Catabolite Repression Resistant Mutant Research Articles

Purification and Characterization of Carboxymethyl Cellulase By A Catabolite Repression Resistant Mutant Strain of Pseudomonas Sp. (Psccrrnt9)

Purification and Characterization of Carboxymethyl Cellulase By A Catabolite Repression Resistant Mutant Strain of Pseudomonas Sp. (Psccrrnt9)

English

  A moderately thermotolerant bacterial strain was isolated from the hot spring of Tatta Pani (AJ and K) Pakistan and was designated as Bacillus subtilis strain GQ 301542 after biochemical, morphological and 16S rDNA sequence analysis. This strain and its catabolite repression resistant mutant CRM197 were utilized for the study of different production kinetic parameters of both endoglucanase and cellobiohydrolase. Time course study on one monomeric (glucose), one dimeric (maltose) and two polymeric substrates (α-cellulose and wheat straw) was carried out at different time intervals (4 - 28 h, after each 4 h) for determining the maximum enzyme productivity on a particular substrate. Maximum rate of endoglucanase production by the mutant (53.1 IU/L/h) was significantly (P = 0.0007) higher than that (23.7 IU/L/h of the parental organism  following their growth on glucose in Dubos salts medium while the optimum product yields (Yp/s) was calculated as 69.0 IU/g S (parent) and 82.3 IU/g S (mutant) for cellobiohydrolase production. Deoxy-D-glucose resistant mutant was significantly (p = 0.03 to 0.0007) improved over its parental strain with respect to some substrate consumption and all product formation parameters and can easily degrade cellulosic biomass for production of fermentable carbohydrates.   Key words: Cellobiohydrolase, endoglucanase, thermotolerant, Bacillus subtilis.

Regulatory mutations affecting the synthesis of cellulase in Pseudomonas fluorescens

Pseudomonas fluorescens , was cultured in basal medium containing carboxymethyl-cellulose (CMC) as inducer and glucose or glycerol as carbon and energy sources. Ethylmethanesulphonate (EMS) was used to mutagenize the wild-type organism to produce mutants. The isolated mutants were screened for the isolation of catabolite repression resistant mutants in the presence of 1% (w/v) glucose as carbon source. A total of fifty mutants were isolated. All the mutants produced cellulase in the presence of CMC as an inducer with specific activity of 0.057, 0.088 and 0.074 units/mg protein for the wild-type, catabolite repression resistant mutant4 (CRRmt 4 ) and catabolite repression resistant mutant24 (CRRmt 24 ), respectively. It was observed that addition of glucose or glycerol as carbon and energy sources to the culture medium resulted into considerable reduction in the cellulolytic activity. However, glycerol appeared to be a better carbon and energy source than glucose which inhibited enzyme expression in most of the strains used in this study. It was also observed that potent cellulase production occurred at the exponential growth phase of the organism. The isolated mutants were grouped into three classes based on their induction ratios namely; unimproved mutants, catabolite repression resistant mutants and mutants with highest induction ratio but sensitive to catabolite repression in the presence of high glucose concentration. The overall results obtained showed that cellulolytic activity in P. fluorescens was regulated by catabolite repression. Key words: Pseudomonas fluorescens , ethylmethanesulphonate, mutants, cellulose, catabolite repression, induction ratio. African Journal of Biotechnology Vol. 4 (8), pp. 838-843

Regulatory mutations affecting the synthesis of cellulase in Pseudomonas fluorescens

Pseudomonas fluorescens , was cultured in basal medium containing carboxymethyl-cellulose (CMC) as inducer and glucose or glycerol as carbon and energy sources. Ethylmethanesulphonate (EMS) was used to mutagenize the wild-type organism to produce mutants. The isolated mutants were screened for the isolation of catabolite repression resistant mutants in the presence of 1% (w/v) glucose as carbon source. A total of fifty mutants were isolated. All the mutants produced cellulase in the presence of CMC as an inducer with specific activity of 0.057, 0.088 and 0.074 units/mg protein for the wild-type, catabolite repression resistant mutant4 (CRRmt 4 ) and catabolite repression resistant mutant24 (CRRmt 24 ), respectively. It was observed that addition of glucose or glycerol as carbon and energy sources to the culture medium resulted into considerable reduction in the cellulolytic activity. However, glycerol appeared to be a better carbon and energy source than glucose which inhibited enzyme expression in most of the strains used in this study. It was also observed that potent cellulase production occurred at the exponential growth phase of the organism. The isolated mutants were grouped into three classes based on their induction ratios namely; unimproved mutants, catabolite repression resistant mutants and mutants with highest induction ratio but sensitive to catabolite repression in the presence of high glucose concentration. The overall results obtained showed that cellulolytic activity in P. fluorescens was regulated by catabolite repression. Key words: Pseudomonas fluorescens , ethylmethanesulphonate, mutants, cellulose, catabolite repression, induction ratio. African Journal of Biotechnology Vol. 4 (8), pp. 838-843

