Structural organization of the Z-line protein, alpha-actinin, in developing skeletal muscle cells

The vegetative growth of the adenylyl cyclase-deficient mutant cr-1 (crisp), of Neurospora crassa, resembled a conidiogenic microcycle. It was demonstrated that an enzyme which is exclusively confined to conidia in wild-type strains, i.e., nicotinamide adenine dinucleotide (phosphate) glycohydrolase (NAD(P)ase; EC 3.2.2.6), was continuously released in the culture medium by the mutant. NAD(P)ase activity of agitated cr-1 cultures was much lower than that of standing cultures; nevertheless, the enzyme was actively produced immediately after agitation was stopped. Supplementation of the growth medium with cyclic AMP normalized the morphological phenotype of the cr-1 mutant and drastically reduced NAD(P)ase production. These results suggest that NAD(P)ase regulation is somehow dependent on cyclic AMP metabolism. However, the effect of the nucleotide over the enzyme does not appear to be direct, since other crisp mutants, cr-2 and cr-3, which also overproduced NAD(P)ase, were completely unresponsive to cyclic AMP. These strains possess normal adenylyl cyclase activity.     

Regulation of extracellular alkaline protease activity by histidine in a collagenolytic Vibrio alginolyticus strain

Vibrio alginolyticus synthesized an inducible extracellular collagenase in a peptone medium during the stationary growth phase. These cultures also possessed extracellular alkaline serine protease activity. The alkaline protease activity did not require a specific inducer and it was produced in tryptone or minimal media. The collagenase was not produced in either the tryptone or minimal media. The alkaline protease activity was sensitive to catabolite repression by a number of carbon sources, including glucose, and by amino acids and ammonium ions. Cyclic AMP, dibutyryl cyclic AMP and cyclic GMP did not relieve catabolite repression. Histidine and urocanic acid stimulated the production of alkaline protease activity in tryptone and minimal media. Other compounds associated with the histidine utilization (hut) pathway did not increase alkaline protease activity. Histidine reversed the repression of alkaline protease activity by glucose of (NH4)2SO4 in minimal medium. Histidine and the compounds associated with the hut pathway inhibited collagenase production.     

Biosynthesis of protease by Pseudomonas aeruginosa MN7 grown on fish substrate

Fish powders and fish protein hydrolysates (FPH) from sardinella (Sardinella aurita) were prepared and tested as growth media for alkaline protease production by Pseudomonas aeruginosa MN7. Cultivated in fish substrate as carbon source, the strain exhibited a slightly greater protease production (about 7800 U ml^–1) than that obtained with commercial peptones (about 7222 U ml^–1). Furthermore, P. aeruginosa MN7 produced the same amount of protease when cultivated in medium containing only fish substrate or that containing all ingredients, indicating that the strain can obtain its carbon and nitrogen requirements directly from whole fish proteins. Moreover, it was found that extensive hydrolysis of fish proteins did not increase protease formation. Protease production in media containing only FPH prepared by Alcalase was about 70% of those obtained with MN7 protease digest of fish protein or with meat-fish powder. These results indicate that sardinella substrates are an excellent carbon and nitrogen source for the growth of P. aeruginosa MN7 and the production of protease.     

Regulation of Exocellular Proteases in Neurospora crassa: Induction and Repression of Enzyme Synthesis

Neurospora crassa strain 74A grown on Vogel's medium containing bovine serum albumin (BSA) as principal carbon source secretes proteolytic enzymes which appear in the culture filtrate. Low concentrations of sucrose (0.1%) are necessary for growth from conidia, as conidia will not germinate on BSA alone. Once growth is initiated, however, protease production begins and at 5 to 6 hr growth and enzyme production are parallel. Higher concentrations of sucrose (0.5-2%) repress protease synthesis. Other metabolizable materials (sugars, amino acids, peptide mixtures) also repress protease synthesis. Some sugars will not sustain growth but allow germination and full induction of protease in the presence of protein. A material found in culture fluids of cells during induction of protease synthesis when added to repressed cultures causes a five-fold increase in the amount of protease production, although this is still approximately half that of normally induced cells. This material appears to be produced by induced cells in as little as 2 hr of culture, which is before detectable levels of protease can be found. It is heat-stable, of low molecular weight, and is not a simple product of protein digestion by the N. crassa proteases.     

