Purification and characterization of a milk clotting protease from Mucor bacilliformis.

An acid protease having milk clotting activity has been isolated from Mucor bacilliformis cultures. The enzyme was basically purified by ionic exchange chromatography. An average yield of 29 mg purified product was obtained from 100 mL crude extract. As purity criteria, SDS-PAGE, reverse-phase HPLC, and N-terminal analysis were performed. The protease is a protein composed of a single polypeptide chain with glycine at the N-terminus. The mol wt is approx 32,000, and its amino acid composition is very similar to those of other fungal proteases. As expected, its clotting activity was drastically inhibited by pepstatin A action. On the other hand, its instability against heat treatment and its clotting/proteolytic activity ratio indicate that it may be considered as a potential substitute for bovine chymosin.

[Selection of bacterial strains for the production of threonine]. / Selección de cepas bacterianas para la producción de treonina.

The production of L-threonine in submerged culture was studied in the following bacterial strains; Brevibacterium flavum ATCC 21269, Corynebacterium acetoacidophilum ATCC 21270, Escherichia coli ATCC 21149, E. coli NRRL 12098, E. coli NRRL 12099 and E. coli NRRL 12100. Erlenmeyer flasks with different volumetric relations liquid/recipient, were used to study the influence of the volumetric oxygen transfer rate. B. flavum reached levels of threonine of 0.72 g/l at 96 hours of culture with a volumetric relation liquid/recipient of 1:5. With a relation 1:25 the maximal level was reached at 48 hours (0.60 g/l of threonine) (Table 1). In addition to threonine this strain accumulated in the culture media glutamic acid (+/- 2 g/l), alanine or glycine and proline. With E. coli ATCC 21149 the aeration favored the production of threonine reaching levels of 0.38 g/l in six day cultures with valine and alanine at levels approximate to 2 g/l. Excepting C. acetoacidophilum, all the strains produced threonine at levels of 0.30 to 3.55 g/l (Table 5). E. coli NRRL 12098 and E. coli NRRL 12100 produced only threonine, the culture medium being free from other aminoacids. With E. coli NRRL 12098 levels of 2 g/l were attained but the production was restricted to the presence of yeast extract in the media (Table 2). E. coli NRRL 12100 was the best strain and the inoculum media influenced the production (Table 3) and the best threonine levels were reached in the best aeration conditions assayed (Table 4) with 3.55 g/l for volumetric relations 1:10 and 1.25 g/l for 1:5.

[Bacterial flora of the digestive tract of specimens of Notothenia neglecta caught in Caleta Potter (South Shetland Archipelago, Antarctica)]. / Flora bacteriana del tracto digestivo de especímenes de Notothenia neglecta pescados en Caleta Potter (Arch. Shetland del Sur, Antártida).

A qualitative and quantitative study of the predominant heterotrophic bacterial flora in the stomach and intestine of the Antarctic fish Notothenia neglecta was carried out: 10 newly caught specimens (Potter cove, King George Island, South Shetland Islands) were analyzed. The cultures were made under aerobic and anaerobic conditions. The stomach flora showed variable results between samples which are probably related to the flora ingested with food. The gut flora was composed almost exclusively of Vibrio spp. These results are in agreement with those attributing to Vibrio the nature of indigenous flora of the intestine of marine teleosts and show that this flora had not changed, not even during the adaptation of these fish to the extreme Antarctic environmental conditions.

[Changes in the bacteriological, chemical and organoleptic characteristics of the Antartic krill (Euphausia superba) during storage at 0-2 degrees C]. / Cambios en las características bacteriológicas, Químics y organolepticas del krill antartico (Euphausia superba) durante el almacenamiento a 0-2 degrees C.

A study was performed on the bacteriological, chemical and organoleptic characteristics of antartic krill (Euphausia superba) stored at 0-2 degrees C. After 6-8 hours of storage a dark color started in the head and legs and spread slowly to the tail. Within 24 hours 17% of the total nitrogen was lost by hepatopancreas autolisis. After 72 hours the krill became inedible due to strong amoniacal odor and flavor. These changes were associated with the multiplication of aerobic psychrophilic bacteria. The bacterial counts of freshly caught krill ranged between 3,7 X 10(2)/g and 2,5 X 10(5)/g at 21 degrees C. During storage at 0-2 degrees C the counts gradually increased and off-odors were produced when they reached values of 10(6)/g at 21 degrees C. The total volatile bases content of freshly caught krill, 0.018 to 0.038%, increased considerably during storage reaching values of approximately 0.100% when off-odors became noticeable and 0.200% or more when the odor was clearly ammoniacal. Pseudomonas spp Gp. II (Shewan) were predominant in the bacterial flora of the freshly caught krill along with Moraxella spp Alcalígenes spp, Vibrio spp, Micrococcus spp and coryneforms. The spoilage flora developed during cold storage consisted mainly of Pseudomonas spp G. II (96-100%). The results were related to the saline composition of medium; however, Pseudomonas spp Gp. II were predominant with both media used.

