RESUMO
The legumain-like cysteine proteinase TvLEGU-1 from Trichomonas vaginalis plays a major role in trichomonal cytoadherence. However, its structure-function characterization has been limited by the lack of a reliable recombinant expression platform to produce this protein in its native folded conformation. TvLEGU-1 has been expressed in Escherichia coli as inclusion bodies and all efforts to refold it have failed. Here, we describe the expression of the synthetic codon-optimized tvlegu-1 (tvlegu-1-opt) gene in Pichia pastoris strain X-33 (Mut+) under the inducible AOX1 promoter. The active TvLEGU-1 recombinant protein (rTvLEGU-1) was secreted into the medium when tvlegu-1-opt was fused to the Aspergillus niger alpha-amylase signal peptide. The rTvLEGU-1 secretion was influenced by the gene copy number and induction temperature. Data indicate that increasing tvlegu-1-opt gene copy number was detrimental for heterologous expression of the enzymatically active TvLEGU-1. Indeed, expression of TvLEGU-1 had a greater impact on cell viability for those clones with 26 or 29 gene copy number, and cell lysis was observed when the induction was carried out at 30 °C. The enzyme activity in the medium was higher when the induction was carried out at 16 °C and in P. pastoris clones with lower gene copy number. The results presented here suggest that both copy number and induction temperature affect the rTvLEGU-1 expression in its native-like and active conformation.
Assuntos
Cisteína Proteases , Expressão Gênica , Pichia/metabolismo , Proteínas de Protozoários , Proteínas Recombinantes de Fusão , Trichomonas vaginalis/genética , Aspergillus niger/enzimologia , Aspergillus niger/genética , Cisteína Proteases/biossíntese , Cisteína Proteases/química , Cisteína Proteases/genética , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Pichia/genética , Sinais Direcionadores de Proteínas/genética , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Trichomonas vaginalis/enzimologia , alfa-Amilases/biossíntese , alfa-Amilases/química , alfa-Amilases/genéticaRESUMO
Alpha-amylases are ubiquitously distributed throughout microbials, plants and animals. It is widely accepted that omnivorous crustaceans have higher α-amylase activity and number of isoforms than carnivorous, but contradictory results have been obtained in some species, and carnivorous crustaceans have been less studied. In addition, the physiological meaning of α-amylase polymorphism in crustaceans is not well understood. In this work we studied α-amylase in a carnivorous lobster at the gene, transcript, and protein levels. It was showed that α-amylase isoenzyme composition (i.e., phenotype) in lobster determines carbohydrate digestion efficiency. Most frequent α-amylase phenotype has the lowest digestion efficiency, suggesting this is a favoured trait. We revealed that gene and intron loss have occurred in lobster α-amylase, thus lobsters express a single 1830 bp cDNA encoding a highly conserved protein with 513 amino acids. This protein gives rise to two isoenzymes in some individuals by glycosylation but not by limited proteolysis. Only the glycosylated isoenzyme could be purified by chromatography, with biochemical features similar to other animal amylases. High carbohydrate content in diet down-regulates α-amylase gene expression in lobster. However, high α-amylase activity occurs in lobster gastric juice irrespective of diet and was proposed to function as an early sensor of the carbohydrate content of diet to regulate further gene expression. We concluded that gene/isoenzyme simplicity, post-translational modifications and low Km, coupled with a tight regulation of gene expression, have arose during evolution of α-amylase in the carnivorous lobster to control excessive carbohydrate digestion in the presence of an active α-amylase.
Assuntos
Proteínas de Artrópodes , Carnivoridade/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Palinuridae , alfa-Amilases , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/genética , DNA Complementar/genética , DNA Complementar/metabolismo , Glicosilação , Isoenzimas/biossíntese , Isoenzimas/genética , Palinuridae/genética , Palinuridae/metabolismo , Proteólise , alfa-Amilases/biossíntese , alfa-Amilases/genéticaRESUMO
The identification of genes related to heat tolerance is fundamental for the development of high-quality seeds that are tolerant to heat stress condition. The objective of this study was to evaluate maize lineages and the gene expression involved in high temperature tolerance during germination using physiological tests, proteomics, and transcriptome analysis. Seeds from six maize lineages (30, 44, 54, 63, 64, and 91) with different levels of tolerance to high temperatures were used. Lineages 54 and 91 were observed to be more tolerant to high temperature conditions. The highest expression of α-amylase was observed in maize seeds from lineages 30 and 91 that were subjected to controlled deterioration. The highest expression of α-amylase was observed in maize seeds from lineages 30 and 91 that were subjected to controlled deterioration; with the controlled deterioration, the highest level of gene expression did not occur in the most tolerant materials; the association of lower expression of genes involved in heat-resistant protein systems was observed in seeds from lineage 44, which were more susceptible to high temperatures, and the highest gene expression of LEA D-34, ZmAN13, and AOX-1 was observed in seeds from lineage 64 when submitted to controlled deterioration.