Synthesis of alkaline protease by catabolite repression-resistant Thermoactinomyces sp. E79 mutant

The production of alkaline protease from Thermoactinomyces sp. E79 was repressed by 0.2% (w/v) glucose in the medium. Catabolite repression-resistant mutant M1 was obtained by combined treatment of UV light and N-methyl- N′-nitro-N-nitrosoguanidine. The glucose uptake studies accomplished by [14C]glucose showed that the mutant has lost its ability for glucose uptake. The protease production by mutant M1 in the enzyme production medium was 62 U/mg, which was twice that of the wild-type strain.

Characterization of glucose-specific catabolite repression-resistant mutants of Bacillus subtilis: identification of a novel hexose:H+ symporter.

Insertional mutagenesis was conducted on Bacillus subtilis cells to screen for mutants resistant to catabolite repression. Three classes of mutants that were resistant to glucose-promoted but not mannitol-promoted catabolite repression were identified. Cloning and sequencing of the mutated genes revealed that the mutations occurred in the structural genes for (i) enzyme II of the phosphoenolpyruvate-glucose phosphotransferase (PtsG), (ii) antiterminator GlcT, which controls PtsG synthesis, and (iii) a previously uncharacterized carrier of the major facilitator superfamily, which we have designated GlcP. The last protein exhibits greatest sequence similarity to the fucose:H+ symporter of Escherichia coli and the glucose/galactose:H+ symporter of Brucella abortus. In a wild-type B. subtilis genetic background, the glcP::Tn10 mutation (i) partially but specifically relieved glucose- and sucrose-promoted catabolite repression, (ii) reduced the growth rate in minimal glucose medium, and (iii) reduced rates of [14C]glucose and [14C]methyl alpha-glucoside uptake. In a delta pts genetic background no phenotype was observed, suggesting that expression of the glcP gene required a functional phosphotransferase system. When overproduced in a delta pts mutant of E. coli, GlcP could be shown to specifically transport glucose, mannose, 2-deoxyglucose and methyl alpha-glucoside with low micromolar affinities. Accumulation of the nonmetabolizable glucose analogs was demonstrated, and inhibitor studies suggested a dependency on the proton motive force. We conclude that B. subtilis possesses at least two distinct routes of glucose entry, both of which contribute to the phenomenon of catabolite repression.

Selective isolation of β-amylase derepressed mutants of Bacillus acidopullulyticus

An experiment was conducted to elucidate the mechanism of the regulation of β-amylase synthesis in Bacillus acidopullulyticus NCIMB11608. It was found that the synthesis of β-amylase was induced by starch and repressed by glucose. β-Amylase derepressed mutants of B. acidopullulyticus NCIMB11608 were isolated by nitrosognanidine mutation, using a starch medium containing 2-deoxyglucose as a repressor for the catabolite repression-resistant mutants. One promising mutant, designated as CR27-9-2, was selected which possessed five times the β-amylase activity of its parental strain. It was also found that the differential rate of enzyme synthesis in mutant CR27-9-2 was constant, regardless of the presence or absence of starch. Therefore, it was concluded that the synthesis of β-amylase in mutant CR27-9-2 was both constitutive and catabolite repression-resistant. In addition, CR27-9-2 was also catabolite repression-resistant for the synthesis of other amylolytic enzymes, such as α-amylase, pullulanase, and glucoamylase.

Beijerinckia indica var. penicillanicum penicillin V acylase: enhanced enzyme production by catabolite repression-resistant mutant and effect of solvents on enzyme activity.

Beijerinckia indica var. penicillanicum mutant UREMS-5, producing 168% more penicillin V acylase, was obtained by successive treatment with UV, gamma-irradiation and ethylmethane sulfonate. Penicillin V acylase production by the mutant strain was resistant to catabolite repression by glucose. Incorporation of glucose, sodium glutamate and vegetable oils in the medium enhanced enzyme production. The maximum specific production of penicillin V acylase was 244 IU/g dry weight of cells. Effect of solvents on hydrolysis of penicillin V by soluble penicillin V acylase and whole cells was studied. Methylene chloride, chloroform and carbon tetrachloride significantly stimulated the rate of penicillin V hydrolysis by whole cells.