[Biomass production and biological purification of distillation slops in a 2-step process]. / Producción de biomasa y depuración biológica de vinaza de destilería en un proceso de dos etapas.

In order to lower the chemical demand (COD) of slops from cane molasses alcohol a treatment of two steps which allows the production of single cell protein of Candida utilis and Paecilomyces variotii has been performed. Its use reduces the treatment cost. In the first step the slops without sterilization supplemented with ammonium sulphate (5 g.l-1) and dipotassium phosphate (0.5 g.l-1) was inoculated with C. utilis and P. variotii. The yield was 24 and 18 g.l-1 of dry biomass and COD reduction of 36 and 75% respectively. In the second step, the remainder effluents were treated with Aspergillus niger. The final COD reduction attained was 93 and 92% respectively.

Factors influencing protease production by two Antarctic strains of Stenotrophomonas maltophilia.

The influence of culture medium buffer capacity, the supplementation of culture medium with L-ala and the requirement of calcium for exoprotease production by Antarctic psychrotrophic Stenotrophomonas maltophilia strains ANT-1-1 and ANT-7-1 were examined. When increasing concentrations of calcium chloride (0 to 0.3 g l-1) were added to culture media, maximum protease production yields increased 70-75% (ANT-1-1) and 50% (ANT-7-1), while biomass levels showed little difference. Calcium was also necessary for optimal activity of proteases. L-ala had no effect on protease production. The reduction in buffer capacity, with the consequent change in external pH, had a positive effect, enhancing protease yields. Secretion of proteases into the medium started at the beginning of the stationary phase, corresponding with a rise in pH values up to pH 8.7 and was maximal at 36 h of culture. These results indicate that the regulation of calcium concentration and buffer capacity and also pH monitoring are factors to be considered when the design of an industrial culture medium and the optimisation of protease production processes using these Antarctic strains are concerned.

Solid state production of a Mucor bacilliformis acid protease.

Acid protease production by a local strain of Mucor bacilliformis was performed by solid state cultivation on different agricultural by-products as substrate. The effects of different parameters on enzyme biosynthesis were studied: Wheat bran wetted at 120% with a 200 mM HCl solution and inoculated with 5 x 10(5) spores/g produced a milk clotting activity of 7500 U/g bran after 72 h cultivation at 24 degrees C.

[Production of a concentrate of Mucor bacilliformis acid protease]. / Produccion de un concentrado de proteasa ácida de Mucor bacilliformis.

A concentrate of milk-clotting enzyme was produced by culture of Mucor bacilliformis on wheat bran medium moistened to 120% water on dry bases with HC1 2 N solution. The wheat bran was autoclaved, spread on trays and inoculated with 5.10(6) spore/gr of dry bran. After 10 days of culture at 21 degrees C, the enzyme produced was extracted with water and adjusted to pH 4.4. The precipitation was performed with ethanol. The precipitate was dissolved in HCl solution (pH 4.5) and it was concentrated by dialysis against polyethylene glycol 20.000. The enzyme solution had a specific activity of 1123 units/mg. and it was tested in the elaboration of cream cheese.

[Production and properties of the milk-clotting enzyme]. / Producción y propiedades de la enzima coagulante de leche.

Milk-clotting enzyme from a strain of Mucor varians Pispek selected in a previous work was obtained by solid culture followed by water extraction. Moistened wheat bran (120% water on dry bases) proved to be a good medium for the production of the milk-clotting enzyme. The production may be related to growth and 4,000 U milk-clotting activity by g of wheat bran was achieved. The milk-clotting enzyme was easily extracted with water from the cultures and could be precipitated by salting out with ammonium sulfate or by mixing with ethanol, methanol or acetone. The crude enzyme is an acid protease having optimal activity at pH: 3.0. Like calf rennet, this crude enzyme from M. varians loses activity with heat treatment. The level of lipolytic activity of the crude enzyme is similar to some commercial preparations and neither an antibiotic nor an amylase activity was demonstrated in the crude extracts.

Respiratory Activity of the Mycelium of Eremothecium ashbyii.