Assuntos
Germinação/genética , Sementes/genética , Estresse Fisiológico/genética , Zea mays/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Sementes/fisiologia , Temperatura , Zea mays/fisiologia , alfa-Amilases/biossínteseRESUMO
Crude glycerol, also known as glycerin, is the main byproduct of the biodiesel industry. It has been estimated that up to 40,000 tons of glycerin will be produced each year by 2020. This study evaluated the value-added use of crude glycerol derived from soybean biodiesel preparation as a carbon source for heterologous protein production using the yeast Pichia pastoris. Eleven glycerin samples were obtained by methanolysis of soybean oil using different acids or bases as catalysts. Cell growth experiments showed that crude glycerol containing either potassium or sodium hydroxide resulted in 1.5-2 times higher final cell densities when compared to glycerol P.A. Finally, crude glycerol containing sodium hydroxide was successfully utilized for constitutive heterologous α-amylase production in P. pastoris. This study demonstrated that crude glycerol without any purification steps may be directly used as carbon source for protein production in P. pastoris.
Assuntos
Biocombustíveis , Carbono/farmacologia , Glicerol/farmacologia , Pichia/metabolismo , Óleo de Soja/química , alfa-Amilases/biossíntese , Aerobiose/efeitos dos fármacos , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/enzimologia , Catálise/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Fermentação/efeitos dos fármacos , Metanol/farmacologia , Pichia/efeitos dos fármacos , Pichia/crescimento & desenvolvimentoRESUMO
A yeast isolate able to produce high levels of extracellular α-amylase was selected from a collection of 385 yeasts and identified as Wickerhamia sp. by the sequence of the D1/D2 domain of the 26 S rDNA gene. Part of the nucleotide sequence of the amy1-W gene was cloned, and a sequence of 191 amino acids deduced from this gene was analyzed. The peptide contains three characteristic well-conserved regions in the active sites of α-amylases (EC 3.2.1.1). The enzyme was purified and in situ activity showed only one band with amylolytic activity. The molecular mass of the α-amylase was estimated at 54 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Enzymatic activity on soluble starch as substrate was optimal at pH 5-6 and 50 °C. This thermostable enzyme was inhibited by EDTA-Na(2) and 1,10-phenanthroline; the activity of the dialyzed enzyme was reactivated with Ca(2+) and Mg(2+) cations, which indicates that the α-amylase is a metalloenzyme. α-Amylase production was induced by starch and maltose and repressed by glucose. The high yield and productivity found in this work makes this Wickerhamia sp. strain a promising candidate for the biotechnological production of α-amylase.
Assuntos
Ascomicetos/enzimologia , Espaço Extracelular/enzimologia , alfa-Amilases/biossíntese , Sequência de Aminoácidos , Ascomicetos/citologia , Ascomicetos/genética , Ascomicetos/crescimento & desenvolvimento , Reatores Biológicos/microbiologia , Genes Fúngicos/genética , Cinética , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Especificidade por Substrato , alfa-Amilases/químicaRESUMO
Larvae of Zabrotes subfasciatus secrete alpha-amylases that are insensitive to the alpha-amylase inhibitor found in seeds of Phaseolus vulgaris. By analyzing amylase activities during larval development on P. vulgaris, we detected activity of the constitutive amylase and the two inducible amylase isoforms at all stages. When larvae were transferred from the non alpha-amylase inhibitor containing seeds of Vigna unguiculata to P. vulgaris, the inducible alpha-amylases were expressed at the same level as in control larvae fed on P. vulgaris. Interestingly, when larvae were transferred from seeds of P. vulgaris to those of V. unguiculata, inducible alpha-amylases continued to be expressed at a level similar to that found in control larvae fed P. vulgaris continuously. When 10-day-old larvae were removed from seeds of V. unguiculata and transferred into capsules containing flour of P. vulgaris cotyledons, and thus maintained until completing 17 days (age when the larvae stopped feeding), we could detect higher activity of the inducible alpha-amylases. However, when larvae of the same age were transferred from P. vulgaris into capsules containing flour of V. unguiculata, the inducible alpha-amylases remained up-regulated. These results suggest that the larvae of Z. subfasciatus have the ability to induce insensitive amylases early in their development. A short period of feeding on P. vulgaris cotyledon flour was sufficient to irreversibly induce the inducible alpha-amylase isoforms. Incubations of brush border membrane vesicles with the alpha-amylase inhibitor 1 from P. vulgaris suggest that the inhibitor is recognized by putative receptors found in the midgut microvillar membranes.
