Induction and regulation of alpha-amylase synthesis in a cellulolytic thermophilic fungus Myceliophthora thermophila D14 (ATCC 48104).

The alpha-amylase enzyme synthesis was higher when M. thermophila D-14 (ATCC 48104) was grown in culture medium incorporated with starch or other carbohydrates containing maltose units. Maximum enzyme production was attained with 1% starch followed by a gradual decrease with increasing concentration. Marked decrease in alpha-amylase synthesis occurred with the addition of glucose to the culture medium and this decreasing activity was proportional to the concentration of glucose. The enzyme synthesis was resumed as soon as the glucose concentration fell below a critical level. The addition of cAMP did not eliminate the repressive activity of glucose. The findings suggest that extracellular alpha-amylase synthesis in M. thermophila D-14 was inducible and subject to catabolite repression.     

Production and characterization of the agarase ofCytophaga flevensis

Cytophaga flevensis produced an inducible agarase which was extracellular under most conditions tested. The effect of cultural conditions on the production of enzyme was studied in batch and continuous culture. In batch culture, production was optimal whenCytophaga flevensis was incubated at 20C in a mineral medium with agar as the sole carbon source and ammonium nitrate as the nitrogen source at an initial pH of 6.6–7.0. The enzyme appeared to be subject to catabolite repression, since its synthesis was repressed when glucose was added to the medium in batch culture. Furthermore, in continuous culture, enzyme production decreased with increasing growth rate. Extracellular agarase was partially purified and the enzyme preparation obtained was very stable. The enzyme has a molecular weight of 26000 daltons. It is a β-agarase which is highly specific for polysaccharides containing neoagarobiose units. The final products of hydrolysis of agarose by the endo-acting enzyme were neoagarotetraose and neoagarobiose. Optimal conditions for its activity were pH 6.3 and 30C. When agarose was used as a substrate, an apparent temperature optimum of 35C was found, due to gelling of the substrate during the assay procedure.     

Multicomponent Substrate Utilization by Natural Populations and a Pure Culture of Escherichia coli

A heterogeneous population, typical of activated sludge, and a prototrophic strain of Escherichia coli were used to test for sequential substrate removal in a glucose-sorbitol medium. Each culture was preacclimated to sorbitol and was studied in the two-component medium under growing and nonproliferating conditions. In all four systems, glucose blocked sorbitol removal. Since large initial inocula were used, the suppression of sorbitol metabolism could not be totally due to repression of enzyme synthesis. The results indicate that glucose may affect the functioning of an existing enzyme system in addition to its established effect on enzyme synthesis. From an applied standpoint, the results indicate that an activated sludge may be completely and immediately prevented from eliminating a waste constituent to which it is acclimated.     

Regulation of Secondary Metabolite Biosynthesis: Catabolite Repression of Phenoxazinone Synthase and Actinomycin Formation by Glucose

Synthesis of the secondary metabolite, actinomycin, and the enzyme, phenoxazinone synthase, involved in the biosynthesis of the antibiotic, were shown to be under severe catabolite repression by glucose. Of a variety of hexoses and carbon compounds examined, glucose, and to a lesser extent, mannose, proved to be the most repressive for enzyme synthesis. The repression by glucose was most evident before production of the antibiotic. In a chemically defined medium suitable for actinomycin production, synthesis of phenoxazinone synthase began at the time the glucose (0.1%) supply was depleted. Soon after, antibiotic synthesis was initiated. Galactose, the major carbon source for growth and antibiotic synthesis, was not utilized until the glucose was consumed. Generally, carbon compounds which supported a rapid rate of growth were most effective in producing catabolite repression.     

Transient responses of <Emphasis Type="Italic">Wickerhamia</Emphasis> sp. yeast continuous cultures to qualitative changes in carbon source supply: induction and catabolite repression of α-amylase synthesis

