS (EJERCEN EFECTOS REGULATORIOS SOBRE LA SINTESIS DE ACIDOS GRASOS SATURADOS E INSATURADOS) Y CON ELLO LA FORMACION DEFECTUOSA DE LA PARED, AUN EN PRESENCIA DE EXCESO DE BIOTINA. 4- ADICION DE PENICILINA O SURFACTANTES QUE PROVOCAN LA FORMACION DE UNA PARED CELULAR DEFICIENTE. 5- CUANDO SE EMPLEAN PARAFINAS COMO UNICA FUENTE DE CARBONO NO PUEDEN EMPLEARSE AUXOTROFOS A BIOTINA U OLEATOS. SOLO AGREGARSE PENICILINA O SURFACTANTES.
- 15. Fuentes de Carbono: Hidrolizados de almidon, melazas de caña o remolacha conteniendo 10-20 % de sacarosa. En Japon se usa Acido acetico. Fuentes de Nitrogeno: Amonio o urea. Se agrega durante el proceso para evitar su derivacion a otros aminoacidos (glutamina, prolina) pH: se mantiene en 8-8.5 por agregado de NH3 Minerales: Fosfatos, Potasio, Sulfatos, Magnesio, Hierro y Manganeso. ♦Evitar el exceso de biotina ♦Limitacion de oxigeno: se acumulan acidos organicos (lactico, succinico) ♦Exceso de NH3: se acumula glutamina PROCESO DE PRODUCCION DE ACIDO GLUTAMICO ♦El uso de penicilina, acidos grasos saturados o tween permite producir glutamico en medios con exceso de biotina (Fuentes de carbono baratas). Tambien el uso de cepas auxotrofas al acido oleico.
- 16. SE EMPLEAN PARA PRODUCIR: 1- INTERMEDIARIOS DE RUTAS BIOSINTETICAS LINEALES A B C D ♦ Para sobreproducir C auxotrofa a D ♦ Para sobreproducir B auxotrofa a C 2- PRODUCTOS FINALES O INTERMEDIARIOS DE RUTAS BIOSINTETICAS RAMIFICADAS ♦ PARA PRODUCIR C , SE UTILIZA UNA CEPA AUXOTROFA A E , G , E +G A B C D F E G ♦ PARA PRODUCIR F, SE UTILIZA UN AUXOTROFO A G, E ♦ PARA PRODUCIR E, SE UTILIZA UN AUXOTROFO A G (o F) PRODUCCION DE AMINOACIDOS CON MUTANTES AUXOTROFAS Ej 1. Produccion de citrulina u ornitina por auxotrofos a Arg GLUTAMATO + N-ACETIL N- ACETIL GLUTAMICO ARGININA SUCCINATO ARGININA CITRULINA ORNITINA N-ACETIL-ORNITINA N- ACETIL GLUTAMIL-P SEMIALDEHIDO N- ACETIL GLUTAMIL-P
- 17. Síntesis de los aminoacidos de la via del aspartato en Corynebacterium ♦RUTA METABOLICA RAMIFICADA CON PRODUCTOS FINALES E INTERMEDIOS DE INTERES: LYS, MET, DAP, HME, THR , ILE. ♦OTRO EJEMPLO ES LA PRODUCCION DE LOS AMINOACIDOS AROMATICOS: TRP, TYR Y PHE-ALA.
