Gamma radiation induced mutagenesis in Aspergillus niger to enhance its microbial fermentation activity for industrial enzyme production.

α- and ß-Galactosidases find application in food processing, health and nutrition. Aspergillus niger is one of the potent producer of these enzymes and was genotypically improved using gamma-ray induced mutagenesis. The mutant-derivative produced two-fold higher α- and ß-galactosidases. For testing genetic variability and its relationship with phenotypic properties of the two organisms, DNA samples of the mutant and parental strains of A. niger were amplified with 28 deca-nucleotide synthetic primers. RAPD analysis showed significantly different pattern between parental and mutant cultures. The mutant derivative yielded homogeneous while parental strain formed heterogeneous amplification patterns. Seven primers identified 42.9% polymorphism in the amplification products, indicating that these primers determined some genetic variability between the two strains. Thus RAPD was found to be an efficient technique to determine genetic variability in the mutant and wild organisms. Both wild and mutant strains were analyzed for their potential to produce galactosidases. Comparison of different carbon sources on enzyme yield revealed that wheat bran is significant (P < 0.01) effective producer and economical source followed by rice bran, rice polishing and lactose. The mutant was significantly better enzyme producer and could be considered for its prospective application in food, nutrition and health and that RAPD can be effectively used to differentiate mutant strain from the parental strain based on the RAPD patterns.

Enhanced production and characterization of a beta-glucosidase from Bacillus halodurans expressed in Escherichia coli.

A putative beta-glucosidase gene from the genome of Bacillus halodurans C-125 was expressed in E. coli under the regulation of T7lac promoter. On induction with isopropyl-beta-D-1-thiogalactopyranoside, the enzyme expressed at approximately 40% of the cell protein producing 238 mg/liter culture. With increase in culture cell density to A(600) 12 in auto-inducing M9NG medium, beta-glucosidase production increased 3-fold. Approximately 70% of the expressed enzyme was in a soluble form, while the rest was in an insoluble fraction of the cell lysate. The soluble and active form of the expressed enzyme was purified by ammonium sulfate precipitation followed by ion-exchange chromatography to a purity >98%. The mass of the enzyme as determined by MALDI-TOF mass spectrometry was 51,601 Da, which is nearly the same as the calculated value. Phylogenetic analysis of the beta-glucosidase of B. halodurans was found to cluster with members of the genus Bacillus. Temperature and pH optima of the enzyme were found to be 45 degrees C and 8.0, respectively, under the assay conditions. K(m) and k(cat) against p-nitrophenyl-beta-D-glucopyranoside were 4 mM and 0.75 sec(-1), respectively. To our knowledge, this is the first report of high-level expression and characterization of a beta-glucosidase from B. halodurans.

Optimization of process variables for minimization of byproduct formation during fermentation of blackstrap molasses to ethanol at industrial scale.

AIMS: To investigate the effect of molasses concentration, initial pH of molasses medium, and inoculum's size to maximize ethanol and minimize methanol, fusel alcohols, acetic acid and aldehydes in the fermentation mash in industrial fermentors. METHODS AND RESULTS: Initial studies to optimize temperature, nitrogen source, phosphorous source, sulfur supplement and minerals were performed. The essential nutrients were urea (2 kg in 60 m(3)), 0.5 l each of commercial phosphoric acid and sulfuric acid (for pH control) added at the inoculum preparation stage only. Yields of ethanol, methanol, fusel alcohols, total acids and aldehydes per 100-l fermentation broth were monitored. Molasses at 29 degrees Brix (degree of dissolved sugars in water), initial pH 4.5, inoculum size 30% (v/v) and anaerobic fermentation supported maximum ethanol (7.8%) with Y(P/S) = 238 l ethanol per tonne molasses (96.5% yield) (8.2% increase in yield), and had significantly lower values of byproducts than those in control experiments. CONCLUSIONS: Optimization of process variables resulted in higher ethanol yield (8.2%) and reduced yield of methanol, fusel alcohols, acids and aldehydes. SIGNIFICANCE AND IMPACT OF THE STUDY: More than 5% substrate is converted into byproducts. Eliminating or reducing their formation can increase ethanol yield by Saccharomyces cerevisiae, decrease the overall cost of fermentation process and improve the quality of ethanol.

Kinetic parameters and thermodynamic values of beta-xylosidase production by Kluyveromyces marxianus.