Production of acetyl xylan esterase by Trichoderma reesei and Schizophyllum commune

Activity of acetyl xylan esterase, an enzyme that removes acetyl groups from acetyl xylan, was coproduced with that of endoxylanase and endoglucanase in two Trichoderma reesei strains and one Schizophyllum commune strain. The levels of activity of extracellular enzymes were measured during the course of cultivation on different carbon sources. The highest activity levels of acetyl xylan esterase were produced by T. reesei QM 9414 in a xylan plus cellulose medium and by S. commune in a cellulose medium. Both strains produced low levels of acetyl xylan esterase activity in glucose, xylose, and cellobiose media. Schizophyllum commune also produced low levels of acetyl xylan esterase activity in xylan and acetyl xylan media. Trichoderma reesei RUT C-30 behaved like a catabolite repression resistant mutant and produced higher enzyme levels than the QM 9414 strain on all carbon sources examined. Analytical gel electrophoresis and isoelectric focusing demonstrated that the acetyl xylan esterase activity of S. commune was represented as one major form (pI 3.4) which also hydrolyzed 4-methylumbelliferyl acetate. The esterase systems of T. reesei strains were found to be more complex than those of S. commune. The pattern of coproduction of the various activities suggested that acetyl xylan esterase is a component of the cellulolytic system of the fungi tested.

Immunoelectron microscopic localization of endoglucanase in the frozen thin sections of a hypercellulolytic mutant of Trichoderma reesei

Endoglucanase (EG) is an enzyme of cellulase complex depolymerizing crystalline cellulose to cellulose microfibril fragments. T. reesei Rut-C30 is a catabolite repression resistant mutant which enhances endoplasmic reticulum (ER) and produces 15-20 times more endoglycanase than the wild type QM6a. About 90% of this enzyme is secreted.Purified secreted EG from three day old culture was used for polyclonal antibody production. The IgG was labeled with 15-18nm colloidal gold. EG secreting mycellia, grown in Vogel's medium with 1% Avicel PH101, were collected by centrifugation. This pellate was washed in sodium cacodylate buffer, fixed in tannic acid-glutaraldehyde mixture and cryoprotected with 30% glycerol. This material was placed on the specimen holders and frozen in liquid freon followed by liquid nitrogen. Frozen thin sections were prepared in a LKB Cryonova microtome using glass knives and collected on sucrose drops.

Regulation and genetic enhancement of beta-amylase production in Clostridium thermosulfurogenes

We studied the general mechanism for regulation of beta-amylase synthesis in Clostridium thermosulfurogenes. beta-Amylase was expressed at high levels only when the organism was grown on maltose or other carbohydrates containing maltose units. Three kinds of mutants altered in beta-amylase production were isolated by using nitrosoguanidine treatment, enrichment on 2-deoxyglucose, and selection of colonies with large clear zones on iodine-stained starch-glucose agar plates. beta-Amylase was produced only when maltose was added to cells growing on sucrose in wild-type and catabolite repression-resistant mutant strains, but the differential rate of enzyme synthesis in constitutive mutants was constant regardless of the presence of maltose. In carbon-limited chemostats of wild-type and catabolite repression-resistant mutant stains, beta-amylase was expressed on maltose but not on glucose or sucrose. beta-Amylase synthesis was immediately repressed by the addition of glucose. Therefore, we concluded that beta-amylase synthesis in C. thermosulfurogenes was inducible and subject to catabolite repression. The addition of cAMP did not eliminate the repressive effect of glucose. The mutants were generally characterized in terms of beta-amylase production, growth properties, fermentation product formation, and alterations in glucose isomerase and glucoamylase activities. A hyperproductive mutant produced eightfold more beta-amylase on starch medium than the wild type and more rapidly fermented starch to ethanol.

Regulation and genetic enhancement of glucoamylase and pullulanase production in Clostridium thermohydrosulfuricum

We studied the general mechanism for regulation of glucoamylase and pullulanase synthesis in Clostridium thermohydrosulfuricum. These amylases were expressed only when the organism was grown on maltose or other carbohydrates containing maltose units. Amylase synthesis was more severely repressed by glucose than by xylose. Catabolite repression-resistant mutants were isolated by using nitrosoguanidine treatment, enrichment on 2-deoxyglucose, and selection of colonies with large clear zones on iodine-stained glucose-starch agar plates. Amylases were produced in both wild-type and mutant strains when starch was added to cells growing on xylose but not when starch was added to cells growing on glucose. In both wild-type and mutant strains, glucoamylase and pullulanase were produced at high levels in starch-limited chemostats but not in glucose- or xylose-limited chemostats. Therefore, we concluded that amylase synthesis in C. thermohydrosulfuricum was inducible and subject to catabolite repression. The mutants produced about twofold more glucoamylase and pullulanase, and they were catabolite repression resistant for production of glucose isomerase, lactase, and isomaltase. The mutants displayed improved starch metabolism features in terms of enhanced rates of growth, ethanol production, and starch consumption.