- 18. PRODUCCI ON DE AMI NOACI DOS CON MUTANTES REGULATORI AS Las mutantes regulatorias pueden seleccionarse por resistencia a analogos estructurales toxicos o pueden ser revertantes de cepas auxotroficas. Analogo estructural de Lys----------------------------aminoetil cisteina 2HC- C- C- C- C- COOH HS- C- C-COOH NH2 NH2 HN- CH2- CH3 PROCESO DE PRODUCCION DE L- LISINA Cepas: Se utilizan cepas de los mismos generos que para la produccion de acido glutamico pero con mutaciones auxotroficas y/o regulatorias a los fines de hacer el proceso mas selectivo (Thr- , Met- , AEC r , Hme- ) Se experimenta tambien con cepas de ingenieria genetica donde se aumenta el dosaje genetico de la enzima DAP sintetasa. Fuentes de carbono: Fermentacion directa a partir de fuentes de carbono sencillas: Melazas, acetato, etanol. Fuentes de nitrogeno: NH3, urea (si tienen ureasa) , sales de Amonio. Si son auxotrofos, deben agregarse los aminoacidos en cantidades limitantes. La biotina no afecta este proceso. Ejemplo de un medio de cultivo industrial: melaza de caña 20 %, hidrolizado de proteina de soja 1.8 % y amoniaco gaseoso como fuente de N y para mantener el pH (aprox. = 7). Tiempo de fermentacion: 60 Hs. Condiciones del proceso: agitacion 150 rpm; aireacion 0.6 vvm, temperatura 0 RENDIMIENTOS DE PRODUCCION DE LISINA FUENTE DE CARBONO ORGANISMO FENOTIPO Y (g/L) Glucosa C. glutamicum Hme - Leu - 34 Glucosa C. glutamicum Hme- Leu- AECr 39 Glucosa B.lactofermentu m AECr Ala- CCLr MLr 70 Acetato B. flavum Hme- Thr- 75 etanol B.lactofermentu m AECr 66 Treonina amino HO valerico
- 19. EL ANALISIS DE FLUJOS A TRAVES DE TODOS LOS PASOS ENZIMATICOS QUE LLEVAN A LA PRODUCCION DE LYS INDICO QUE UN FACTOR LIMITANTE ESTA EN EL PUNTO DE RAMIFICACION DEL PEP. ADP pyk AA PEP ATP ppck pps CO2 PIRUVATO TCA Ppc pyc ASPARTATO OAA odc LYS decarboxilasa OBJETIVOS: ♦Amplificar ppc ♦Eliminar ppck ♦Eliminar pyk (exceso de ATP)
- 20. Split Pathway with Corynebacterium glutamicum Mutante dap D( sobreexpresion) Wild type
- 21. Export de lisina por la proteina transportadora (Lys E) Tambien actuaria como control intracelular del nivel de Lys y otros aminoacidos
- 22. L- treonina Se emplea una cepa de E. coli manipulada geneticamente. La mutante inicial se selecciono por resistencia a AHV. (Mutante regulatoria) E. coli tiene 3 AK reguladas por Thr e Ile (AKI), Met (AKII) y Lys (AKIII) – (Obtencion de auxotrofos a Met , Lys) E. coli tiene 2 ASD reprimidas por Lys, Thr y Met El operon Thr ABC esta regulado por atenuacion transcripcional Thr deaminasa y Thr deshidrogenasa (ilv A y tdh) son tambien puntos de control (Auxotrofos a Ile)
- 23. Caracteristicas genotipicas y fenotipicas de una Cepa recombinante productora de Treonina (65 g/L) rel A Gen que codifica para el factor ppGpp que aumenta la transcripcion de los genes trna de los AA thr C Mutante con thr C inactivado ilvA Mutante de treonina deaminasa que reduce el nivel de atenuacion del operon thr suc Uso de sacarosa rht 23A Gen relacionado a eflux: Aumenta la excrecion tdh Mutante sin treonina deshidrogenasa: disminuye la degradacion ppc PEP carboxilasa insensible al aspartato : aumenta el flujo de carbonos hacia OAA y Asp pVC40 Plasmido con el operon thr clonado y con una mutacion thrA* (mutacion AKI)
Notas del editor