The purpose of this study was to evaluate the inductive effect of starch and maltose, and the repressive/inhibitory effect of glucose, on amy-1 gene expression and α-amylase production by Wickerhamia sp., using continuous culture under transient-state conditions at a dilution rate (D) of 0.083 h^?1. Induction and repression kinetics of α-amylase were studied by changing the medium feed from glucose to maltose or starch in the induction experiments and vice versa in the repression experiments. Expression levels of amy-1 gene were measured by RT-qPCR. Results showed that starch was a more efficient inducer of α-amylase synthesis compared to maltose, with maximum accumulation rate constants of 0.424 and 0.191 h^?1, respectively. In contrast, α-amylase synthesis in starch and maltose cultures was partially repressed by glucose as indicated by a specific activity close to basal levels and a decay constant rate (??0.065 and ??0.069 h^?1, respectively) higher than ??D. A linear dependence of the specific rate of α-amylase production on mRNA relative abundance of amy-1 gene was observed. An inhibitory effect of glucose was not observed even at a concentration of 30 g L^?1. In conclusion, the transient continuous culture is a useful tool to determine the qualitative and quantitative effects of maltose and starch on α-amylase induction and of glucose on enzyme repression, as well as to obtain a detailed understanding of the dynamic behavior of the yeast culture. Furthermore, results showed that amylaceous substrates can be very effective carbon sources for the production of α-amylase without being inhibited by glucose.     

Induction of D-galactonate dehydratase in Mycobacterium butyricum

The induced synthesis of D-galactonate dehydratase in Mycobacterium butyricum has been studied initially after addition or removal of inductor or inhibitor. The enzyme was induced by galactonate and galactose; the system reached half-maximal effect of synthesis at 3.3 mM of galactonate. The lag of about 30 min between the addition of the inductor and the appearance of the enzyme at 37 degrees C was noted. The lag was dependent on temperature and independent of inductor concentration. After the withdrawal of the inductor the expression of a supposed galactonate dehydratase-coding messenger takes place which can be blocked by streptomycin or chloramphenicol. Both the messenger (the mean life of about 38 min) and the enzyme appeared relatively stable. The enzyme synthesis was found to be under strong catabolite repression caused by glucose and several other compounds and cyclic AMP failed to increase the enzyme synthesis or to overcome the repression. Zinc ions at concentration below 1 mM proved to have no effect on the enzyme synthesis but inhibited the enzyme itself that can be restored by EDTA.     

Regulatory factors affecting alpha-amylase production in bacillus licheniformis.

Possible factors regulating alpha-mylase synthesis in wild-type Bacillus licheniformis and in mutants producing elevated levels of the enzyme were studied in terms of catabolite repression, apparent temperature-sensitive repression, induction, and culture age. The synthesis of alpha-amylase in the parent strain occurred long after the culture reached the stationary phase of growth as a result of de novo protein synthesis, occurred only at high temperature around 50 C and not below 45 C, appeared to be induced in the presence of oligosaccharides with some linkage of alpha-1,4-, beta-1,4, beta-1,6-glucosyl glucose, or alpha-1,6-galactosyl glucose, and was repressed by the addition of exogenous glucose or low-molecular-weight metabolites. The addition of cyclic adenosine 3',5'-monophosphate stimulated alpha-amylase accumulation in growing cultures of the parent strain, but neither shortened the long lap period prior to the start of alpha-amylase synthesis nor mitigated the repressive effect of glucose. Mutant strains derived from the parent strain showed variation in the pattern of alpha-amylase synthesis, and some of them such as F-12s and F-14 produced alpha-amylase constitutively and without sensitivity to catabolite repression or transient repression from the moment of cell growth. These results are discussed in relation to possible regulatory mechanisms that might account for the observed characteristics of alpha-amylase synthesis in this facultative thermophilic microorganism.     

Glucose control of staphylococcal enterotoxin A synthesis and location is mediated by cyclic AMP

The regulation of staphylococcal enterotoxin A (SEA) synthesis in a defined medium was studied using continuous culture techniques. SEA production was repressed by glucose and repression could be overcome by addition of exogenous cyclic AMP. As well as this classical catabolite repression control, addition of glucose to de-repressed steady-state cultures resulted in rapid disappearance of toxin from the medium (also mediated by loss of cyclic AMP). When the toxin dissappeared from the medium, it was taken up again by the bacteria without apparent modification.     

Molecular weight of the undegraded polypeptide chain of Pseudomonas amyloderamosa isoamylase

Crystalline isoamylase of Pseudomonas amyloderamosa was found to be contaminated with a trace of proteolytic enzyme. This contaminant digested the isoamylase under neutral or alkaline conditions, especially in the presence of sodium dodecyl sulfate (SDS). A reliable molecular weight of the enzyme was obtained by SDS-polyacrylamide gel electrophoresis and by gel filtration on Sepharose-6B in 6 M guanidine-hydrochloride after heat inactivation of the contaminant. The molecular weight of the undergraded polypeptide chain of the isoamylase was about 90 000. The lower molecular weight and the subunit structure of the enzyme reported previously are incorrect.     

