Influence of various fermentation variables on exo-glucanase production in Cellulomonas flavigena

The influence of carbon and nitrogen sources on the production of exo-glucanase was investigated. The enzyme production was variable according to the carbon or nitrogen source used. Levels of beta-cellobiohydrolase (CBH) were minimal in the presence of even low concentrations of glucose. Enzyme production was stimulated by other carbohydrates and thus is subject to carbon source control by easily metabolizable sugars. In Dubos medium, on cellobiose, the cellobiohydrolase titres were 2-to 110-fold higher with cells growing on monomeric sugars and 2.7 times higher than cells growing on other disaccharides. alpha-Cellulose was the most effective inducer of beta-cellobiohydrlase and filter paperase (FPase) activities, followed by kallar grass straw. Exogenously supplied glucose inhibited the synthesis of the enzyme in cultures of Cellulomonas flavigena. Nitrates were the best nitrogen sources and supported greater cell mass, cellobiohydrolase and FPase production. During growth on alpha-cellulose containing 8-fold sodium nitrate concentration, maximum volumetric productivities (Qp) of beta-cellobiohydrolase and FPase were 87.5 and 79.5 IU/l./h respectively and are significantly higher than the values reported for some other potent fungi and bacteria

Influence of various fermentation variables on exo-glucanase production in Cellulomonas flavigena

The influence of carbon and nitrogen sources on the production of exo-glucanase was investigated. The enzyme production was variable according to the carbon or nitrogen source used. Levels of \u3b2-cellobiohydrolase (CBH) were minimal in the presence of even low concentrations of glucose. Enzyme production was stimulated by other carbohydrates and thus is subject to carbon source control by easily metabolizable sugars. In Dubos medium, on cellobiose, the cellobiohydrolase titres were 2-to 110-fold higher with cells growing on monomeric sugars and 2.7 times higher than cells growing on other disaccharides. \u3b1-Cellulose was the most effective inducer of \u3b2-cellobiohydrlase and filter paperase (FPase) activities, followed by kallar grass straw. Exogenously supplied glucose inhibited the synthesis of the enzyme in cultures of Cellulomonas flavigena . Nitrates were the best nitrogen sources and supported greater cell mass, cellobiohydrolase and FPase production. During growth on \u3b1-cellulose containing 8-fold sodium nitrate concentration, maximum volumetric productivities (Qp) of \u3b2-cellobiohydrolase and FPase were 87.5 and 79.5 IU/l./h respectively and are significantly higher than the values reported for some other potent fungi and bacteria

Kinetics and Regulation Studies of the Production of β-Galactosidase from Kluyveromyces marxianus Grown on Different Substrates

Lactose-intolerance is manifested in 50 % of the world’s population. This can be remediated by removing lactose from the diet or converting it into glucose and galactose with β-galactosidase (EC 3.2.1.23). In this work, batch production of this enzyme in the presence of lactose, galactose, cellobiose, xylose, arabinose, sucrose and glucose was investigated using Kluyveromyces marxianus in shake flask culture studies. Substrate type and temperature were the independent variables that directly regulated the specific growth and β-galactosidase production rates. Lactose (2 %) supported the maximum specific product yield (YP/X), followed by galactose, sucrose, cellobiose, xylose, arabinose and glucose. Its synthesis was regulated by an induction and a growth-dependent repression mechanism. The optimum temperature for the production was found to be 35–37 °C. The highest volumetric productivity of enzyme (80.0 IU/L/h) occurred on lactose-corn steep liquor medium. This was significantly higher than the calculated values reported in the literature. Thermodynamic studies revealed that the cells provided a defence mechanism against thermal inactivation. The enzyme was stable at 60 °C and pH=5.0–7.0, and it may find application in commercial lactose hydrolysis

Enhanced sporulation and toxin production by a mutant derivative of Bacillus thuringiensis

Bacillus thuringiensis (Bt) strains along with insecticidal crystal proteins (ICP) also produce proteases, and chitinases during growth and sporulation. These both potentate the activity of ICPs. This work aimed to obtain bioinsecticide over-production and thermotolerant mutant through classical mutagenesis of vegetative cells of Bt by using UV-mutation multiple times. The isolated survivors were screened on the basis of their production of β-glucosidase, delta-endotoxins and biomass in glucose-based medium. Maximum spore and crystal proteins were produced at 40°C with corn steep liquor as nitrogen source and hydrol as a carbon source. The best mutant MUV7 supported significantly (P = 0.0001) higher values of all kinetic parameters than those supported by the wild culture. Lower demand of activation energy (47.4 kJ mol-1) for crystal protein formation revealed that the best mutant was comparable with those of thermophilic group of organisms.Key words: Bioinsecticide, biomass, delta-endotoxin, insecticidal crystal proteins, kinetics, spores

Hyper-production of a thermotolerant \u3b2-xylosidase by a deoxy-D-glucose and cycloheximide resistant mutant derivative of Kluyveromyces marxianus PPY 125