- Glutamato (caldos, salsas, etc) Aspartato + alanina (jugos de fruta) SABORIZANTES glicina (edulcorantes) Aspartato + fenilalanina (endulzante: Aspartame “Nutrasweet”) Cisteina (panificacion, jugos de fruta = antioxidante) CALIDAD DE PRODUCTO : Triptofano + histidina (leches en polvo= antioxidantes) Fibras de polialanina FABRICACION DE POLIMEROS Resinas de isociananto de Lys Cueros sinteticos con polimetilglutamato COSMETICOS incluyendo champues, bronceadores, dentifricos, surfactantes FARMACOS Tirosina como precursor de L- Dopa, bloqueantes de receptores,etc
- ALGUNOS DATOS ECONOMICOS The biggest producer of methionine is Degussa AG, the Frankfurt (West Germany) chemical and precious metals company, which produced 75,000 tons/year of that essential amino acid, which is used in pharmaceuticals, medical infusion solutions, and special diet foodstuffs. Japan, primarily Ajinomoto and Kyowa Hakko, produces about two-thirds of the world volume of amino acids (Kieslich, 1985). Another significant producer of amino acids is China with a total output of 75,000 tons annually (Han Ying-Shan, 1991). A world wide market survey of amino acid production shows that the synthetic DL-methionine and its hydroxy a nalog used as feed supplements are both produced commercially from petrochemicals, not via fermentation. L-lysine and L-tryptophan, on the other hand, are usually produced by fermentation (Eldib et al., 1985). The Japanese companies Ajinomoto and Bio-Kyowa were the only producers of L-lysine in the early 1970's. Since then, the South Korean firm Miwon, the French company Eurolysine and the Mexican company Fermex have joined the ranks. Currently, no American company produces L-lysine. In fact in the 1960's and early 1970's Du Pont, Monsanto, and Stauffer Chemical Co. were the only large American companies producing any amino acids. Stauffer closed its monosodium glutamate plant in 1983 because of severe competition from foreign suppliers (Eldib et al., 1985). L-lysine, L-tryptophan and L- or DL-methionine are the most common amino acid animal feed supplements, and their world-wide demand is increasing both in the health food industry and in bioresearch. The Japanese companies Ajinomoto, Kyowa Hakko, Tanabe Seiyaku, Mitsui Toatsu, and Showa Denko produce L- or DL-tryptophan on an industrial scale. In Europe tryptophan is produced by Degussa only in semi-commercial quantities. There are no U.S. companies that produce tryptophan (Eldib et al., 1985). In 1983 two Japanese firms announced plans to produce L-lysine at plants in the U.S.A.: Bio-Kyowa, in Cape Giradeau, Missouri (projected yearly production capacity of 15,000 tons) and Ajinomoto, in Eddyville, Iowa, (projected yearly production capacity of 6,000 tons). The current U.S. demand for L-lysine is 24,000 tons/year. American producers sell lysine at the current market price of $1.40/pound ($3.11/kg), and L-tryptophan at almost $9/pound ($20/kg) (Process Biochem., 1985). 468. In a Rohm GmbH pilot process N-acetyl-DL-amino acids are converted to L-amino acids in a packed bed reactor at 37°C by Plexazym AC (see paragraph 459). N-acetyl-DL-methionine, 0.7 M at pH 8, is hydrolyzed 80% at a space velocity of 6 hr-1. The rate of hydrolysis decreases to about 30% when the space velocity is increased to 25 hr-1. A yield of 500 kg L-methionine/kg Plexazym AC is reached in a 90 day reactor run (Plainer & Sprossler, 1982).
