Catabolite Repression

A newly isolated haloalkaliphilic Bacillus sp., Ve1, produced substantial levels of extracellular alkaline protease. Enzyme production corresponded with growth and reached a maximum level (410 U/ml) during the early stationary phase along with a brick red pigmentation. Protease production was maximum (397 U/ml) in gelatin broth. While growth and protease production was optimum at 10% (w/v) NaCl, only marginal growth without enzyme production was evident in the absences of salt. Ve1 could grow and produce protease at pH 7-9, the optimum being at 8 and 9, respectively. Among the organic nitrogen sources used, growth was best supported by a combination of peptone and yeast extract, while the optimum protease production was with casamino acid followed by gelatin. Enzyme production was highly reduced in soya peptone, trader's protein and tryptone. Inorganic nitrogen sources proved to be less favourable. Strong catabolic repression on protease production was observed with glucose and ammonia. The study assumes significance in the light of increasing emphasis on biocatalysis under more than one extreme condition. #

In view of rising prices of crude oil due to increasing fuel demands, the need for alternative sources of bioenergy is expected to increase sharply in the coming years. Among potential alternative bioenergy resources, lignocellulosics have been identiWed as the prime source of biofuels and other value-added products. Lignocelluloses as agricultural, industrial and forest residuals account for the majority of the total biomass present in the world. To initiate the production of industrially important products from cellulosic biomass, bioconversion of the cellulosic components into fermentable sugars is necessary. A variety of microorganisms including bacteria and fungi may have the ability to degrade the cellulosic biomass to glucose monomers. Bacterial cellulases exist as discrete multi-enzyme complexes, called cellulosomes that consist of multiple subunits. Cellulolytic enzyme systems from the Wlamentous fungi, especially Trichoderma reesei, contain two exoglucanases or cellobiohydrolases (CBH1 and CBH2), at least four endoglucanases (EG1, EG2, EG3, EG5), and one-glucosidase. These enzymes act synergistically to catalyse the hydrolysis of cellulose. DiVerent physical parameters such as pH, temperature, adsorption, chemical factors like nitrogen, phosphorus, presence of phenolic compounds and other inhibitors can critically inXuence the bioconversion of lignocellulose. The production of cellulases by microbial cells is governed by genetic and biochemical controls including induction, catabolite repression, or end product inhibition. Several eVorts have been made to increase the production of cellulases through strain improvement by mutagenesis. Various physical and chemical methods have been used to develop bacterial and fungal strains producing higher amounts of cellulase, all with limited success. Cellulosic bioconversion is a complex process and requires the synergistic action of the three enzymatic components consisting of endoglucanases, exoglucanases and-glucosidases. The co-cultivation of microbes in fermentation can increase the quantity of the desirable components of the cellulase complex. An understanding of the molecular mechanism leading to biodegradation of lignocelluloses and the development of the bioprocessing potential of cellulolytic microorganisms might eVectively be accomplished with recombinant DNA technology. For instance, cloning and sequencing of the various cellulolytic genes could economize the cellulase production process. Apart from that, metabolic engineering and genomics approaches have great potential for enhancing our understanding of the molecular mechanism of bioconversion of lignocelluloses to value added economically signiWcant products in the future.

Maltooligosaccharides Marinobacter sp. Solvent stable a b s t r a c t Maltooligosaccharides especially maltotriose and maltotetraose producing amylases are highly desirable for application in bread making and other food industries. A maltotriose and maltotetraose producing amylase from moderately halophilic Marinobacter sp. EMB8 is described. Under optimized culture conditions, 48.0 IU/mL amylase was obtained. The enzyme was purified to homogeneity by ultrafiltration, DEAE cellulose and Sephadex G-75 column chromatography with 52% yield and 76-fold purification. It was a monomeric protein of 72 kDa. The amylase had many novel features viz. stability up to 20% NaCl, 80°C temperature, pH 6.0-11.0 and in wide range of organic solvents at high concentrations. The enzyme efficiently hydrolyzed starch into maltooligosaccharides rich in maltotriose and maltotetraose. These novel properties make the Marinobacter sp. amylase a potentially useful enzyme.

Aspergillus niger NCIM 1207 produces high levels of extracellular /~-glucosidase and xylanase activities in submerged fermentation. Among the nitrogen sources, ammonium sulfate, ammonium dihydrogen orthophosphate, and corn-steep liquor were the best for the production of cellulolytic enzymes by A. niger. The optimum pH and temperature for cellulase production were 3.0-5.5 and 28~ respectively. The cellulase complex of this strain was found to undergo catabolite repression in the presence of high concentrations of glucose. Glycerol at all concentrations caused catabolite repression of cellulase production. The addition of glucose (up to 1% concentration) enhanced the production of cellulolytic enzymes, but a higher concentration of glucose effected the pronounced repression of enzymes. Generally the growth on glucose-or glycerol-containing medium was accompanied by a sudden drop in the pH of the fermentation medium to 2.0. Index Entries: Aspergillus niger; catabolite repression; cellulase inactivation. INTRODUCTION Cellulose is degraded by the synergistic action of three kinds of enzymes in the cellulase complex, namely, exo-l,4-fi-D-glucanase, endo-l,4fi-D-glucanase and fl-glucosidase. Trichoderma reesei is known to produce *Author to whom all correspondence and reprint requests should be addressed.