Kinetic parameters for production of beta-xylosidase by Kluyveromyces marxianus were determined in growth media containing glucose, xylose, cellobiose, sucrose and lactose as carbon sources. K. marxianus achieved maximum beta-xylosidase specific product yield (Y(P/X)) when grown on xylose. Basal level of activity was achieved in cultures grown on glucose. Kinetic parameters of enzyme production and cell mass formation were correlated. Enzyme synthesis was regulated by an induction mechanism and growth-dependent repression mechanism. Thermodynamic analysis revealed that the cell system exerted protection against thermal inactivation. A partially purified enzyme showed good stability when incubated at 60 degrees C and was quite stable at a pH of 5.0-7.0 and may be exploited for commercial applications.

Kinetic studies of the native and mutated intracellular beta-glucosidases from Cellulomonas biazotea.

The mutation, conferring streptomycin and deoxyglucose resistance on cells, had profound effect on the kinetic and thermodynamic parameters inferring thermostabilization of beta-glucosidase from mutant 51 SM(r) of Cellulomonas biazotea. Free energy of activation for substrate binding, enthalpy and entropy of activation for irreversible denaturation of mutant-derived enzyme were decreased compared with enzyme from wild organism suggesting that the mutation partly stabilized the enzyme and that mutation made it more reactive.

Kinetics and thermodynamics of the native and mutated extracellular endo-glucanases from Cellulomonas biazotea.

The mutation had dramatic effect on the kinetic and thermodynamic parameters inferring thermostability of endo-glucanase from Cellulomonas biazotea mutant 51 SM(r). The denaturation activation energies of native and mutated enzymes were 73.3 and 68.8 kJ/mol respectively. They showed compensation effect at 55 degrees C. Both enthalpy and entropy values of irreversible thermal inactivation for mutated enzyme were decreased suggesting that the mutation partly stabilized the enzyme.

A simple and nondestructive method for the separation of polysaccharides from beta-glucosidase produced extracellularly by Aspergillus niger.

An apparatus based on electrophoresis has been devised that removes noncovalently bound polysaccharides from extracellular proteins of Aspergillus niger with concomitant partial beta-glucosidase purification and concentration. The apparatus consists of a series of three chambers separated by polyacrylamide gels. Dialyzed and concentrated crude extract of Aspergillus niger containing beta-glucosidase was poured into the middle chamber, while smaller anodic and cathodic chambers contained buffer. When electric current was applied, negatively charged protein-polysaccharide complexes moved toward the anode. Most of the negatively charged proteins, including beta-glucosidase, crossed the gel barrier into the anodic compartment, while neutral polysaccharides were either trapped in the gels or remained in the middle chamber. In this way, 125 ml of dialyzed and concentrated crude extract of Aspergillus niger was processed. Therefore, after 24 h of electrophoresis, 68% of the proteins and 90% of the beta-glucosidase activity, but only negligible amounts of polysaccharide, were transferred to the anodic chamber. The removal of high-molecular-weight polysaccharide from beta-glucosidase had a detrimental effect on the stability of the enzyme.

Production of ethanol and xylitol from corn cobs by yeasts.

Saccharomyces cerevisiae and Candida tropicalis were used separately and as co-culture for simultaneous saccharification and fermentation (SSF) of 5-20% (w/v) dry corn cobs. A maximal ethanol concentration of 27, 23, 21 g/l (w/v) from 200 g/l (w/v) dry corn cobs was obtained by S. cerevisiae, C. tropicalis and the co-culture, respectively, after 96 h of fermentation. However, theoretical yields of 82%, 71% and 63% were observed from 50 g/l dry corn cobs for the above cultures, respectively. Maximal xylitol concentration of 21, 20 and 15 g/l from 200 g/l (w/v) dry corn cobs was obtained by C. tropicalis, co-culture, and S. cerevisiae, respectively. Maximum theoretical yields of 79.0%, 77.0% and 58% were observed from 50 g/l of corn cobs, respectively. The volumetric productivities for ethanol and xylitol increased with the increase in substrate concentration, whereas, yield decreased. Glycerol and acetic acid were formed as minor by-products. S. cerevisiae and C. tropicalis resulted in better product yields (0.42 and 0.36 g/g) for ethanol and (0.52 and 0.71 g/g) for xylitol, respectively, whereas, the co-culture showed moderate level of ethanol (0.32 g/g) and almost maximal levels of xylitol (0.69 g/g).

Induction, production, repression, and de-repression of exoglucanase synthesis in Aspergillus niger.