- Aspergillus ochraceus produce una aminoacilasa que se inmobiliza en DEAE-Sephadex y que hidroliza selectivamente los N-acil L Aminoacidos obteniendose el L-AA libre. El N-acil D-AA que no es hidrolizado se separa por cristalizacion diferencial. El procedimiento se usa para separar (D-L) Met, Ala, Gly obtenidos por via quimica A basically similar approach has been applied by Snamprogetti (Italy) for batchwise resolution of a racemic mixture of N-acetyl-DL-tryptophan in a small pilot plant (Marconi & Morisi, 1979; Bartoli et al., 1978). Amino acid acylase entrapped in cellulose triacetate fibers was used to produce L-tryptophan and N-acetyl-D-tryptophan. In the pilot process the feed tank contains 9.85 kg acetyl-DL-tryptophan, 23.8 g CoCl2x6H2O, 1.6 kg NaOH (for pH 7.0) and 200 liters water at 45°C. This solution is recycled for 5.5 hr through the enzyme reactor at flow velocity of 350 l/hr (after which the hydrolysis is practically complete). The reactor is 77 by 19 cm with 4 kg of dry fiber, containing 0.28 kg protein/kg dry fiber. The hydrolyzed material is evaporated to 15 liters under vacuum and separated, based on solubility differences, to yield 3.9 kg of L-tryptophan (95% yield) and 15 liters of acetyl D-tryptophan solution. The latter is mixed with 3 l of acetic anhydride and held for 5 hr at 45°C to yield 4.7 kg of racemized and precipitated product (yield 96%). The racemized acetyl-D-tryptophan is recycled to the feed tank. The small pilot plant, having a capacity of about 1 kg of L-tryptophan/hr, has operated for several months. The activity of the aminoacylase fibers decreased by 20% during this period, and their productivity was about 400 kg of L-tryptophan per kilogram of fiber. The same approach was also used for the resolution of N-acetyl derivatives of DL-valine and DL-methionine (Marconi & Morisi, 1979). 466. In 1983 Degussa AG (West Germany) reported a plan to develop a new technology for the production of optically and chemically pure L-amino acids, i.e., arginine, isoleucine, threonine, proline, and tryptophan, by 1985-1988 (Harper, 1983). For the optical resolution of racemates the company uses a membrane reactor fed with the soluble catalyst aminoacylase instead of immobilized enzymes. A Degussa-produced N-acetyl racemic amino acid solution is cycled through the membrane reactor where acylase, produced by Amano Pharmaceutical Co., Ltd. in Nagoya, Japan, produces deacylated L-amino acids. The latter, of lower molecular weight as compared with N-acetyl-D- and N-acetyl-L-amino acids, pass through the reactor's membrane. Because of its high molecular weight, the enzyme is also retained in the vessel (McGraw Hill's Biotechnology Newswatch, 1982c). Separated L-amino acids are collected by ion exchange or crystallization. After racemization, the remaining solution is recycled with more of the original N-acetyl-DL-amino acid substrate. According to a Degussa representative, the continuous-production process with the membrane reactor is more efficient than other methods, including those with carrier-fixed enzymes (McGraw Hill's Biotechnology Newswatch, 1982c). 4.
- OPTICAL RESOLUTION OF AMINO ACIDS BY IMMOBILIZED AMINOACYLASE Chemical synthesis of amino acids generally produces an optically inactive racemic mixture, i.e. both the L- and D-isomers. To obtain natural L-amino acid from the chemically synthesized DL-form, optical resolution is required. Among the many optical resolution methods, the enzymatic method with microbial aminoacylase is one of the most advantageous procedures, yielding optically pure L-amino acids. The enzyme is specific for the L-form and thus a chemically synthesized acyl-DL-amino acid is hydrolyzed asymmetrically by aminoacylase to give L-amino acid and unhydrolyzed acyl-D-amino acid. Both products are separated