The respiratory activity of the mycelium of Eremothecium ashbyii from submerged cultures was manometrically determined at different stages of its development and the results were statistically analyzed. The experiments were performed in a manner designed to diminish the endogenous respiration without affecting the response to the addition of an exogenous substrate. Lactose was the carbohydrate tested that produced the lowest oxygen consumption. The oxidation of maltose, which was high at 24 hr, decreased by more than 50% at 48 and 55 hr. Glucose and sucrose were actively oxidized by mycelium of three ages. From the intermediates of carbohydrate metabolism, 24-hr mycelium did not produce oxygen consumption with malate, lactate, citrate, fumarate, and alpha-ketoglutarate. At 48 hr, mycelium did not oxidize either lactate or citrate; 55-hr mycelium showed oxygen consumption with all intermediates tested. Acetate and pyruvate always produced high oxygen consumption. Ethyl alcohol produced high oxygen consumption with mycelium of all tested ages.

Purification and characterization of an acid proteinase from mesophilic Mucor sp. solid-state cultures.

The fourth-day extract of a solid-state culture of the mesophilic Mucor sp. (M-105) strain showed a high milk-clotting activity and a clotting/proteolytic activity ratio similar to that of commercial preparations from microbial origin used in cheese manufacture. After ultrafiltration of the crude extract, the milk-clotting proteinase was purified in two steps: ion-exchange followed by size-exclusion chromatography. Enzyme homogeneity was assessed by HPLC, SDS-PAGE and N-terminal residue determination. A pI value of 4.21 was obtained and a molecular weight of 33 kDa was calculated from size-exclusion chromatography and SDS-PAGE data. The optimum pH for proteolytic activity towards dimethylcasein was in the 3.0-3.5 range. The proteinase retained 26 and 13% of its proteolytic activity after a 30-min incubation period, at pH 5.0 and 50 and 60 degrees C, respectively. This evidenced a lower heat stability than that of the thermophilic enzymes currently used in the cheese industry and also than that of bovine chymosin. The enzyme was fully inhibited by pepstatin A and no effect was observed with PMSF, p-CMPS or EDTA. The N-terminal amino acid sequence: GTGTVPVTDDGNLNEYYXTVTVGXP was compared with those from other fungal enzymes.

Selección de cepas bacterianas para la producción de treonina / [Selection of bacterial strains for the production of threonine]

The production of L-threonine in submerged culture was studied in the following bacterial strains; Brevibacterium flavum ATCC 21269, Corynebacterium acetoacidophilum ATCC 21270, Escherichia coli ATCC 21149, E. coli NRRL 12098, E. coli NRRL 12099 and E. coli NRRL 12100. Erlenmeyer flasks with different volumetric relations liquid/recipient, were used to study the influence of the volumetric oxygen transfer rate. B. flavum reached levels of threonine of 0.72 g/l at 96 hours of culture with a volumetric relation liquid/recipient of 1:5. With a relation 1:25 the maximal level was reached at 48 hours (0.60 g/l of threonine) (Table 1). In addition to threonine this strain accumulated in the culture media glutamic acid (+/- 2 g/l), alanine or glycine and proline. With E. coli ATCC 21149 the aeration favored the production of threonine reaching levels of 0.38 g/l in six day cultures with valine and alanine at levels approximate to 2 g/l. Excepting C. acetoacidophilum, all the strains produced threonine at levels of 0.30 to 3.55 g/l (Table 5). E. coli NRRL 12098 and E. coli NRRL 12100 produced only threonine, the culture medium being free from other aminoacids. With E. coli NRRL 12098 levels of 2 g/l were attained but the production was restricted to the presence of yeast extract in the media (Table 2). E. coli NRRL 12100 was the best strain and the inoculum media influenced the production (Table 3) and the best threonine levels were reached in the best aeration conditions assayed (Table 4) with 3.55 g/l for volumetric relations 1:10 and 1.25 g/l for 1:5.