Regulation of Exocellular Proteases in Neurospora crassa: Role of Neurospora Proteases in Induction

Cells of Neurospora crassa strain 74A, grown on sucrose for 12 h and transferred to a medium containing protein as sole carbon source, would not produce exocellular protease in significant amounts. When a filtrate from a culture induced to make protease by normal growth on a medium containing protein as principal carbon source was added to an exponential-phase culture in protein medium, exocellular protease was made in amounts similar to those made during normal induction. The material in the culture filtrate that participated in the induction process was identified as protease by its heat lability, molecular weight, and the dependence of induction rate on units of proteolytic activity added to the exponential-phase culture. Induction of the formation of exocellular protease by exponential-phase cells appears to require a protein substrate, added proteolytic activity, and protein synthesis. The protease produced by induced exponential-phase cells was as efficient in promoting induction as normally induced enzyme, whereas constitutive intracellular enzyme was only 50% as efficient. The bacterial protease thermolysin was able to induce exocellular protease at 90.7% of the rate observed with added N. crassa exocellular protease.     

Influence of dilution rate on NAD(P) and NAD(P)H concentrations and ratios in a Pseudomonas sp. grown in continuous culture.

A freshwater Pseudomonas sp. was grown in continuous culture under steady-state conditions in L-lactate-, succinate-, glucose- or ammonium-limited media. Under carbon limitation, the NAD(H) (i.e. NAD + NADH) concentration of the organisms increased exponentially from approximately 2 to 7 mumol/g dry wt as the culture dilution rate (D) was decreased from 0.5 to 0.02 h-1. Organisms grown at a given D in any of the carbon-limited media possessed very similar levels of NAD(H). Therefore, under these conditions, cellular NAD(H) was only a function of the culture O and was independent of the nature of the culture carbon source. D had no influence on the NAD(H) content of cells grown under ammonium limitation. In contrast, cellular NADH concentration was not influenced by D in carbon- or ammonium-limited media. In L-lactate-limited medium, bacteria possessed 0.14 mumol NADH/g dry wt; very similar levels were found in organisms grown in the other media. The results are consistent with those of Wimpenny & Firth (1972) that bacteria rigidly maintain a constant NADH level rather than a constant constant NADH: NAD ratio. NADP(H) (i.e. NADP + NADPH) and NADPH levels were also not influenced by changes in the culture carbon source or in D; in L-lactate-limited medium these concentrations were 0.97 and 0.53 mumol/g cell dry wt, respectively. The NADPH:NADP(H) ratio was much higher than the NADH:NAD(H) ratio, averaging 55% in carbon-limited cells.     

Identification of a New Marine Bacterial Strain SD8 and Optimization of Its Culture Conditions for Producing Alkaline Protease

While much attention has been given to marine microorganisms for production of enzymes, which in general are relatively more stable and active compared to those from plants and animals, studies on alkaline protease production from marine microorganisms have been very limited. In the present study, the alkaline protease producing marine bacterial strain SD8 isolated from sea muds in the Geziwo Qinhuangdao sea area of China was characterized and its optimal culture conditions were investigated. Strain SD8 was initially classified to belong to genus Pseudomonas by morphological, physiological and biochemical characterizations, and then through 16S rDNA sequence it was identified to be likely Pseudomonas hibiscicola. In addition, the culture mediums, carbon sources and culture conditions of strain SD8 were optimized for maximum production of alkaline protease. Optimum enzyme production (236U/mL when cultured bacteria being at 0.75 mg dry weight/mL fermentation broth) was obtained when the isolate at a 3% inoculum size was grown in LB medium at 20 mL medium/100mL Erlenmeyer flask for 48h culture at 30°C with an initial of pH 7.5. This was the first report of strain Pseudomonas hibiscicola secreting alkaline protease, and the data for its optimal cultural conditions for alkaline protease production has laid a foundation for future exploration for the potential use of SD8 strain for alkaline protease production.     

Dynamics of the NADP+ and NAD+ levels in the mycelium of P. nigricans Thom strains of varying productivity depending on the carbon source]

The levels of NADP+ and NAD+ in the mycelium of the highly productive strain 117 and low productive strain B of P. nigricans were studied by the 2nd, 5th, 9th and 13th days of development on the mineral medium in the presence of glucose, succinate or acetate. It was found that at the beginning of the growth the levels of NADP+ in both strains in the presence of the same carbon source were the same, just as the levels of NAD+ in the presence of glucose or succinate. The same strain had different levels of NADP+ in the presence of different carbon sources. The levels of NAD+ depended on both the carbon source and the strain. In the presence of glucose both nucleotides were accumulated by the end of the culture development and in greater amounts by strain 117. In the presence of succinate the maximum levels were observed at the beginning of the culture growth, while in the presence of accetate the maximum levels were recorded by the end of the culture development (strain 117) and by the 19th day (strain B). It is supposed that NAD+ is transformed into adenylates in the fungi.     