Production of \u3b2-xylosidaseby a cycloheximide and 2-deoxy-D-glucose-resistant mutant of Kluyveromyces marxianus PPY125 was studied when cultured on growth media containing galactose, glucose, xylose, cellobiose, sucrose and lactose as carbon sources. Xylose, cellobiose, lactose and sucrose were the key substrates. Both K. marxianus PPY125 and its mutant (M 125) supported maximum \u3b2-xylosidase specific product yield (YP/X) following growth on xylose. Basal level of activity was observed in non-induced cultures grown on glucose. The mutant produced 1.5 to 2-fold more \u3b2-xylosidase than that produced by the wild cells. Synthesis of \u3b2-xylosidase was regulated by an induction mechanism in both wild and mutant cells. Addition of glucose did not inhibit the synthesis of \u3b2-xylosidase in both parental and mutant cultures in the presence of corn steep liquor. Partially purified enzyme showed good stability when incubated at 60\ub0C and was quite stable at pH 5.0-7.0. Thermodynamic studies revealed that the enzyme derived by the mutant M125 was more thermostable as evidenced by higher midpoint inactivation temperature, lower activation energy demand for \u3b2-xyloside hydrolysis, as well as lower enthalpy and entropy demand for reversible denaturation of enzyme

Fungal Biomass Protein Production from Trichoderma harzianum Using Rice Polishing

Industrially important enzymes and microbial biomass proteins have been produced from fungi for more than 50 years. High levels of crude protein as much as 45% are present in fungal biomass with balanced essential amino acids. The aim of this study was to access the potential of Trichoderma harzianum to produce fungal biomass protein from rice polishings. Maximum biomass yield was obtained at 5% (w/v) rice polishings after 72 h of incubation at 28 ∘ C at pH 4. Carbon and nitrogen ratio of 20 : 1 gave significantly higher production of fungal biomass protein. The FBP in the 75 L fermenter contained 49.50% crude protein, 32.00% true protein, 19.45% crude fiber, 9.62% ash, 11.5% cellulose content, and 0.325% RNA content. The profile of amino acids of final FBP exhibited that all essential amino acids were present in great quantities. The FBP produced by this fungus has been shown to be of good nutritional value for supplementation to poultry. The results presented in this study have practical implications in that the fungus T. harzianum could be used successfully to produce fungal biomass protein using rice polishings

Proizvodnja i karakterizacija α-galaktozidaze fermentacijom na čvrstoj podlozi s pomoću višestruko mutiranog soja Aspergillus niger

α-Galactosidase is applied in the sugar industry to enhance sugar recovery from sugar beet syrup and to improve nutritional value of the soymilk. In the present investigation, the influence of process variables on the production of this important enzyme has been explored in a newly isolated multiple mutant strain of Aspergillus niger in solid-state fermentation (SSF). Defined fermentation parameters include substrate type (pure lactose and by-products of rice and flour mills as prime substrates), nitrogen source, incubation time, initial pH of the medium and incubation temperature. Extracellular α-galactosidase reached the value of 135.4 IU/g of dry substrate (IU/g) after 96 h of fermentation. Supplementation with 2 g of glucose and 3 g of corn steep liquor significantly increased the enzyme production, and maximum value of product yield (318 IU/g) by the mutant strain was significantly higher than that reported by the wild type (this work), or other A. niger mutants, recombinants and yeasts reported in literature as producers of elevated levels of α-galactosidase. Among three α-galactosidases, one possessing high subunit molecular mass proteins (99 and 100 kDa) has been characterized in both wild and mutant organisms. Thermal properties of the purified enzymes indicate that the mutation decreased the values of activation energy for the formation of enzyme-substrate (ES) complex, enthalpy, Gibbs free energy demand for substrate binding, and transition state stabilization. A thermodynamic study of irreversible inactivation of enzymes suggests that the mutant–derived enzyme is more thermostable than the native enzyme, which is attributable to amino acids involved in active catalysis. Because of these properties, the mutant organism is a novel organism and may be exploited for bulk production of thermostable α-galactosidase for the above industrial and nutritional applications.U industriji se α-galaktozidaza primjenjuje radi povećanja prinosa šećera iz sirupa šećerne repe te poboljšanja hranjive vrijednosti sojinog mlijeka. U radu je ispitan utjecaj varijabli procesa na proizvodnju ovog važnog enzima fermentacijom na čvrstoj podlozi s pomoću novoizoliranog višestruko mutiranog soja Aspergillus niger. Procijenjeni su sljedeći parametri: supstrat (čista laktoza te nusprodukti meljave riže i brašna), izvor dušika, vrijeme inkubacije, početna pH-vrijednost podloge i temperatura inkubacije. Nakon 96 sati fermentacije dobiveno je 135,4 IU ekstracelularne galaktozidaze po g suhe podloge. Dodatak od 2 g glukoze i 3 g kukuruznog ekstrakta značajno je povećao proizvodnju enzima. S pomoću mutantnog soja postignut je kudikamo veći maksimalni prinos (318 IU/g) nego s divljim sojem (u ovom radu) ili drugim u literaturi navedenim mutantnim sojevima A. niger, rekombinantnim vrstama ili kvascima koji proizvode α-galaktozidazu. Ispitane su tri α-galaktozidaze, od kojih je ona što sadrži podjedinice proteina velike molekularne mase (99 i 100 kDa) karakterizirana i u divljem i u mutantnom soju. Toplinska svojstva pročišćenih enzima pokazuju da je mutacija smanjila energiju aktivacije potrebnu za nastajanje kompleksa enzim-supstrat, entalpiju, količinu Gibbsove slobodne energije utrošene za vezivanje supstrata i stabilizaciju prijelaznog stanja. Termodinamičkim ispitivanjem ireverzibilne inaktivacije enzima zaključeno je da enzim izoliran iz mutantnog soja ima veću termostabilnost od prirodnog enzima zbog aminokiselina u aktivnom katalitičkom procesu. Zbog toga bi se svojstva mutantni organizam mogao upotrijebiti u proizvodnji veće količine termostabilne α-galaktozidaze, za njezinu primjenu u industriji šećera, te radi poboljšanja hranjive vrijednosti proizvoda