easily by their differing charges and solubilities. The acyl-D-amino acids are then racemized by heat treatment into a racemic mixture of acyl-D- and acyl-L-amino acids, and reused for the resolution procedure. Since the substrate specificity of mold aminoacylase is broad and attacks acyl-L-amino acids with various side-chains, the enzymatic resolution of racemates can be applied to various amino acids (Chibata, 1980). From 1954 to 1969, this enzymatic resolution method was employed by Tanabe Seiyaku Co. Ltd. using soluble Aspergillus oryzae aminoacylase for the industrial production of several L-amino acids. The enzyme reaction is carried out batchwise. This procedure, however, had some disadvantages inherent to a batch process using soluble enzymes in that in order to isolate L-amino acids from the reaction mixture, it is necessary to remove enzyme protein by pH and/or heat treatments (Chibata, 1978a). This results in uneconomical use of enzyme, lowered yield of L-amino acids, and increased labor. Therefore, as a result of extensive studies of the continuous optical resolution of DL-amino acids using immobilized aminoacylase, the industrial production of L-amino acids was switched to the immobilized enzyme process in 1969 to produce L-methionine. 2. Commercial preparations of immobilized aminoacylase As in the case of immobilized glucose isomerase covalently bonded enzymes are not prevalent among preparations of immobilized aminoacylase intended for industrial application. The best known preparations include aminoacylase immobilized by ionic binding to DEAE-Sephadex (developed by Tanabe Seiyaku Co.), in which the enzyme is trapped by means of fiber wet spinning (developed by Snamprogetti, see also paragraph 408) into fibers of cellulose triacetate as microdroplets of its aqueous solution. Tanabe indicates that the preparation of their immobilized enzyme is easy, the activity is "stable and high", and the regeneration of deteriorated immobilized enzyme preparations is possible. This last point is particularly important since DEAE-Sephadex is a very expensive carrier. The immobilized enzyme is prepared as follows. 1,000 liters of DEAE Sephadex A-25 and 1,100-1,700 liters of aqueous solution of aminoacylase are mixed and stirred at 35°C, pH 7.0, for 10 hr, before filtration and washing with water. The yield of activity compared with that of the initial enzyme preparation is 50-60%. The half-life of the DEAE-Sephadex aminoacylase is equal to 65 days at 50°C, as compared with 48 days at 37°C for aminoacylase entrapped in polyacrylamide gel (Chibata, 1978a). According to Snamprogetti's data (Marconi & Morisi, 1979), the entrapped preparations of aminoacylase from hog kidney and microorganisms show "very good" stability under operating conditions in the course of resolving racemic mixtures of N-acetylmethionine in that the loss of enzyme activity is only 25-30% after 50 days of operation. Rohm GmbH (Darmstadt, FRG) used macroporous beads made of plexiglas-like material to immobilize amino acid acylase. This carrier has a porosity of 3-4 mg/g and the enzyme is bound covalently by oxirane groups. Because of their rigid structure, the 0.1-0.3 mm diameter beads are pressure stable and show "good flow properties". The brand name of the immobilized aminoacylase is Plexazym AC, and 1 g of it is as effective as 0.4 g of the original preparation under operating conditions (Plainer & Sprossler, 1982). Recently Chinese researchers reported the immobilization of aminoacylase from A. oryzae by adsorption on synthetic modified polyacrylamide. "High immobilized enzyme activity and high operational stability" is mentioned regarding the immobilized preparation (loss of activity at 30 min at 70°C is 90% for soluble enzyme, 37% for the immobilized enzyme) (Wang et al., 1992).