Mutants of Neurospora deficient in nicotinamide adenine dinucleotide (phosphate) glycohydrolase.

A new screening technique has been developed for the rapid identification of Neurospora crassa mutants that are deficient in nicotinamide adenine dinucleotide glycohydrolase (NADase) and nicotinamide adenine dinucleotide phosphate glycohydrolase (NADPase) activities. Using this procedure, five single-gene mutants were isolated whose singular difference from wild type appeared to be the absence of NAD(P)ase (EC 3.2.2.6). All five mutants were found to be genetically allelic and did not complement in heterocaryons. This gene, nada [NAD(P)ase], was localized in linkage group IV. One of the nada alleles was found to specify an enzyme that was critically temperature sensitive and had altered substrate affinity. Mutations at the nada locus did not affect the genetic program for the expression of NAD(P)ase during cell differentiation, nor did they have a general effect on NAD catabolism. Nada mutations did not have simultaneous effects on other glycohydrolase activities. Tests of dominance (in heterocaryons) and in vitro mixing experiments did not provide evidence that nada mutations alter activators or inhibitors of NAD(P)ase. Thus, the nada gene appears to specify only the structure of N. crassa NAD(P)ase.     

Regulation of extracellular protease production in Candida lipolytica

Production of extracellular protease by Candida lipolytica NRRL Y-1094 was depressed upon transfer to carbon-, nitrogen- or sulphur-free medium but not upon transfer to phosphorus-free medium. The protease activities produced under the three nutrient limitations had alkaline pH optima and similar substrate and inhibitor specificities. Any one of the following three conditions wass found to be sufficient for depression of extracellular protease: (1) “poor” carbon source, (b) cysteine intracellular pool below 0.5 μmol/g dry weight cells and (c) ammonia intracellular pool below 10 μmol/g dry weight cells. Thus, extracellular protease production in C. lipolyutica was subject to at least three different regulatory controls, carbon, sulphur and nitrogen repression. Intracellular cysteine and ammonia appeared to be the metabolic signals for sulphur and nitrogen repression, respectively. Anabolic glutamate dehydrogenase did not act as a regulatory protein mediating nitrogen repression. Exogenous protein had an inductive effect on extracellular protease production.     

Repeated batch production of alkaline protease by solid-state fermentation using urethane foam as carriers

By immobilizing fungi on a urethane foam carrier (UFC), a novel solid-state fermentation system was developed in order to produce repeatedly industrial useful enzymes. In this study, alkaline protease was produced by growing Aspergillus oryzae 460 (ATCC 20386) in a flask scale. Repeated batch production of alkaline protease was carried out by exchanging a part of the culture broth with fresh medium every 12 hr. The effects of feeding medium composition, and feeding volume were examined. Alkaline protease production stopped in the early phase at high values of soluble starch feeding rate and culture broth dilution rate. The enzyme production continued longer when 10 to 30 g/l polypepton was added to the feeding medium. Using soluble starch solution as feeding medium, a maximum activity of 520,000 U/l-bulk volume alkaline protease was obtained at culture time of 168 hr where the culture conditions of soluble starch concentration and feeding volume were 100 g/l and 0.025 l/l-bulk volume/dose, respectively. Production of the enzyme continued for 300 hr and total aklaline protease activity reached 870,000 U/l-bulk volume by adding 30 g/l polypepton to the fresh medium.     

Developmental regulation of nicotinamide adenine dinucleotide (phosphate) glycohydrolase in Neurospora crassa.

The formation of nicotinamide adenine dinucleotide (phosphate) glycohydrolase [NAD(P)ase; EC 3.2.2.6] in Neurospora crassa was found to be both spatially and temporally programmed. Ascospores were devoid of the enzyme. Vegetative hyphae contained little or no NADase activity. During the differentiation of aerial cell types (aerial hyphae and macroconidia), the specific activity of the enzyme increased by at least three orders of magnitude. Although transiently associated with young aerial hyphae, the enzyme became an integral and stable part of the mature macroconidia. NAD(P)ase could also be “derepressed” under conditions that permitted aerialogenesis in the absence of conidiation. The increase in the specific activity of NAD(P)ase during cell differentiation required concomitant RNA and protein synthesis; in vitro mixing experiments revealed no cell-specific activators or inhibitors of enzyme activity. The temperature-critical period for the in vitro inactivation of a temperature-sensitive enzyme variant was restricted to the period of actual enzyme expression.The data reported in this paper combined with data reported in a previous paper (Nelson et al., 1975b) underscore an important distinction in studies of development, namely, developmental regulation of a macromolecule versus regulation of development by a macromolecule. This paper provides evidence that NAD(P)ase is developmentally regulated. The previous paper provides evidence that the appearance of this enzyme need not regulate development.     