Assuntos
Proteínas de Insetos/biossíntese , Lectinas de Plantas/farmacologia , Gorgulhos/enzimologia , alfa-Amilases/biossíntese , Animais , Indução Enzimática , Fabaceae/metabolismo , Comportamento Alimentar , Proteínas de Insetos/antagonistas & inibidores , Isoenzimas/biossíntese , Larva/enzimologia , Larva/crescimento & desenvolvimento , Larva/fisiologia , Phaseolus/metabolismo , Gorgulhos/efeitos dos fármacos , Gorgulhos/fisiologia , alfa-Amilases/antagonistas & inibidoresRESUMO
Nitric oxide (NO) is a freely diffusible, gaseous free radical and an important signaling molecule in animals. In plants, NO influences aspects of growth and development, and can affect plant responses to stress. In some cases, the effects of NO are the result of its interaction with reactive oxygen species (ROS). These interactions can be cytotoxic or protective. Because gibberellin (GA)-induced programmed cell death (PCD) in barley (Hordeum vulgare cv Himalaya) aleurone layers is mediated by ROS, we examined the effects of NO donors on PCD and ROS-metabolizing enzymes in this system. NO donors delay PCD in layers treated with GA, but do not inhibit metabolism in general, or the GA-induced synthesis and secretion of alpha-amylase. alpha-Amylase secretion is stimulated slightly by NO donors. The effects of NO donors are specific for NO, because they can be blocked completely by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The antioxidant butylated hydroxy toluene also slowed PCD, and these data support our hypothesis that NO is a protective antioxidant in aleurone cells. The amounts of CAT and SOD, two enzymes that metabolize ROS, are greatly reduced in aleurone layers treated with GA. Treatment with GA in the presence of NO donors delays the loss of CAT and SOD. We speculate that NO may be an endogenous modulator of PCD in barley aleurone cells.
Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Hordeum/fisiologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/metabolismo , Penicilamina/análogos & derivados , Sementes/fisiologia , Ácido Abscísico/metabolismo , Ácido Abscísico/farmacologia , Antioxidantes/metabolismo , Benzoatos/farmacologia , Hidroxitolueno Butilado/farmacologia , Catalase/biossíntese , Catalase/genética , Sobrevivência Celular/efeitos dos fármacos , Corantes Fluorescentes , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Giberelinas/metabolismo , Giberelinas/farmacologia , Hordeum/efeitos dos fármacos , Hordeum/genética , Imidazóis/farmacologia , Doadores de Óxido Nítrico/metabolismo , Nitroprussiato/metabolismo , Nitroprussiato/farmacologia , Consumo de Oxigênio/efeitos dos fármacos , Penicilamina/metabolismo , Penicilamina/farmacologia , Protoplastos/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sementes/efeitos dos fármacos , Sementes/genética , Superóxido Dismutase/biossíntese , Superóxido Dismutase/genética , alfa-Amilases/biossíntese , alfa-Amilases/genéticaRESUMO
Thermostable amylolytic enzymes have been currently investigated to improve industrial processes of starch degradation. Studies on production of alpha-amylase by Nocardiopsis sp., an endophytic actinomycete isolated from yam bean (Pachyrhizus erosus L. Urban), showed that higher enzyme levels were obtained at the end of the logarithmic growth phase after incubation for 72 h at pH 8.6. Maximum activity of alpha-amylase was obtained at pH 5.0 and 70 degrees C. The isolated enzyme exhibited thermostable properties as indicated by retention of 100% of residual activity at 70 degrees C, and 50% of residual activity at 90 degrees C for 10 min. Extracellular enzyme from Nocardiopsis sp. was purified by fractional precipitation with ammonium sulphate. After 60% saturation produced 1130 U mg-1 protein and yield was 28% with purification 2.7-fold. The enzyme produced by Nocardiopsis sp. has potential for industrial applications.