The influence of carbon and nitrogen sources on the production of cellulases was investigated. The enzyme production was variable according to the carbon source. Levels of beta-cellobiohydrolase (CBH) were minimal in the presence of even low concentrations of glucose. Enzyme production was stimulated by other carbohydrates. The enzyme is subject to carbon source control by easily metabolizable sugars. Wheat bran and cellulose were the most effective promoters of beta-cellobiohydrolase and filter paperase (FPase) activities respectively, followed by rice bran. Exogenously supplied glucose inhibited the synthesis of the enzyme in cultures of A. niger growing on wheat bran. In defined medium with cellobiose, the cellobiohydrolase titres were 2- to 110-fold higher with cells growing on monomeric sugars and 1.5 times higher than cells growing on other disaccharides. It appeared that synthesis of beta-cellobiohydrolase varied under an induction mechanism, and a repression mechanism which changed the rate of synthesis of beta-cellobiohydrolase and FPase in induced over non-induced cultures. In this organism, substantial synthesis of beta-cellobiohydrolase can be induced by cellobiose, cellodextrin, cellulose or cellulose and hemi-cellulose containing substrates which showed low volumetric substrate uptake rate. The organism required limiting concentration of carbon, nitrogen or phosphorous for production of beta-cellobiohydrolase and FPase. During growth of A. niger on wheat bran, maximum volumetric productivities (Qp) of beta-cellobiohydrolase and FPase were 39.6 and 32.5 IU/lh and were significantly higher than the values reported for some other potent fungi and bacteria. The addition of actinomycin D (a repressor of transcription) and cycloheximide, (a repressor of translation) completely repressed CBH/FPase biosynthesis, suggested that the regulation of CBH synthesis in this organism occurs at both transcriptional and translational level. Thermodynamic studies revealed that the culture exerted protection against thermal inactivation when exposed to different fermentation temperatures.

Influence of carbon and nitrogen sources and temperature on hyperproduction of a thermotolerant beta-glucosidase from synthetic medium by Kluyveromyces marxianus.

The effect of carbon source and its concentration, inoculum size, yeast extract concentration, nitrogen source, pH of the fermentation medium, and fermentation temperature on beta-glucosidase production by Kluyveromyces marxianus in shake-flask culture was investigated. These were the independent variables that directly regulated the specific growth and beta-glucosidase production rate. The highest product yield, specific product yield, and productivity of beta-glucosidase occurred in the medium (pH 5.5) inoculated with 10% (v/v) inoculum of the culture. Cellobiose (20 g/L) significantly improved beta-glucosidase production measured as product yield (YP/S) and volumetric productivity (QP) followed by sucrose, lactose, and xylose. The highest levels of productivity (144 IU/[L.h]) of beta-glucosidase occurred on cellobiose in the presence of CSL at 35 degrees C and are significantly higher than the values reported by other researchers on almost all other organisms. The thermodynamics and kinetics of beta-glucosidase production and its deactivation are also reported. The enzyme was substantially stable at 60 degrees C and may find application in some industrial processes.

Enhanced production of cellulases byCellulomonas strains grown on different cellulosic residues.

Cellulomonas strains consumed commercial cellulose, cellulosic residues, xylan, cellobiose and carboxymethyl cellulose (CMC) as carbon sources in liquid culture, the growth being the most on cellobiose medium. All three components of the cellulase complex ofCellulomonas were produced when the organisms utilized all substrates as sole carbon and energy sources. The filter-paper cellulase (FPase) and endo-glucanase (CMCase) activities were higher in media containing alpha-cellulose and cellulosic residues than in media containing CMC, cellobiose, and xylan. Cell-free supernatants of all organisms exhibited greater CMC hydrolyzing activity than filter paper and beta-glucoside hydrolyzing activities. All strains synthesized beta-glucosidase maximally on cellobiose followed by commercial cellulose and cellulosic residues.C. biazotea produced the highest FPase and CMCase activity during growth on alpha-cellulose. It was followed byC. flavigena, C. cellasea, andC. fimi. Endo-glucanase and FPase from all organisms were secreted into the medium; 10-13 % became adsorbed on the surface of the insoluble substrates and could be successfully eluted using Tween 80. beta-Glucosidase was located in cell extracts from all organisms.C. biazotea produced FPase and beta-glucosidase activities several-fold greater than those produced by many other strains ofCellulomonas and some other cellulolytic bacteria and fungi.

Cloning of endoglucanase genes from Cellulomonas biazotea into E. coli and S. cerevisiae using shuttle vector YEp24.