Suppression of temperature sensitive mutations in oncogene-related CDC genes in Saccharomyces cerevisiae by catabolite repression resistance and cytoplasmic petite mutations.

The "start" cell division control genes CDC36 and CDC28 have been reported to contain a certain sequence homology to tissue oncogenes (ets and some protein kinase encoding oncogenes respectively). Here we report that temperature sensitive mutations in these genes are suppressed in cytoplasmic "petite" mutants and catabolite repression resistant mutants.

Ethanol from Whey: Continuous Fermentation with a Catabolite Repression-Resistant Saccharomyces cerevisiae Mutant

An alternative method for the conversion of cheese whey lactose into ethanol has been demonstrated. With the help of continuous-culture technology, a catabolite repression-resistant mutant of Saccharomyces cerevisiae completely fermented equimolar mixtures of glucose and galactose into ethanol. The first step in this process was a computer-controlled fed-batch operation based on the carbon dioxide evolution rate of the culture. In the absence of inhibitory ethanol concentrations, this step allowed us to obtain high biomass concentrations before continuous fermentation. The continuous anaerobic process successfully incorporated a cell-recycle system to optimize the fermentor productivity. Under conditions permitting a low residual sugar concentration (</=1%), maximum productivity (13.6 g liter h) was gained from 15% substrate in the continuous feed at a dilution rate of 0.2 h. Complete fermentation of highly concentrated feed solutions (20%) was also demonstrated, but only with greatly diminished fermentor productivity (5.5 g liter h).

Cellulase biosynthesis in a catabolite repression-resistant mutant of Thermomonospora curvata.

A catabolite repression-resistant mutant of the thermophilic actinomycete Thermomonospora curvata was obtained by treatment with ethyl methanesulfonate and UV light. Cellulase biosynthesis was undiminished by glucose, 2-deoxyglucose, or alpha-methyl glucoside, which are potent repressors in the wild type. Intracellular cyclic AMP levels were higher in the mutant in both the absence and the presence of repressors.

Enrichment technique for the selection of catabolite repression-resistant mutants of Trichoderma as producers of cellulase

The enrichment technique for the preparation of catabolite repression-resistant producers of cellulase from Trichoderma reesei is based on the submerged cultivation of mutagenized conidia on 2% (w/v) cellobiose or carboxymethyl-cellulose and in the presence of 0.5% (w/v) 2-deoxyglucose as the catabolite repressor. Conidia that are resistant towards the catabolite repressor can produce enzymes necessary for hydrolysis of used substrates and grow under the given conditions. They can be separated from the ungerminated conidia by filtration and used for the production of new conidia which are already enriched with catabolite repression-resistant mutants.

Enrichment technique for the selection of catabolite repression-resistant mutants of Trichoderma as producers of cellulase

The enrichment technique for the preparation of catabolite repression-resistant producers of cellulase from Trichoderma reesei is based on the submerged cultivation of mutagenized conidia on 2% (w/v) cellobiose or carboxymethyl-cellulose and in the presence of 0.5% (w/v) 2-deoxyglucose as the catabolite repressor. Conidia that are resistant towards the catabolite repressor can produce enzymes necessary for hydrolysis of used substrates and grow under the given conditions. They can be separated from the ungerminated conidia by filtration and used for the production of new conidia which are already enriched with catabolite repression-resistant mutants.

Increased endoplasmic reticulum content of a mutant of Trichoderma reesei (RUT-C30) in relation to cellulase synthesis

The structure and content of endoplasmic reticulum (ER) were studied in the shake cultures of two strains of Trichoderma reesei, one wild type (QM6a) and the other a cellulase (EC 3.2.1.4) hyper-producing and catabolite repression resistant mutant (RUT-C30). The results of quantitative electron microscopic and biochemical assays were correlated. At the stage of growth when cellulase secretion was high the ER in RUT-C30 cells was highly developed and more abundant than the ER in QM6a cells. A process regulating ER biogenesis may have been deactivated by mutation in RUT-C30 cells and thus the potential for extracellular enzyme synthesis and secretion has been enhanced.

Improvement of T. reesei strain through mutation and selective screening techniques

A short note on a strain improvement program of Trichoderma reesei QM 9414 initiated in 1976. A mutant, D1-6, was isolated and found to be a distinctly superior strain compared to QM 9414 and several other progenies of this parent. The note presents the possibilities of isolating not only hyperproducing strains but also those which are catabolic repression resistant. In fact, D1-6 is both a hyperproducer and a catabolite repression resistant mutant. (Refs. 7).