Produccion de enzimas coagulantes de leche por cultivo sumergido aireado de una cepa de Mucor mucedo. / Milk clotting enzymes production by aerated submerged culture of a strain of Mucor mucedo

Mucor mucedo produce enzimas coagulantes de leche al desarrollar en substrato solido (afrecho humedecido) o en cultivo sumergido, aireado. En esta ultima condicion de cultivo, el empleo de caseina o suero de leche como unica fuente nitrogenada permitio detectar actividad coagulante, alcanzandose los mejores niveles de produccion para concentraciones de caseina entre 0,5 y 0,7%. La adicion de glucosa mejoro los rendimientos, considerandose optima una concentracion del 1%, con un aumento del 113% en la produccion. El efecto de los cationes Fe++, Cu++, Mn++ y Zn++ fue variable; su ausencia simultanea deprimio la produccion en un 40%, mientras que su efecto individual era muy ligero o nulo. Los aniones sulfato y fosfato presentaron efectos variables, dependiendo de su concentracion; sus niveles optimos fueron 2x10(-4) M y 2,2x10(-2) M, respectivamente. De los inoculos ensayados se selecciono el constituido por micelio vegetativo desagregado de 72 horas, al 17% (v/v), esto en razon de que se adelanto el pico de produccion y se elevaron y mantuvieron por periodos mas prolongados, los niveles de actividad coagulante. En condiciones optimas se logro como produccion maxima 190 U/ml de caldo

Producción de biomasa y depuración biológica de vinaza de destilería en un proceso de dos etapas / [Biomass production and biological purification of distillation slops in a 2-step process]

In order to lower the chemical demand (COD) of slops from cane molasses alcohol a treatment of two steps which allows the production of single cell protein of Candida utilis and Paecilomyces variotii has been performed. Its use reduces the treatment cost. In the first step the slops without sterilization supplemented with ammonium sulphate (5 g.l-1) and dipotassium phosphate (0.5 g.l-1) was inoculated with C. utilis and P. variotii. The yield was 24 and 18 g.l-1 of dry biomass and COD reduction of 36 and 75

respectively. In the second step, the remainder effluents were treated with Aspergillus niger. The final COD reduction attained was 93 and 92

Factors influencing protease production by two Antarctic strains of Stenotrophomonas maltophilia

The influence of culture medium buffer capacity, the supplementation of culture medium with L-ala and the requirement of calcium for exoprotease production by Antarctic psychrotrophic Stenotrophomonas maltophilia strains ANT-1-1 and ANT-7-1 were examined. When increasing concentrations of calcium chloride (0 to 0.3 g l-1) were added to culture media, maximum protease production yields increased 70-75 (ANT-1-1) and 50 (ANT-7-1), while biomass levels showed little difference. Calcium was also necessary for optimal activity of proteases. L-ala had no effect on protease production. The reduction in buffer capacity, with the consequent change in external pH, had a positive effect, enhancing protease yields. Secretion of proteases into the medium started at the beginning of the stationary phase, corresponding with a rise in pH values up to pH 8.7 and was maximal at 36 h of culture. These results indicate that the regulation of calcium concentration and buffer capacity and also pH monitoring are factors to be considered when the design of an industrial culture medium and the optimisation of protease production processes using these Antarctic strains are concerned.

Produccion de un concentrado de proteasa acida de Mucor bacilliformis. / Production of an acid protease concentrate from Mucor bacilliformis

Se obtuvo un concentrado de enzimas congulantes de leche por cultivo de Mucor bacilliformis en afrecho de trigo humectado a 120% de agua sobre base seca con solucion de HC1 0,2 N. El afrecho esterilizado fue dispuesto en bandejas, e inoculado con 5.10 (6) esporos/gr de afrecho seco. Luego de 10 dias de incubacion se extrajo el afrecho enmohecido con agua, se ajusto el pH a 4,4 y se precipito el sobrenadante limpido con etanol. El precipitado fue disuelto en agua clorhidrica (pH 4,5) y concentrado por dialisis contra polietilenglicol 20000. La solucion con una actividad especifica de 1128 U/mg, se utilizo en dos ensayos de elaboracion de queso tipo cremoso

Produccion y propiedades de la enzima coagulante de leche. / Produccion and properties of milk-clotting enzyme

Se obtuvieron enzimas coagulantes de leche en cultivo en sustrato solido con una cepa de Mucor varians Pispek, previamente seleccionada. El afrecho de trigo humedecido (120% de agua sobre base seca) demostro ser un buen medio de produccion, alcanzando niveles de 4.000 U de actividad coagulante de leche por g de afrecho. La enzima fue extraida de los cultivos con agua y precipitada con sulfato de amonio o con solventes como etanol, metanol o acetona.La enzima cruda es una proteasa acida con actividad optima a pH 3,0. Como la quimiosina de ternero, esta enzima cruda de M.varians pierde actividad con el tratamiento termico. El nivel de actividad lipolitica de la enzima cruda es similar al de algunas preparaciones comerciales y no se demostro actividad antibiotica, ni amilolitica en los extractos crudos