Assuntos
Actinomycetales/enzimologia , Fabaceae/microbiologia , Plantas Medicinais , alfa-Amilases/biossíntese , Actinomycetales/isolamento & purificação , Biodegradação Ambiental , Biomassa , Reatores Biológicos , Biotecnologia , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Amido/metabolismo , Temperatura , alfa-Amilases/isolamento & purificação , alfa-Amilases/metabolismoRESUMO
In this paper the influence of the amaranth seed meal and the aeration conditions on the alpha-amylase production by Aspergillus niger NRRL 3112 were studied. The assays of selection of culture medium were carried out in a rotary shaker at 250 rpm and 2.5 cm stroke. The aeration conditions were studied in a mechanically stirred fermentor New Brunswick type. A concentration of alpha-amylase of 2750 U.Dun/ml was achieved at 120 h with a dry weight of 8.0 g/l, using a base medium with 5.0 g/l Amaranthus cruentus seed meal. In the experiment performed in a New Brunswick fermentor, the highest value was 2806 U.Dun/ml. This result was obtained after 120 h, operating at 300 rpm and an airflow of 1 l/l. min. in a limited dissolved oxygen concentration. It was determined that the increase in the agitation rate was not favorable to the enzyme production, despite that an increase was verified in the dissolved oxygen. The morphology of the microorganism, in long and ramified hyphae, was the critical factor to obtain higher levels of alpha-amylase.(AU)
Assuntos
Corante Amaranto/farmacologia , Aspergillus niger/efeitos dos fármacos , Meios de Cultura/química , Corantes/farmacologia , Proteínas Fúngicas/biossíntese , Indicadores e Reagentes/farmacologia , alfa-Amilases/biossíntese , Aspergillus niger/enzimologia , Aspergillus niger/crescimento & desenvolvimento , FermentaçãoRESUMO
In this paper the influence of the amaranth seed meal and the aeration conditions on the alpha-amylase production by Aspergillus niger NRRL 3112 were studied. The assays of selection of culture medium were carried out in a rotary shaker at 250 rpm and 2.5 cm stroke. The aeration conditions were studied in a mechanically stirred fermentor New Brunswick type. A concentration of alpha-amylase of 2750 U.Dun/ml was achieved at 120 h with a dry weight of 8.0 g/l, using a base medium with 5.0 g/l Amaranthus cruentus seed meal. In the experiment performed in a New Brunswick fermentor, the highest value was 2806 U.Dun/ml. This result was obtained after 120 h, operating at 300 rpm and an airflow of 1 l/l. min. in a limited dissolved oxygen concentration. It was determined that the increase in the agitation rate was not favorable to the enzyme production, despite that an increase was verified in the dissolved oxygen. The morphology of the microorganism, in long and ramified hyphae, was the critical factor to obtain higher levels of alpha-amylase.
Assuntos
Corante Amaranto/farmacologia , Aspergillus niger/efeitos dos fármacos , Corantes/farmacologia , Meios de Cultura/química , Proteínas Fúngicas/biossíntese , Indicadores e Reagentes/farmacologia , alfa-Amilases/biossíntese , Aspergillus niger/enzimologia , Aspergillus niger/crescimento & desenvolvimento , FermentaçãoRESUMO
A strain of Aspergillus tamarii, a filamentous fungus isolated from soil, was able to produce both Ó-amylase and glucoamylase activities in mineral media supplemented with 1(per cent) (w/v) starch or maltose as the carbon source. Static cultivation led to significantly higher yields than those obtained using shaking culture. The production of amylases was tolerant to a wide range of initial culture pH values (from 4 to 10) and temperature (from 25 to 42degree C). Two amylases, once Ó-amylase and one glucoamylase, were separated by ion exchange chromatography. Both partially purified enzymes had optimal activities at pH values between 4.5 and 6.0 and were stable under acid conditions (pH 4.0-7.0). The enzymes exhibited optimal activities at temperatures between 50(degree) and 60(degree) C and wete stable for more than ten hours at 55(degree) C.
Assuntos
Aspergillus/metabolismo , Glucana 1,4-alfa-Glucosidase/biossíntese , alfa-Amilases/biossíntese , Aspergillus/enzimologia , Glucana 1,4-alfa-Glucosidase/química , alfa-Amilases/químicaRESUMO
The influence of aeration and automatic pH control on the production of alpha-amylase by a strain of "Bacillus subtilis" NRRL 3411 from acid cheese whey was studied. Tests were carried out in a rotary shaker and in mechanically stirred ferments. Alpha-maylase was analysed according to DUN's method. Oxygen absorption rate was determined by Cooper's method. Cell oxygen demand was determined as oxygen consumption in a Warburg respirometer. The level of dissolved oxygen was mesured by means of a galvanic silver-lead electrode. Results suggest the possibility of industrial use of acid cheese whey as a carbon source for alpha-amylase production, since the yiels was similar to that produced with lactose. The highest alpha-amylase levels 100,000 DUN/ml units were not attained at highr aeration rates -431 mLO2/L.h-. The indicated value correspond to a 96 h process with automatic pH enzyme production was directly related to growth in the form of cell aggregates.