A parametric study ot protease production in batch and fed-batch cultures of Bacillus firmus

Proteolytic enzymes produced by Bacillus species find a wide variety of applications in brewing, detergent, food, and leather industries. Owing to significant differences normally observed in culture conditions promoting cell growth and those promoting production of metabolites such as enzymes, for increased efficacy of bioreactor operations it is essential to identify these sets of conditions (including medium formulation). This study is focused on formulation of a semidefined medium that substantially enhances synthesis and secretion of an alkaline protease in batch cultures of Bacillus firmus NRS 783, a known superior producer of this enzyme. The series of experiments conducted to identify culture conditions that lead to improved protease production also enables investigation of the regulatory effects of important culture parameters including pH, dissolved oxygen, and concentrations of nitrogen and phosphorous sources and yeast extract in the medium on cell growth, synthesis and secretion of protease, and production of two major nonbiomass products, viz., acetic acid and ethanol. Cell growth and formation of the three nonbiomass products are hampered significantly under nitrogen, phosphorous, or oxygen limitation, with the cells being unable to grow in an oxygen-free environment. Improvement in protease production is achieved with respect to each culture parameter, leading in the process to 80% enhancement in protease activity over that attained using media reported in the literature. Results of a few fed-batch experiments with constant feed rate, conducted to examine possible enhancement in protease production and to further investigate repression of protease synthesis by excess of the principal carbon and nitrogen sources, are also discussed. The detailed investigation of stimulatory and repressory effects of simple and complex nutrients on protease production and metabolism of Bacillus firmus conducted in this study will provide useful guidelines for design of bioreactors for production of protease and bulk chemicals by this bacterium.     

Regulation of the formation of protease byBacillus megaterium

The formation of protease takes place in washed cells ofBacillus megaterium incubated in a nitrogen-free medium. The rate of enzyme synthesis is decreased much less than that of cell proteins as compared with growing cells. The synthesis of protease in a nitrogen-free medium requires the presence of glucose. The omission of glucose results in stopping of the enzyme formation and substantial decrease of the rate of protein synthesis. Protease is not synthesized when the washed cells are incubated in a phosphate, free medium. The incubation of the cells in a nitrogen-free medium results in a decrease of the concentration of amino acids in the pool. In a phosphate-free medium the content of free amino acids increases temporarily and decreases again later. When the culture grown in the medium containing threonine or threonine and isoleucine in addition to NH₄ ions is transferred into the medium without amino acids, no protease formation is found during derepression of enzymes synthesizing both amino acids. The cells grown in a medium containing casamino acids begin to form the enzyme after a short lag period when transferred into the medium containing NH₄ as a sole nitrogen source or into a nitrogen-free medium.     

Regulation of two extracellular proteases of Neurospora crassa by induction and by carbon-nitrogen and sulfur-metabolite repression.

Synthesis and release by Neurospora crassa 74A (FGSC 262) of a neutral and an alkaline protease have been studied by experiments in which mycelia grown in Vogel's minimal medium were transferred to media containing protein inducer and deficient in carbon, nitrogen, and/or sulfur sources. The kinetics of accumulation of each of the two proteases in filtrates of induced, metabolite-deprived (derepressed) cultures were found to be similar and the same two electrophoretically separable enzymes were elicited by each of the three induction and derepression treatments. Moreover, antiserum specific for the major protease (Protease 2) elicited by one of the treatments gave a reaction of identity with the major proteases elicited by the other two treatments. It would therefore appear that these two proteases are coordinately regulated by a single system of induction and repression in which successful induction by exogenous protein depends upon the lifting of any one of carbon, nitrogen, or sulfur metabolite repressions.     