Enhancing the cyclodextrin production by synchronous utilization of isoamylase and α-CGTase

Cyclodextrins (CD) are cyclic α-1,4-glucans composed of glucose units, and they have multiple applications in food, pharmaceuticals, cosmetics, agriculture, chemicals, etc. CD are usually produced by cyclodextrin glycosyltransferase (CGTase) from starch. In the present study, a simultaneous conversion approach was developed to improve the yield of CD from starch by conjunction use of isoamylase with α-CGTase. The isoamylase of Thermobifida fusca was cloned and expressed in Escherichia coli BL21(DE3). The biochemical characterization of the enzyme showed that the optimum temperature and pH of the recombinant enzyme was 50 °C and 5.5, respectively, and it maintained 60 %, 85 % and 78 % relative activity at 30 °C, 40 °C and 60 °C, respectively. When the recombinant isoamylase and α-CGTase were used simultaneously to convert potato starch (15 %, w/v) into CD, the optimum conditions were found to be: 10 U of α-CGTase and 48 U of isoamylase per gram of substrate, with reaction temperature of 30 °C and pH 5.6. On the optimum condition, the total yield of CD reached 84.6 % (w/w) after 24 h, which was 31.2 % higher than transformation with α-CGTase alone. This is the first report of synchronous bioconversion of CD by both α-CGTase and isoamylase, and represents the highest efficiency of CD production reported so far.     

Production and Catabolite Repression of the Constitutive Polygalacturonic Acid trans-Eliminase of Aeromonas liquefaciens

Production of polygalacturonic acid (PGA) trans-eliminase was greatly stimulated under conditions of restricted growth of Aeromonas liquefaciens. This was accomplished either by substrate restriction in a continuous-feeding culture or by restricting divalent cations in a batch culture, with the use of PGA as the sole source of carbon in a chemically defined medium containing inorganic nitrogen. Slow feeding of glucose, glycerol, or PGA to carbon-limited cultures allowed PGA trans-eliminase to be formed at a maximum differential rate 500 times greater than in batch cultures with excess substrate present. The differential rate of enzyme formation obtained by slow feeding of these three substrances or of a mixture of PGA plus glucose was observed to be the same. Therefore, PGA trans-eliminase produced by A. liquefaciens, contrary to the current view, appears to be constitutive. These observations also indicate that production of PGA trans-eliminase is subject to catabolite repression and that limiting the substrate reverses this repression. It was also found that, under conditions of unrestricted growth, any compound which the bacteria can use as a source of carbon and energy repressed constitutive PGA trans-eliminase production. The heritable reversal of catabolite repression of PGA trans-eliminase synthesis was demonstrated by isolation of mutant strain Gc-6 which can readily synthesize the constitutive catabolic enzyme PGA trans-eliminase while growing in the presence of excess substrate.     

The expression of β-galactosidase by Escherichia coli during continuous culture

We have used the technique of continuous culture to study the expression of β-galactosidase in Escherichia coli. In these experiments the cultures were grown on carbon-limited media in which half of the available carbon was supplied as glycerol, glucose, or glucose 6-phosphate, and the other half as lactose. Lactose itself provided the sole source of inducer for the lac operon. The steady-state specific activity of the enzyme passed through a maximal value as a function of dilution rate. Moreover, the rate at which activity was maximal (0.40 h^?1) and the observed specific activity of the enzyme at a given growth rate were found to be identical in each of the three media tested. This result was unexpected, since the steady-state specific activity can be shown to be equal to the differential rate of enzyme synthesis, and since it is known that glycerol, glucose, and glucose-6-P-cause different degrees of catabolite repression in batch culture. The differential rate of β-galactosidase synthesis was an apparently linear function of the rate of lactose utilization per milligram protein regardless of the composition of the input medium. That is, it is independent of the rate of metabolism of substrates other than lactose which are concurrently being utilized and the enzyme level appears to be matched to the metabolic requirement for it. If this relationship is taken to indicate the existence of a fundamental control mechanism, it may represent a form of attenuation of the rate of β-galactosidase synthesis which is independent of cyclic AMP levels.     