Assuntos
Bacillus subtilis/metabolismo , Queijo/microbiologia , alfa-Amilases/biossíntese , Bacillus subtilis/enzimologia , Meios de Cultura Livres de Soro/análise , Consumo de OxigênioRESUMO
Amylase production by a strain of Aureobasidium pullulans isolated in the laboratory was evaluated in liquid media (complex and synthetic) and in solid medium (wheat bran). There was an inhibitory effect in amylase production or amylase secretion by glucose. Asparagine was the best nitrogen source for amylase production (4-6 g/l). Only chlamidospores and melanin but not, amylase activity, were obtained with ammonium sulfate. Amylase production in solid culture was higher than the production obtained in the liquid media assayed. Optimum initial moisture content in solid culture ranged between 57 and 74%. No difference was observed in amylase production between solid media inoculated with cells grown in liquid or solid media.
Assuntos
Proteínas Fúngicas/biossíntese , Glucana 1,4-alfa-Glucosidase/biossíntese , Fungos Mitospóricos/enzimologia , alfa-Amilases/biossíntese , Meios de Cultura/farmacologia , Indução Enzimática , Fungos Mitospóricos/efeitos dos fármacos , Fungos Mitospóricos/crescimento & desenvolvimentoRESUMO
We report here a methodology that allows the identification of glycosylation sites by a combination of protein enzymatic digestion, glycopeptide separation on a reverse-phase HPLC column, and further recognition in a dot-blot system using concanavalin A-horseradish peroxidase. Wheat germ agglutinin-horseradish peroxidase is used as the recognition system for peptides generated after proteolytic digestion of endoglycosidase H deglycosylated protein. Glycosylation sites were confirmed by automatic Edman degradation and fast atom bombardment mass spectrometry. This methodology was applied to a model glycoprotein, alpha-amylase from Bacillus licheniformis, which is unglycosylated in its natural host and appears highly glycosylated when expressed in the methylotrophic yeast Pichia pastoris.
Assuntos
Concanavalina A , Peroxidase do Rábano Silvestre , Processamento de Proteína Pós-Traducional , Conjugado Aglutinina do Germe de Trigo-Peroxidase do Rábano Silvestre , alfa-Amilases/genética , Sequência de Aminoácidos , Bacillus , Sequência de Carboidratos , Glicosilação , Hexosaminidases , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Saccharomyces cerevisiae , alfa-Amilases/biossínteseRESUMO
The stabilization locus parB was subcloned into the broad host range plasmid pAP2, which contains the alpha-amylase gene from Bacillus subtilis, and introduced into Xanthomonas campestris pv campestris and X.c.pv manihotis. Analysis of the stability of plasmid pAP2 (parB-) and pAP23 (parB+) showed that the parB locus decreased significantly the plasmid loss rate mainly by X.c.pv campestris. The lower efficiency of stabilization in X.c.pv manihotis was probably due to the incompatibility system between the native plasmids and the newly introduced pAP23. Although parB had conferred higher stability, it determined a lower rate of alpha-amylase activity even by the strain Cm where its stabilization rate was higher.