Delta-endotoxin production capacity of several Bacillus thuringiensis (Bt) strains exhibiting various larvicidal activities towards either lepidoptera or diptera was investigated in gruel and fish meal media. Diptera-specific strains produced less delta-endotoxins (1246-1998 mg l −1 ) than lepidoptera-specific ones (3060-3301 mg l −1 ). Carbon catabolite repression regulated delta-endotoxins synthesis for all strains, but no relationship existed between the specificity of delta-endotoxins and their sensitivity to such repressive regulation. The production of proteolytic activities by the isolate BNS3 and the commercial strain HD1, was reduced in 0.5 g l −1 sodium chloride and 0.1% Tween-80 but not by other strains. Moreover, in the case of the diptera-specific strain BUPM98, there was a need for additional nitrogen sources and to some extent, several amino acids from yeast extract, leading to an increase of 22% in delta-endotoxin production. Sporulation of BUPM98 was never reached in the rich Luria Broth (LB) medium, compared with that of the lepidoptera-specific strain BNS3. In shake flask cultures at 10 g l −1 , sodium acetate notably increased (38-79%) delta-endotoxins production by the diptera-specific strains, with a strong decrease in proteolytic activities. Similar delta-endotoxin production and high proteolytic activities were obtained with or without 10 g l −1 sodium acetate with an excess of aeration in a 2-l fermenter. In a less aerated medium, a strong effect of acetate was observed, decreasing production of proteolytic activities and exhibiting high catabolite repression on delta-endotoxin synthesis. The effect of sodium acetate was not directly related to sporulation and protease synthesis but to acetate metabolism. Culture conditions were defined for the screening of Bt strains producing high counts of delta-endotoxins specific to either lepidoptera or diptera.

Over-production of delta-endotoxin by Bacillus thuringiensis, strain BNS3, entomopathogenic towards lepidoptera through overcome of catabolic repression of its synthesis, was investigated into full-controlled 3 l fermenter using simple and complex media. Equilibrium between density of the vegetative cells and their ability to synthesize toxins during sporulation was shown to be necessary to take into account. By application of various dissolved oxygen profiles during fermentation, it was clear that the level of dissolved oxygen saturation in the fermentation broth affected cell density as well as delta-endotoxin synthesis. Indeed, the adequate dissolved oxygen profiles to be followed throughout the fermentation for high production of bioinsecticides were determined using the two different media. It was found out that aeration rates corresponding to 60% and 70% oxygen saturation during the first 6 h of fermentation should be applied into 15 g l −1 glucose-based medium and 42 g l −1 gruel-based one, respectively. Then, 40% oxygen saturation should be ensured up to the end of fermentation, independently of the carbon source origin. With higher oxygen saturation values, cell densities were increased, but delta-endotoxin synthesis yields were strongly reduced. Cells produced with low aeration into the medium acquired higher capacities to synthesize toxins during sporulation. Interestingly, by following adequate oxygen profiles, it was possible to use increased initial glucose or gruel concentrations, without significant decrease of delta-endotoxin production yields. Consequently, the adequate control of dissolved oxygen in the culture media of B. thuringiensis allowed at least partial overcome of the carbon catabolite repression which seemed to be an apparent regulation, mostly due to the rate of use of energy generated during a first step of high growth of B. thuringiensis cells. These results are of great importance from practical point of view, since it could be possible to produce large quantities of insecticidal crystal proteins during large-scale fermentation, contributing to the reduction of B. thuringiensis insecticides production cost. Interestingly, in bioinsecticides produced with adequate aeration profiles, spores have lower yield, which is in favor of the dissemination of less spores into the environment.

A catabolite derepressed Bacillus subtilis strain KCC103 was used to produce a-amylase in medium containing sugarcane bagasse hydrolysate (SBH). Addition of SBH (1% reducing sugar (w/v)) to the nutrient medium supported maximum a-amylase production of 67.4 U ml À1. HPLC analysis of SBH showed the presence of glucose, xylose and arabinose in the ratio of 0.9:1.0:0.16 (w/w/w). In SBH-medium glucose and xylose were consumed completely while arabinose remained unutilized. Uptake rate of glucose was 2-folds higher than xylose but rate of a-amylase production with xylose was 1.5-folds higher than glucose. Arabinose had no effect on growth and a-amylase synthesis. Further, a-amylase production in SBH-medium was enhanced to 144.5 U ml À1 (2.2-fold) by response surface methodology where the levels of SBH, and other media components were varied. The modified medium consisted of (in g l À1) SBH: 24; peptone: 17.43; yeast extract: 1.32 and beef extract: 1.82. High level of SBH showed no significant inhibition of a-amylase synthesis. The derepressed strain KCC103 is useful to produce a-amylase economically in short time (30-36 h).

