Optimiranje proizvodnje inulinaze s Kluyveromyces bulgaricus

The present work is based on observation of the effects of pH and temperature of fermentation on the production of microbial enzyme inulinase by Kluyveromyces marxianus var. bulgaricus. Inulinase hydrolyzes inulin, a polysaccharide which can be isolated from plants such as Jerusalem artichoke, chicory or dahlia, and transformed into pure fructose or fructooligosaccharides. Fructooligosaccharides have great potential in food industry because they can be used as calorie-reduced compounds and noncariogenic sweeteners as well as soluble fibre and prebiotic compounds. Fructose formation from inulin is a single step enzymatic reaction and yields are up to 95 % the fructose. On the contrary, conventional fructose production from starch needs at least three enzymatic steps, yielding only 45 % of fructose. The process of inulinase production was optimised by using experimental design method. pH value of the cultivation medium showed to be the most significant variable and it should be maintained at optimum value of 3.6. The effect of temperature was slightly lower and optimal values were between 30 and 33 °C. At a low pH value of the cultivation medium, the microorganism was not able to producem enough enzyme and enzyme activities were low. Similar effect was caused by high temperature. The highest values of enzyme activities were achieved at optimal fermentation conditions and the values were: 100.16–124.36 IU/mL (with sucrose as substrate for determination of enzyme activity) or 8.6–11.6 IU/mL (with inulin as substrate), respectively. The method of factorial design and response surface analysis makes it possible to study several factors simultaneously, to quantify the individual effect of each factor and to investigate their possible interactions. As a comparison to this method, optimisation of a physiological enzyme activity model depending on pH and temperature was also studied.Ovaj se rad temelji na ispitivanju utjecaja pH i temperature na proces biosinteze enzima inulinaze s kvascem Kluyveromyces bulgaricus (prije Kluyveromyces marxianus). Mikrobni enzim inulinaza hidrolizira inulin, polisaharid izoliran najčešće iz biljaka (jeruzalemska artičoka, cikorija i dalija), do fruktoze ili fruktooligosaharida. Fruktooligosaharidi se uvelike primjenjuju u prehrambenoj industriji jer se mogu koristiti kao niskokalorični i zaslađivači koji sprečavaju karijes, te kao topljiva vlakna i prebiotički spojevi. Potpuna je hidroliza inulina jednostupanjska enzimska reakcija koju katalizira inulinaza uz 95 %–tni prinos fruktoze. Nasuprot tome, konvencionalna proizvodnja fruktoze iz škroba zahtijeva barem tri stupnja, s tri različita enzima, a prinos je tek 45 % fruktoze. Proces proizvodnje inulinaze optimiran je metodom planiranja pokusa (»experimental design«). pH-vrijednost hranjive podloge pokazala se kao najva`niji parametar u proizvodnji inulinaze i optimalna je vrijednost iznosila 3,6. Temperatura je imala nešto slabiji utjecaj, a optimalne su vrijednosti bile između 30 i 33 °C. Pri niskoj pH-vrijednosti podloge mikroorganizam nije uspijevao proizvesti dovoljnu količinu enzima, pa su i enzimske aktivnosti male. Slično djelovanje uzrokovano je i pri visokim temperaturama uzgoja. Najviše vrijednosti enzimske aktivnosti, postignute pri optimalnim uvjetima, iznosile su: 100,16 do 124,36 U/mL (sa saharozom kao supstratom za određivanje enzimske aktivnosti) ili 8,6 do 11,6 U/mL (s inulinom kao supstratom). Metode planiranja pokusa i analize odzivnih ploha omogućuju promatranje utjecaja nekoliko parametara istodobno, te utvrđivanje intenziteta tih utjecaja, a i njihovih mogućih interakcija. Usporedo je provedeno optimiranje fiziološkog modela enzimske aktivnosti ovisno o pH i temperaturi

Utjecaj organskih otapala na značajke slobodne i imobilizirane inulinaze, izolirane iz kvasca Kluyveromyces marxianus ATCC 16045

The aim of this work is to evaluate the effects of the butyl acetate concentration on the characteristics of free and immobilized inulinase from Kluyveromyces marxianus ATCC 16045. The mass fractions of organic solvent (OS) in sodium acetate buffer (0.1 M) were studied in the range from 25 to 70 %. The characteristics of both free and immobilized enzymes were not significantly affected by the OS mass fraction. The optimal temperature for the free enzyme was 55 °C at all OS mass fractions studied, whereas for the immobilized enzyme the optimum was 55 °C at 70 % of butyl acetate, and in the range from 50 to 60 °C at 25 and 50 % of OS. The optimum pH values, at all OS mass fractions, were 4.8 and 4.4 for the free and immobilized enzymes, respectively. The immobilized enzyme showed more stability at 50 °C and pH=4.8 for the whole range of OS mass fractions, since its stability was improved about 3 times. The kinetics parameters were calculated using Lineweaver-Burk plots. For the free enzyme, the vmax values were 12.5, 58.5 and 37.6 U/mL and the Km values 17.5, 280.7 and 210.4 mM at butyl acetate mass fractions of 25, 50 and 70 %, respectively. Similarly, for the immobilized enzyme, the vmax values were 38.9, 59.5 and 72.5 U/mL and the Km values 3.1, 5.4 and 14.0 mM at the same butyl acetate mass fractions, respectively.Svrha je ovoga rada bila procijeniti utjecaj koncentracije butilnog acetata na značajke slobodne i imobilizirane inulinaze, izolirane iz kvasca Kluyveromyces marxianus ATCC 16045. Upotrijebljeni su maseni udjeli organskih otapala u acetatnom puferu od 25 do 70 %, te je utvrđeno da maseni udjeli otapala nije bitno utjecao na značajke enzima, Optimalna je temperatura za aktivnost slobodnog enzima bila 55 °C pri svim udjelima otapala, a za imobilizirani enzim 55 °C uz dodataka 70 % butilnog acetata, te u rasponu od 50 do 60 °C uz dodatak 25 i 50 % otapala. Utvrđena je optimalna pH-vrijednost od 4,8 za slobodni, te 4,4 za imobilizirani enzim. Stabilnost imobiliziranog enzima bila je trostruko veća pri 50 °C i pH=4,8, pri svim udjelima otapala. Određeni su kinetički parametri primjenom dijagrama Lineweaver-Burk, te utvrđene ove vrijednosti za slobodni enzim: vmax od 12,5 U/mL pri 25 %, 58,5 U/mL pri 50 % i 37,6 U/mL pri 70 % otapala, te Km od 17,5 mM pri 25 %, 280,7 mM pri 50 %, te 210,4 mM pri 70 % otapala. Vrijednosti za imobilizirani enzim iznosile su: vmax od 38,9 U/mL pri 25 %, 59,5 U/mL pri 50 % i 72,5 U/mL pri 70 % otapala, a Km od 3,1 mM pri 25 %, 5,4 mM pri 50 % i 14,0 mM pri 70 % butilnog acetata