Assuntos
Expressão Gênica/genética , Plasmídeos/genética , Xanthomonas campestris/genética , Alelos , Clonagem Molecular , Expressão Gênica/fisiologia , Xanthomonas campestris/crescimento & desenvolvimento , alfa-Amilases/biossínteseRESUMO
Se estudió la producción de alfa-amilasa empleando una cepa de Bacillus subtilis NRRL 3411. Las variables estudiadas incluyeron shock término, concentración y naturaleza de la fuente de carbono, nitrógeno y factores. Además se realizaron experiencias sobre estabilización de los caldos enzimáticos libres de células. Se determinó que utilizando esporos sometidos a un shock térmico de 100-C de 10 min, en un medio con lactosa, caseína, extracto de levadura y minerales, se alcanzaron valores de 140 unidades de alfa-amilasa en 72 h de proceso. También se demostró que el agregado de 20% de glicerol y 1% de benzoato de sodio aseguran caldos estabilizados a 20-C, por un período de 30 días
Assuntos
alfa-Amilases/isolamento & purificação , Bacillus subtilis/enzimologia , Proteínas de Bactérias/isolamento & purificação , alfa-Amilases/biossíntese , Bacillus subtilis/efeitos dos fármacos , Carbono/metabolismo , Meios de Cultura/farmacologia , Indução Enzimática , Temperatura Alta , Nitrogênio/metabolismo , Proteínas de Bactérias/biossíntese , Esporos BacterianosRESUMO
Se estudió la producción de alfa-amilasa empleando una cepa de Bacillus subtilis NRRL 3411. Las variables estudiadas incluyeron shock término, concentración y naturaleza de la fuente de carbono, nitrógeno y factores. Además se realizaron experiencias sobre estabilización de los caldos enzimáticos libres de células. Se determinó que utilizando esporos sometidos a un shock térmico de 100-C de 10 min, en un medio con lactosa, caseína, extracto de levadura y minerales, se alcanzaron valores de 140 unidades de alfa-amilasa en 72 h de proceso. También se demostró que el agregado de 20% de glicerol y 1% de benzoato de sodio aseguran caldos estabilizados a 20-C, por un período de 30 días (AU)
Assuntos
Estudo Comparativo , alfa-Amilases/isolamento & purificação , Bacillus subtilis/enzimologia , Proteínas de Bactérias/isolamento & purificação , alfa-Amilases/biossíntese , Bacillus subtilis/efeitos dos fármacos , Proteínas de Bactérias/biossíntese , Carbono/metabolismo , Meios de Cultura/farmacologia , Indução Enzimática , Temperatura Alta , Nitrogênio/metabolismo , Esporos BacterianosRESUMO
We studied alpha-amylase production by Bacillus subtilis strain NRRL 3411, the effect of heat shock, concentration and nature of the growth factors, carbon and nitrogen source. We also considered the stability of the cell-free enzymatic culture by using spores heat shocked at 100 degrees C for 10 minutes in media containing lactose, casein, yeast extract and minerals. We achieved a final value of 1400 units of alpha-amylase after 72 h of process. It was also found that by adding 20% glycerol and 1% sodium benzoate, cultures were stable at 20 degrees C for 30 days.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/isolamento & purificação , alfa-Amilases/isolamento & purificação , Bacillus subtilis/efeitos dos fármacos , Proteínas de Bactérias/biossíntese , Carbono/metabolismo , Meios de Cultura/farmacologia , Indução Enzimática , Temperatura Alta , Nitrogênio/metabolismo , Esporos Bacterianos , alfa-Amilases/biossínteseRESUMO
Foram estudadas algumas características morfológicas e fisiológicas do B. amyloliquefaciens ATCC 23842 visando ao melhoramento genético da linhagem para o aumento da produçäo de alfa-amilase. A linhagem mostrou bom crescimento em meio ágar nutriente, apresentando tempo de geraçäo de 49,2 minutos. Neste meio, o Bacillus apresentou menor secreçäo de muco e cadeia contendo de 4 a 6 células, independentemente da fase de crescimento. Em meio mínimo, contendo amido solúvel como substrato, a produçäo de enzima iniciou-se após quatro horas de incubaçäo, coincidindo com a fase logarítmica de crescimento. De 8 a 12 horas de incubaçäo observou-se um aumento na concentraçäo da enzima enquanto a concentraçäo de açúcares redutores permanecu constante. Verificou-se que a luz ultravioleta näo se mostrou adequada para a obtençäo de mutantes com maior capacidade de síntese da enzima, provavelmente em virtude das características morfológicas apresentadas pela linhagem, que dificultaram uma absorçäo uniforme da radiaçäo. Estudo realizados para detecçäo de plasmídos, por meio de eletroforese em gel de agarose a 1% revelaram a ausência desses elementos
Assuntos
alfa-Amilases/biossíntese , Bacillus/enzimologia , Bacillus/genética , Meios de Cultura , Eletroforese em Gel de Ágar , Indução Enzimática , Mutagênese/efeitos da radiação , Raios UltravioletaRESUMO
Utilizando-se Bacillus amyloliquefaciens ATCC 2342 e o B. natto, isolado do produto comercial "natto", respectivamente, bons produtores de alfa-amilase e proteases, conseguiu-se aumentar os níveis de produçäo destas enzimas através das técnicas de DNA transformante e fusäo de protoplastos. O melhoramento genético por transformaçäo foi mais eficiente do que aquele por fusäo de protoplastos