Among the lignocellulosic substrates tested, wheat bran supported a high xylanase (EC 3.2.1.8) secretion by Humicola lanuginosa in solid-state fermentation (SSF). Enzyme production reached a peak in 72 h followed by a decline thereafter. Enzyme production was very high (7832 U/g of dry moldy bran) when wheat bran was moistened with tap water at a substrate-to-moistening agent ratio of 1:2.5 (w/v) and an inoculum level of 3 × 10 6 spores/10 g of wheat bran at a water activity (a w ) of 0.95. Cultivation of the mold in large enamel trays yielded a xylanase titer comparable with that in flasks. Parametric optimization resulted in a 31% increase in enzyme production in SSF. Xylanase production was approx 23-fold higher in SSF than in submerged fermentation (SmF). A threshold constitutive level of xylanase was secreted by H. lanuginosa in a medium containing glucose as the sole carbon source. The enzyme was induced by xylose and xylan. Enzyme synthesis was repressed beyond 1.0% (w/v) xylose in SmF, whereas it was unaffected up to 3.0% (w/w) in SSF, suggesting a minimization of catabolite repression in SSF.

Two phosphate solubilizing bacteria (PSB), M3 and SP1, were obtained from the rhizosphere of mungbean and sweet potato, respectively and identified as strains of Pseudomonas aeruginosa. Their rock phosphate (RP) solubilizing abilities were found to be due to secretion high amount of gluconic acid. In the presence of malate and succinate, individually and as mixture, the P solubilizing ability of both the strains was considerably reduced. This was correlated with a nearly 80% decrease in the activity of the glucose dehydrogenase (GDH) but not gluconate dehydrogenase (GAD) in both the isolates. Thus, GDH enzyme, catalyzing the periplasmic production of gluconic acid, is under reverse catabolite repression control by organic acids in P. aeruginosa M3 and SP1. This is of relevance in rhizospheric conditions and is a new explanation for the lack of field efficacy of such PSB.

A microorganism isolated from the Sundarbans region of the Bay of Bengal, India, showed potent antimicrobial activity against gram-positive and gramnegative bacteria, molds, yeast and several multiple-drugresistant (MDR) bacteria, including methicillin-resistant Staphylococcus aureus (MRSA). The isolate grew in the presence of 20% (w/v) NaCl, antibiotic production being maximum with 5% (w/v) NaCl in the production medium. Natural seawater stimulated antibiotic biosynthesis. The absence of catabolite repression during the synthesis of the antimicrobial substance was demonstrated by the utilization of glucose by this isolate. The 16S rRNA gene of this aerobic, gram-positive, mycelium-and spore-forming microorganism was amplified, and molecular phylogenetic analysis of the DNA sequence showed less than 93% similarity with its closest relative, indicating differentiation at the genus level. The highly stable, active principle was purified by butyl acetate extraction and silica-gel chromatography and a single compound was found to posses the broad-spectrum activity. Molecular characterization showed that the active compound is a lipid.

Citric acid production from sugar cane molasses byAspergillus niger NIAB 280 was studied in a batch cultivation process. A maximum of 90 g/L total sugar was utilized in citric acid production medium. From the parental strainA. niger, mutant strains showing resistance to 2-deoxyglucose in Vogal's medium containing molasses as a carbon source were induced by γ-irradiation. Among the new series of mutant strains, strain RP7 produced 120 g/L while the parental strain produced 80 g/L citric acid (1.5-fold improvement) from 150 g/L of molasses sugars. The period of citric acid production was shortened from 10 d for the wild-type strain to 6–7 d for the mutant strain. The efficiency of substrate uptake rate with respect to total volume substrate consumption rate,Q s (g per L per h) and specific substrate consumption rate,q s (g substrate per g cells per h) revealed that the mutant grew faster than its parent. This indicated that the selected mutant is insensitive to catabolite repression by higher concentrations of sugars for citric acid production. With respect to the product yield coefficient (Y p/x), volume productivity (Q p) and specific product yields (q p), the mutant strain is significantly (p≤0.05) improved over the parental strain.