We constructed a SmaI genomic library of Cellulomonas biazotea DNA in E. coli and in the S. cerevisiae shuttle vector, YEP 24. Three clone were identified that conferred the ability for E. coli or S. cerevisiae transformants to produce carboxymethylcellulase (CMCase). Cells transformed with these clones were compared with one another and with nontransformed cells for hyper-production of CMCase. In vivo and in vitro studies indicated that the CMCase genes were fully expressed and the enzyme activity was located extracellularly. The optimum pH and temperature for the CMCase thus cloned were pH 7 and 50 degrees C, respectively, as was the case for the donor.

Kinetic analysis of the active site of an intracellular beta-glucosidase fromCellulomonas biazotea.

Purified beta-glucosidase fromCellulomonas biazotea had an apparentK (m) andV for 2-nitrophenyl beta-D: -glucopyranoside (oNPG) of 0.416 mmol/L and 0.22 U/mg protein, respectively. The activation energy for the hydrolysis of pNPG of beta-glucosidase was 65 kJ/mol. The inhibition by Mn(2+) vs. oNPG of parental beta-glucosidase was of mixed type with apparent inhibition constants of 0.19 and 0.60 micromol/L for the enzyme and enzyme-substrate complex, respectively. Ethanol at lower concentrations activated while at higher concentrations it inhibited the enzyme. The determination of apparent pK (a)'s at different temperatures and in the presence of 30 % dioxane indicated two carboxyl groups which control theV value. The thermal stability of beta-glucosidase decreased in the presence of 10 % ethanol. The half-life of beta-glucosidase in 1.75 mol/L urea at 35 degrees C was 145 min, as determined by 0-9 mol/L transverse urea gradient-PAGE.

Purification and the effect of manganese ions on the activity of carboxymethylcellulases from Aspergillus niger and Cellulomonas biazotea.

Carboxymethylcellulases (CMCases) from Aspergillus niger and Cellulomonas biazotea were purified by a combination of ammonium sulfate precipitation, anion-exchange and gel-filtration chromatography with a 12- and 9-fold increase in the purification factor. The native and subunit molar mass of CMCase from A. niger were 40 and 25-57 kDa, respectively, while those from C. biazotea were 23 and 20-30 kDa, respectively. Low concentrations of Mn2+ activated the enzymes from both organisms (mixed activation) with apparent activation constants of 0.80 and 0.45 mmol/L of CMCases from A. niger and C. biazotea, respectively, while at higher CMC concentrations Mn2+ inhibited the enzymes (mixed and partial uncompetitive inhibition). The reason for this complex behavior is that more than one Mn2+ bind to the same enzyme form with the apparent average inhibition constants of 2.7 and 1.3 mmol/L for CMCases from A. niger and C. biazotea, respectively.

Stability and identification of active-site residues of carboxymethylcellulases from Aspergillus niger and Cellulomonas biazotea.

Determination of the apparent pKa's of purified carboxymethylcellulases from Aspergillus niger and Cellulomonas biazotea at different temperatures and in the presence of dioxane indicated two side chain carboxyl groups which controlled the limiting rate in both organisms. The thermostability of both enzymes slightly decreased with increasing pH from 5 to 75 but was unaffected in the presence of 0.5 mmol/L Mn2+. The CMCase from C. biazotea had an activation energy of 35 kJ/mol and a half-life of 89 min in the presence of 8 mol/L urea at 40 degrees C. The half-life of CMCase from A. niger in 8 mol/L urea and at 37 degrees C was 125 min as determined by a 0-9 mol/L transverse urea gradient PAGE. The CMCases from A. niger and C. biazotea had the same thermostabilities in the absence of CMC although the enzyme from the former was more thermostable in the presence of the substrate. The CMCase from A. niger was also more efficient in hydrolyzing CMC than the enzyme from C. biazotea.

Cloning and expression of beta-glucosidase genes in Escherichia coli and Saccharomyces cerevisiae using shuttle vector pYES 2.0.