- Economic estimations A comparison of the cost for production of L-amino acids by soluble and immobilized amino acid acylase in shown in Figure 8. With the immobilized enzyme, the purification procedure for product become simpler and the yield is higher than in the case of the soluble enzyme. Therefore, less substrate is required for the production of a unit amount of L-amino acid. As the immobilized aminoacylase is stable, the cost of enzyme is reduced markedly compared with that of the soluble enzyme. In the case of immobilized enzyme, the process is controlled automatically, and the labor cost is also reduced substantially. As a result the overall production cost of the immobilized enzyme process is about 60% of that of a conventional batch process using soluble enzyme (Chibata, 1978a). The cost of the enzyme in this process is only approximately 1-2 per cent of the overall operating budget, as estimated in 1984 (Chaplin, 1984). 5. Scale of the processes By 1971 the reported capacity of the Tanabe Seiyaku process (using immobilized aminoacylase) was greater than 700 kg of L-amino acids per day (Suckling, 1977). Immobilized aminoacylase columns usually produce up to 750 kg of product per day (Chaplin, 1984). Based on 1984 data, this enzyme technology can be used to produce over 50,000 tons of L-amino acids annually (Chaplin, 1984). However, it was reported in 1984 that presumably less than 250 tons of L-amino acids is produced by this technology per year, and the estimated immobilized enzyme amount is less than 1 ton/year (Poulsen, 1985). Amano (Japan) also uses immobilized aminoacylase in industry (Poulsen, 1985). USO DE CETOACIDOS La enzima FDH se emplea para la produccion de L- alanina, L-leucina y L- fenilalanina (mas importante ). Requiere NADH como cofactor por lo que se prefieren las celulas inmovilizadas en poliacrilamida o carragenanos para evitar su adicion. O un sistema de regenracion de cofactor reducido El sistema puede operar hasta 680 dias a 37 0 C (pH 8.5) Another process developed by Degussa became the first industrial application of a two-enzyme system with regeneration of a cofactor and the production of some L-amino acids from cheap keto acids. The regeneration of cofactors like NAD or ATP, which dissociate from their apoenzymes, is a serious problem in enzyme engineering, since dissociating coenzymes must retain a degree of mobility in order to have access to the active centers of the two apoenzymes. The alternative, i.e. continuous replacement of the cofactor, would be very expensive. In the Degussa process NAD+ is coupled to polyethylene glycol and retained in the membrane reactor with keto acid and formate dehydrogenase (Hartmeier, 1985).
- Recently, Tanabe Seiyaku announced a new approach for the continuous production of L-alanine from L-aspartic acid developed by the company (Chibata, 1982). That approach used immobilized Pseudomonas dacunhae cells with high L-aspartate beta-decarboxylase activity. The decarboxylase enzyme shows high enantiomer selectivity reacting only with L-aspartic acid. Thus, L-alanine and D-aspartic acid can be produced simultaneously from D,L-aspartic acid. D-Aspartic acid is used as an important intermediate for semisynthetic penicillin. However, this continuous decarboxylase system generates problems associated with evolution of CO2 gas during the reaction. It was difficult both to maintain the plug flow of the substrate solution under normal pressure and to keep a constant pH of the reaction mixture in the reactor because of CO2 effervescence. The company therefore designed a closed column reactor that performs the enzyme reaction at an elevated pressure of 10 kg/cm2. Since liberated CO2 gas is mixed into the reaction mixture, the complete plug-flow of the substrate solution is maintained and the pH of the reaction mixture is not changed appreciably. Moreover, the efficiency of immobilized cells to produce L-alanine in the closed column system at high pressure increases by 1.5 times (from 250 to 360 mmole/l/hr) as compared with the efficiency at the normal pressure conditions; the stability of the immobilized cells is not affected by pressure elevation (Chibata, 1982). Tanabe Seiyaku established a continuous production process for L-alanine in 1982 and succeeded in making it a functioning commercial enterprise. In 1981 Degussa installed an experimental 5 ton/year plant in Konstanz, West Germany, producing L-alanine, -methionine, -valine, -phenylalanine, and -tryptophan. In 1982 the production volume of the plant increased to 60 tons/year as a result of a new method of separating amino acids from protein hydrolysates by means of ion exchangers. The company planned to install a new 6,000 tons/year plant for L-lysine production by fermentation method at Valencia de Don Juan, Spain in 1984 (Harper, 1983; McGraw Hill's Biotechnology Newswatch, 1982c), and produced 10-15 tons per month of L-methionine, L-valine and L-phenylalanine by means of the two-enzyme system with cofactor regeneration (Hartmeier, 1985) (see also par. 467). Aldehido + (NH4)CO3+ KCN Ejemplo: produccion de D aspartico para sintesis de penicilinas