Glucose transport and catabolite repression in Endomycopsis fibuligera yeasts

The role of systems for glucose transport in the manifestation of carbon catabolite repression of glucoamylase synthesis was studied in the yeast Endomycopsis fibuligera. Experimentas were conducted with its mutant AB-192 defective in the system of transport universal for glucose and 2-deoxy-D-glucose (2-DG). The nature of the mutation was established from the following data: (1) transport of labeled glucose into the mutant cells was twice as low in comparison with the parent culture 20-9; (2) transport of labeled 2-DG was suppressed almost entirely; (3) no competition was found between glucose and 2-DG for penetration into the mutant cells. Glucoamylase synthesis in the mutant AB-192 was not sensitive to catabolite repression by glucose. This was confirmed by the resistance of the AB-192 cells to the inhibition by glucose and their complete resistance to the repression by 2-DG. Moreover, an addition of cAMP did not stimulate glucoamylase synthesis by the mutant culture in the presence of glucose and 2-DG. It can be concluded therefore that the resistance of the yeast to catabolite repression by the glucose is caused by the mutation in the system for carbohydrate transport. The results suggest that the system of glucose transport plays an important role in the manifestation of carbon catabolite repression in the yeast Endomycopsis fibuligera.     

Gene sequence analysis and properties of EGC, a family E (9) endoglucanase from Fibrobacter succinogenes BL2

Abstract β-Glucosidase in Aspergillus nídulans was found to be both intracellular and extracellular. The intracellular β-glucosidase was synthesized after the exhaustion of carbon source in the medium. The extracellular enzyme appeared with autolysis of the mycelium. Biosynthesis of β-glucosidase was not induced by various carbohydrates but repressed to varying extents in the presence of glucose, glycerol, and 2-deoxyglucose. This repression was not relieved by addition of cAMP. The repression was relieved much more by mutations in the creA gene than by one in the creC gene. Thus, β-glucosidase synthesis in A. nidulans is subject to carbon catabolite repression.     

Characteristics of Protease Production by Cephalosporium sp

We investigated protease formation by Cephalosporium sp. strain KM388, which produced trypsin inhibitor in the same cultures, in medium containing polypeptone, meat extract, and glucose (natural medium) and in medium containing NaNO₃, glucose, and yeast extract (semisynthetic medium). In natural medium, protease was secreted into the culture broth after cessation of growth caused by consumption of the polypeptone, the growth-limiting substrate. Enzyme formation in the stationary growth phase was due to de novo and so-called preferential synthesis, because cycloheximide immediately inhibited enzyme formation. In semisynthetic medium, protease was produced in parallel with mycelial growth, but production was repressed by the addition of polypeptone to the medium; protease production began after the added polypeptone was consumed. On the other hand, if glucose was eliminated from natural medium, the lag period of initiation of enzyme production was reduced until the late exponential phase. The addition of phosphate up to a concentration of 1.0% to natural medium also shortened the lag period and damped the pH change of the broth during cultivation.     

Production and secretion of a multifunctional ß-glucosidase by Humicola grisea var. thermoidea: effects of L-sorbose

The effects of L-sorbose on growth, morphology and production of a multifunctional ß-glucosidase by the thermophilic fungus Humicola grisea var. thermoidea were investigated. Sorbose increased the lag phase period 3-fold and drastically altered the morphology of the fungal hyphae. Cellobiose and lactose were good inducers of the enzyme. The addition of 5 % sorbose to cultures containing 1 % cellobiose enhanced the extracellular levels of the ß-glucosidase 3.3-fold with constant cytosolic and cell-wall bound levels, demonstrating stimulation of both enzyme synthesis and secretion. The stimulation of enzyme production by sorbose was dependent on the presence of cellobiose as inducer, since 2- to 3-fold inhibition was observed in lactose and glucose. Production and secretion of phosphatases and endoglucanases was not stimulated by sorbose, which did not affect the subcellular distribution of the ß-glucosidase also. However, it reduced the uptake rates of glucose and cellobiose. Taken together, the results discarded increased non-specific enzyme secretion and/or increased release of the enzyme from the cell-wall as possible molecular mechanisms for the effects of sorbose on the production of the multifunctional ß-glucosidase by H. grisea. An alternative mechanism, based on a prolonged action of cellobiose as inducer associated with a decreased catabolic repression by glucose, was discussed.     