Application of statistical experimental design for optimization of alkaline protease production from Bacillus sp. RGR-14

Statistical optimization of culture conditions for production of a widely suited detergent protease from Bacillus sp. RGR-14 was carried out using a two-step approach. A quick identification of the important factors with simple screening experiment was followed by application of complex response surface design for further optimization. The production of extracellular alkaline protease by Bacillus sp. was favored in the presence of complex carbon and nitrogen sources, viz. starch, casamino acid and soybean meal. A reduced quadratic model was found to fit the alkaline protease production. Response surface analysis revealed the significant role of phosphate ions in determining alkaline protease production. A steep, stretched out response surface showed direct relation between the level of protease production and casamino acid and starch concentration in the medium. A 12.85 fold increase in protease production could be obtained within the design space. Protease production was found to be repressed in the presence of high concentrations of casamino acid. The model could be validated in up to 2 l shake flasks (3914 U ml⁻¹). The same statistical design could explain economic protease production in cost-effective medium as well.     

Flower-Inducing Activity of Lysine in Lemna paucicostata 151 Cultured on Nitrogen-Deficient Medium

Lemna paucicostata 151, a weakly responsive short-day plant,flowers even under continuous illumination when cultured onnitrogen-free medium for more than 72 hours with subsequentculture on nitrogen-rich medium. During the nitrogen-free culture,the protease activity and protein content of the plant increasedand decreased, respectively. The plant contained a protein(s)that induced flowering of the plant when added to the medium.The level of this protein(s) also decreased during the nitrogen-freeculture. The total amount of free amino acids in plants culturedon nitrogen-free medium for 96 hours decreased to about 15%of that in plants at the start of nitrogen-free culture, butlevels of some amino acids increased. These amino acids wereexamined for their effects on flowering of plants cultured onnitrogen-deficient or nitrogen-free medium. Most of the aminoacids had no effect on flowering. However, profuse floweringwas induced when lysine was added to the medium. Lysine promotedthe flower-inductive process(es) rather than the developmentof flower buds. These results suggest that nitrogen deficiency-induced floweringof the plants is induced by lysine, which is generated froma specific protein(s) by proteolysis. (Received May 11, 1992; Accepted July 30, 1992)     

Extracellular acid and alkaline proteases from Candida olea

Candida olea 148 secreted a single acid protease when cultured at acidic pH. In unbuffered medium, the culture pH eventually became alkaline and a single alkaline protease was produced. This was the only proteolytic enzyme produced when the organism was grown in buffered medium at alkaline pH. Both proteolytic enzymes were purified to homogeneity (as assessed by SDS-PAGE). The Mr of the acid protease was 30900, the isoelectric point 4.5; optimum activity against haemoglobin was at 42 degrees C and pH 3.3. This enzyme was inactivated at temperatures above 46 degrees C and was inhibited by either pepstatin and diazoacetyl-norleucine methyl ester but was insensitive to inhibition by either 1,2-epoxy-3-(p-nitrophenoxy)-propane or compounds known to inhibit serine, thiol or metallo proteases. The acid protease contained 11% carbohydrate. The alkaline protease had an Mr of 23400 and isoelectric point of 5.4. The activity of this enzyme using azocoll as substrate above 42 degrees C and was inhibited by phenylmethyl-sulphonyl fluoride and irreversible inactivated by EDTA. The enzyme was also partially inhibited by DTT but was insensitive to either pepstatin or p-chloromercuribenzoic acid.     

Processing of an NH2-terminally extended thermostable alpha-amylase by Bacillus subtilis alkaline protease

To analyze the processing of extracellular enzymes of Bacillus subtilis, an NH2-terminally extended hybrid alpha-amylase [pTUBE638-alpha-amylase (E24)] was purified from the periplasm of E. coli(pTUBE638) as the substrate for the in vitro processing reaction, in which a 21-amino-acid extra-peptide was added at the NH2-terminus of the mature thermostable alpha-amylase. The extended peptide in pTUBE638-alpha-amylase (E24) was completely processed by the extracellular alkaline protease of B. subtilis alone at pH 7.5 to 10.0. The processing was inhibited by 2 mM PMSF. In contrast, the neutral protease did not process the extended peptide. The processing activity of the purified alkaline protease was fully active in 100 mM phosphate and glycine-NaCl-NaOH buffer while it was partially active in 100 mM Tris-HCl or MOPS buffer. The optimum pH of the activity ranged from 8.0 to 9.0, although the optimum pH of the alkaline protease activity toward casein and Azocoll was 10.5. The NH2-terminal amino acid sequences of the enzymes processed in vitro coincided with those of the mature extracellular thermostable alpha-amylases in the culture medium of B. subtilis (pTUBE638). The appearance of the processing activity of alkaline protease was correlated with the changes of the pH in the culture medium.