At 28 °C, Streptomyces lavendulae produced high levels of penicillin V acylase (178 IU/l of culture) when grown on skim milk as the sole nutrient source for 275 h. The enzyme showed catabolite repression by glucose and was produced in the stationary phase of growth. Penicillin V was a good inducer of penicillin V acylase formation, while phenoxyacetic acid, the side-chain moiety of penicillin V, did not alter enzyme production significantly. The enzyme was stable between pH 6 and 11 and at temperatures from 20 °C to 55 °C. This extracellular enzyme was able to hydrolyse natural penicillins and unable to hydrolyse penicillin G.

When Saccharomyces cerevisiae are grown on a mixture of glucose and another fermentable sugar such as sucrose, maltose or galactose, the metabolism is diauxic, i.e. glucose is metabolized ®rst, whereas the other sugars are metabolized when glucose is exhausted. This phenomenon is a consequence of glucose repression, or more generally, catabolite repression. Besides glucose, the hexoses fructose and mannose are generally also believed to trigger catabolite repression. In this study, batch fermentations of S. cerevisiae in mixtures of sucrose and either glucose, fructose or mannose were performed. It was found that the utilization of sucrose is inhibited by concentrations of either glucose or fructose higher than 5 g/l, and thus that glucose and fructose are equally capable of exerting catabolite repression. However, sucrose was found to be hydrolyzed to glucose and fructose, even when the mannose concentration was as high as 17 g/l, indicating, that mannose is not a repressing sugar. It is suggested that the capability to trigger catabolite repression is connected to hexokinase PII, which is involved in the in vivo phosphorylation of glucose and fructose.

Sugars are excellent carbon sources for all yeasts. Since a vast amount of information is available on the components of the pathways of sugar utilization in Saccharomyces cerevisiae it has been tacitly assumed that other yeasts use sugars in the same way. However, although the pathways of sugar utilization follow the same theme in all yeasts, important biochemical and genetic variations on it exist. Basically, in most non-conventional yeasts, in contrast to S. cerevisiae, respiration in the presence of oxygen is prominent for the use of sugars. This review provides comparative information on the different steps of the fundamental pathways of sugar utilization in non-conventional yeasts: glycolysis, fermentation, tricarboxylic acid cycle, pentose phosphate pathway and respiration. We consider also gluconeogenesis and, briefly, catabolite repression. We have centered our attention in the genera Kluyveromyces, Candida, Pichia, Yarrowia and Schizosaccharomyces, although occasional reference to other genera is made. The review shows that basic knowledge is missing on many components of these pathways and also that studies on regulation of critical steps are scarce. Information on these points would be important to generate genetically engineered yeast strains for certain industrial uses. ß

Various agro-industrial residues in combination with peptone, NH 4 Cl and/or soy bran were screened as substrates for extracellular bglucosidase (BGL) production by Monascus purpureus NRRL1992 on submerged fermentations (SmF). Higher BGL production was achieved when the agro-industrial residues were combined with peptone, and the utilization of NH 4 Cl (inorganic nitrogen source) had not supported high enzyme production. The combination between grape waste and peptone was the best for enzyme production, and was selected as the growth substrate for further investigations. The evaluation of the effects of the medium components on enzyme production showed that the influence of peptone was more important than grape waste. The production of extracellular BGL by M. purpureus was inducible and controlled by carbon (glucose) catabolite repression. #

Chaetomium erraticum was capable of producing all the three components of a cellulase enzyme system including exoglucanase, endoglucanases, and β-glucosidase extracellularly. However, the cultivation conditions and the medium composition markedly affected the ability of microorganism to synthesize various enzymes. Exoglucanase was highest under static conditions, while endoglucanase and β-glucosidase were maximized under shake conditions. Among the various defined substrates, CMC proved to be the best inducer for exoglucanase under static conditions and β-glucosidase under shake conditions. MCC induced maximum endoglucanase under shake conditions. The biosynthesis of all three components of cellulases was repressed with different concentrations of glucose, puromycin, actinomycin, and actidione, while the supplementation of exogenous cyclic-AMP was fully capable of releasing the catabolite repression for production of all three components.

Penicillin-G fermentation with industrial media in 1 m3 stirred tank bioreactors was studied. A model based on the Bajpai-Reuss model structure was developed. Under typical production conditions catabolite repression is nonidentifiable and extensive mycelium differentiation occurs. Thus, the original model was reformulated, neglecting glucose repression of penicillin production and including biomass autolysis. The multi-substrate nature of industrial media was critically analysed. By combining the two most important carbon substrates present, a simple and applicable model was obtained. Model predictions agreed well with experimental data and reproduced the general characteristics observed in the fermentations. The predictive power of the model was tested for fermentations with different sugar feed rate profiles and raw materials (corn-steep liquor and sugar syrup). Several aspects of parameter estimation and model development are discussed on the basis of direct experimental data inspection and a sensitivity analysis of model parameters.