On the regulation of adenosine 3', 5'-monophosphate synthesis in bacteria. I. Effect of carbon source variation on cyclic AMP synthesis in Escherichia coli B/r.

1. The effect of carbon source variation in bacterial growth media on their growth rate, inducible enzyme and cyclic AMP synthesis was examined: an inverse relationship between the culture's growth rate and its differential rate of inducible enzyme (tryptophanase and beta-galactosidase), and cyclic AMP synthesis was found. 2. The effect of the culture's growth phase on its sensitivity or resistance to glucose catabolite repression was determined in the wild type and a catabolite insensitive mutant (ABDROI): the wild type's sensitivity to glucose repression was not affected, whereas the insensitivity of the mutant was found to be limited to its early logarithmic phase of growth. At late log, or stationary phase, the mutant was found to be sensitive to glucose repression. 3. Examination of the kinetics of glucose uptake by the mutant, using alpha-[1 4-C] methyl-glucoside showed evidence for two transport systems each with a different affinity to glucose. A low affinity transport system (apparent Km of 3.4-10-minus 5 M) which appears mostly at the early logarithmic phase of growth. A high affinity transport system (apparent Km of 1.2-10-minus 5 M) which appears mostly at the late log and stationary phases of growth. 4. The effect of the culture density variation on its sensitivity to glucose repression showed that sensitivity to glucose catabolic repression is primarily a reflection of the formation of an allosteric effector molecule between glucose and its specific transport molecule which in turn regulates the activity of the adenylate cyclase.     

Production of alkaline protease from an alkaliphilic actinomycete

The repression of alkaline protease synthesis from alkaliphilic actinomycete was studied by using glucose, peptone, yeast extract, KH2PO4 and amino acids; tyrosine, tryptophan, lysine, and arginine. There was a critical limit of stimulation of enzyme production by these components. Crude components such as molasses, wheat flour, and wheat bran were found to be effective for growth and enzyme production. The high level of enzyme production using agro-industrial by-products is commercially significant due to cheap nature of these sources. The findings are quite attractive, as only few actinomycetes, particularly alkaliphilic ones, have so far been explored for their enzymatic potential and regulation of enzyme synthesis.     

The regulation of agarase production by resting cells of Cytophaga flevensis

The regulation of the synthesis of extracellular agarase by Cytophaga flevensis was studied in resting-cell suspensions. Enzyme synthesis was strictly dependent on the presence of a suitable inducer. Enzyme production was maximal at 20 C in phosphate buffer pH 6.9 in the presence of 1.3mm calcium chloride, 0.03% casamino acids and inducer. Enzyme production was virtually the same at 15 and 20 C, reduced to 50% at 25 C and was not detectable at 30 C. It was highly stimulated by the presence of 0.03% of casamino acids in the incubation mixture and was also favoured by the presence of 1.3mm calcium ions. Of a variety of compounds tested, only melibiose or neoagaro-oligosaccharides were effective inducers. Among the neoagaro-oligosaccharides, neoagarotetraose was the best inducer. At higher concentrations of inducer compounds catabolite repression of enzyme synthesis was apparent. This was also found when glucose was added to the incubation mixture. This repression was not relieved by the addition of cyclic AMP. Indications were found that the excretion process was limiting the rate of production of extracellular enzyme.     

产碱性纤维素酶菌株的选育和酶合成基本特性

芽孢杆菌x－6菌株经甲基磺酸乙酯（EMS）和紫外线（UV）复会诱变,从其万古霉素（Vm）抗性突变体中选育获得一突变株EV23,所产生的碱性核甲基纤维素酶（CMCase）酶活力由原来的0．84u／ml提高到3.53u／ml。EV23菌株所产该酶基本为组成性地合成纤维素酶,酶合成明显表现出抗降解物阻遏的特点,以葡萄糖为碳源培养,4％浓度时酶合成水平最高。酶合成效率受菌体生长速率影响较大。在高浓度易代谢基质和三羧酸循环中间物存在下,酶合成将受到一定程度的阻遏。酶合成还与能量代谢有关,探讨了外源ATP、cAMP对