Kinetics of endoglucanase and cellobiohydrolase production by parent and mutant derivative of moderately thermotolerant Bacillus subtilis GQ 301542 on optimized medium

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 (-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

Maksimalna proizvodnja intracelularne lipaze s pomoću mutanta Rhizopus oligosporus DGM 31: kinetička studija

Regulation and maximization of lipase production in a mutant derivative of R. oligosporus has been investigated using different substrates, inoculum sizes, pH of the medium, temperature, and nitrogen sources in shake flask experiments and batch fermentation in a fermentor. The production of intracellular lipase was improved 3 times following medium optimization involving one-at-a-time approach and aeration in the fermentor. Interestingly, intracellular lipase was poorly induced by oils, instead its production was induced by sugars, mainly starch, lactose, sucrose, xylose, glucose and glycerol. Dependent variables studied were cell mass, lipase activity, lipase yield, lipase specific and volumetric rate of formation. It was confirmed that lipase production in the derepressed mutant is sufficiently uncoupled from catabolite repression. The results of average specific productivities at various temperatures worked out according to the Arrhenius equation revealed that mutation decreased the magnitude of enthalpy and entropy demand in the inactivation equilibrium during product formation, suggesting that mutation made the metabolic network of the organism thermally more stable. The highest magnitudes of volumetric productivity (QP=490 IU/(L·h)) and other product attributes of lipase formation occurring on optimized medium in the fermentor are greater than the values reported by other workers. The purified enzyme is monomeric in nature and exhibits stability up to 80 °C and pH=6.0–8.0. Activation energy, enthalpy and entropy of catalysis at 50 °C, and magnitudes of Gibbs free energy for substrate binding, transition state stabilization and melting point indicated that this lipase is highly thermostable.Primjenom različitih supstrata, veličina inokuluma, pH-vrijednosti podloge, temperature i izvora dušika regulirana je i maksimizirana proizvodnja lipaze s pomoću mutanta Rhizopus oligosporus DGM 31 u pokusima na tresilici i šaržnim procesom u reaktoru. Proizvodnja intracelularne lipaze povećala se 3 puta optimiranjem podloge i prozračivanjem reaktora. Dodatak ulja malo je poboljšao proizvodnju lipaze, a znatno je poboljšana dodatkom šećera, osobito škroba, laktoze, saharoze, ksiloze, glukoze i glicerola. Ispitane su ove zavisne varijable: masa stanica, aktivnost i prinos lipaze, te specifična i volumetrijska brzina nastajanja enzima. Dokazano je da proizvodnja lipaze s pomoću mutanta ne ovisi o represiji s katabolitima. Vrijednosti prosječne specifične produktivnosti pri različitim temperaturama, izračunate prema Arrheniusovoj jednadžbi, pokazuju da mutacija smanjuje količinu entalpije i entropije potrebne za postizanje ravnotežne inaktivacije tijekom formiranja produkta, te da je omogućila odvijanje toplinski puno stabilnijeg metaboličkog procesa u organizmu. Korištenjem optimirane podloge u reaktoru postignuta je dosad najveća volumetrijska produktivnost lipaze (QP=7,3 IU/g škroba/h). Pročišćeni je enzim monomeran i stabilan čak i do 80 °C i pri pH-vrijednosti od 6,0 do 8,0. Veliku termostabilnost lipaze dokazuju: energija aktivacije, entalpija i entropija potrebne za provedbu katalize pri 50 °C, Gibbsova slobodna energija utrošena za vezanje supstrata, stabilnost prijelaznog stanja i točka taljenja