Genes for beta-glucosidase (Bgl) isolated from a genomic library of the cellulolytic bacterium, Cellulomonas biazotea, were cloned in pUC18 in its SacI cloning site and transformed to E. coli. Ten putative recombinants showed blackening zones on esculin plates, yellow zones on pNPG plates, in liquid culture and on native polyacrylamide gel electrophoresis activity gels. They fell into three distinct groups. Three representative E. coli clones carried recombinant plasmids designated pRM54, pRM1 and pRM17. The genes were located on 5.6-, 3.7- and 1.84-kb fragments, respectively. Their location was obtained by deletion analysis which revealed that 5.5, 3.2, and 1.8 kb fragments were essential to code for BglA, BglB, and BglC, respectively, and conferred intracellular production of beta-glucosidase on E. coli. Expression of the bgl genes resulted in overproduction of beta-glucosidase in the three clones. Secretion occurred into the periplasmic fractions. Three inserts carrying bgl genes from the representative recombinant E. coli were isolated with SacI, ligated in the shuttle vector pYES 2.0 in its SacI site and transformed to E. coli and S. cerevisiae. The recombinant plasmids were redesignated pRPG1, pRPG2 and pRPG3 coding for BglA1, BglB1 and BglC1. The cloned genes conferred extracellular production of beta-glucosidase on S. cerevisiae and enabled it to grow on cellobiose and salicin. The gall promoter of shuttle vector pYES 2.0 enabled the organisms to produce twice more beta-glucosidase than that supported by the lacZ-promoter of pUC18 plasmid in E. coli. The cloned gene can be used as a selection marker for introducing recombinant plasmids in wild strains of S. cerevisiae. The enzyme produced by bgl+ yeast and E. coli recombinants resembles that of the donor with respect to temperature and pH requirement for maximum activity. Other enzyme properties of the beta-glucosidases from S. cerevisiae were substantially the same as those from C. biazotea.

Novel thermodynamics of xylanase formation by a 2-deoxy-d-glucose resistant mutant of Thermomyces lanuginosus and its xylanase potential for biobleachability.

Production of xylanases by Thermomyces lanuginosus wild type and its deoxyglucose resistant mutant M7 on different substrates was investigated. The mutation conferred catabolite repression resistance as M7 supported xylanase production on glucose-based medium that was 8.8-fold higher than that of wild type. Product formation parameters were highest in media containing a combination of corncob and corn steep liquor. Among the soluble substrates, xylose was the best inducer. In the presence of glucose, the wild type produced 26 IU of xylanase per g of glucose while the xylanase yield of the mutant was 224 IU per g of glucose. Thermodynamic studies showed that M7 required lower Gibbs free energy, enthalpy and entropy for product formation than the wild type. In biobleaching studies an 18.6% decrease in kappa number and 2.63% increase in brightness for enzyme-treated pulp was observed. Moreover prebleaching with M7-derived enzyme resulted in a 27.3% reduction in chlorine demand as compared with that of 18.5% decrease when wild organism-derived enzyme was employed. These improvements indicate that the mutant-derived enzymes possessed a useful prebleaching potential and could be exploited for large-scale application.

Kinetics and thermodynamics of ethanol production by a thermotolerant mutant of Saccharomyces cerevisiae in a microprocessor-controlled bioreactor.

AIMS: The present investigation deals with the development of thermotolerant mutant strain of yeast for studying enhanced productivity of ethanol from molasses in a fully controlled bioreactor. METHODS AND RESULTS: The parental culture of Saccharomyces cerevisiae ATCC 26602 was mutated using UV treatment. A single thermotolerant mutant was isolated after extensive screening and optimization, and grown on molasses medium in liquid cultures. The mutant was 1.45-fold improved than its wild parent with respect to ethanol productivity (7.2 g l-1 h-1), product yield (0.44 g ethanol g-1 substrate utilized) and specific ethanol yield (19.0 g ethanol g-1 cells). The improved ethanol productivity was directly correlated with titres of intracellular and extracellular invertase activities. The mutant supported higher volumetric and product yield of ethanol, significantly (P

Kinetic of improved production and carboxymethyl cellulose hydrolysis by an endo-glucanase from a derepressed mutant of Cellulomonas biazotea.

The maximum product yield of endo-glucanase (650 IU g(-1) substrate) from Cellulomonas biazotea mutant 51 Sm(r) was 1.5- to 2.5-fold more than was produced by the wild type cells and was twice that reported by previous researchers. Mutation substantially improved the enthalpy (DeltaH (*)) and entropy of activation (DeltaS (*)) for product formation, turnover number, specificity constant activation energy, free energies for transition state formation and substrate binding for CMC hydrolysis respectively.

Kinetics of improved production and thermostability of an intracellular beta-glucosidase from a mutant-derivative of Cellulomonas biazotea.

The highest productivity (20 IU l(-1) h(-1)) of beta-glucosidase by a mutant of Cellulomonas biazotea was 2.5-fold more than that of the parent organism. The enzyme had a lower activation energy (57 kJ mol(-1)) than the native enzyme (68 kJ mol(-1)). The enzyme from the mutant had enthalpy and entropy values for irreversible intactivation of 95.6 kJ mol(-1) and 60 J.mol(-1) K(-1) compared with 108 kJ mol(-1) and 86 J mol(-1) K(-1) for the native enzyme suggesting that the mutation had stabilized the enzyme.