Utjecaj organskih otapala na značajke slobodne i imobilizirane inulinaze, izolirane iz kvasca Kluyveromyces marxianus ATCC 16045

The aim of this work is to evaluate the effects of the butyl acetate concentration on the characteristics of free and immobilized inulinase from Kluyveromyces marxianus ATCC 16045. The mass fractions of organic solvent (OS) in sodium acetate buffer (0.1 M) were studied in the range from 25 to 70 %. The characteristics of both free and immobilized enzymes were not significantly affected by the OS mass fraction. The optimal temperature for the free enzyme was 55 °C at all OS mass fractions studied, whereas for the immobilized enzyme the optimum was 55 °C at 70 % of butyl acetate, and in the range from 50 to 60 °C at 25 and 50 % of OS. The optimum pH values, at all OS mass fractions, were 4.8 and 4.4 for the free and immobilized enzymes, respectively. The immobilized enzyme showed more stability at 50 °C and pH=4.8 for the whole range of OS mass fractions, since its stability was improved about 3 times. The kinetics parameters were calculated using Lineweaver-Burk plots. For the free enzyme, the vmax values were 12.5, 58.5 and 37.6 U/mL and the Km values 17.5, 280.7 and 210.4 mM at butyl acetate mass fractions of 25, 50 and 70 %, respectively. Similarly, for the immobilized enzyme, the vmax values were 38.9, 59.5 and 72.5 U/mL and the Km values 3.1, 5.4 and 14.0 mM at the same butyl acetate mass fractions, respectively.Svrha je ovoga rada bila procijeniti utjecaj koncentracije butilnog acetata na značajke slobodne i imobilizirane inulinaze, izolirane iz kvasca Kluyveromyces marxianus ATCC 16045. Upotrijebljeni su maseni udjeli organskih otapala u acetatnom puferu od 25 do 70 %, te je utvrđeno da maseni udjeli otapala nije bitno utjecao na značajke enzima, Optimalna je temperatura za aktivnost slobodnog enzima bila 55 °C pri svim udjelima otapala, a za imobilizirani enzim 55 °C uz dodataka 70 % butilnog acetata, te u rasponu od 50 do 60 °C uz dodatak 25 i 50 % otapala. Utvrđena je optimalna pH-vrijednost od 4,8 za slobodni, te 4,4 za imobilizirani enzim. Stabilnost imobiliziranog enzima bila je trostruko veća pri 50 °C i pH=4,8, pri svim udjelima otapala. Određeni su kinetički parametri primjenom dijagrama Lineweaver-Burk, te utvrđene ove vrijednosti za slobodni enzim: vmax od 12,5 U/mL pri 25 %, 58,5 U/mL pri 50 % i 37,6 U/mL pri 70 % otapala, te Km od 17,5 mM pri 25 %, 280,7 mM pri 50 %, te 210,4 mM pri 70 % otapala. Vrijednosti za imobilizirani enzim iznosile su: vmax od 38,9 U/mL pri 25 %, 59,5 U/mL pri 50 % i 72,5 U/mL pri 70 % otapala, a Km od 3,1 mM pri 25 %, 5,4 mM pri 50 % i 14,0 mM pri 70 % butilnog acetata

Intrinsic Activity of Inulinase from Kluyveromyces marxianus ATCC 16045 and Carbon and Nitrogen Balances

Kvasac Kluyveromyces marxianus ATCC 16045 šaržno je uzgajan na minimalnoj podlozi za maksimalnu proizvodnju inulinaze. Fermentirana podloga je pročišćena brzom proteinskom tekućinskom kromatografijom i ultrafiltracijom, pa je dobivena čista frakcija enzima. Pomocu SDS-PAGE elektroforeze izračunata je molekularna masa od 59 kDa, a mikro Kjeldahlovom metodom određivanja dušika procijenjena je prava aktivnost inulinaze od 879 IU/mg. Ti su rezultati zatim upotrijebljeni za postizanje ravnoteže u procesu fermentacije, što je dovelo do zaključka da se manje od 0,1 % izvora ugljika ili dušika iz podloge ugradilo u ekstracelularnu inulinazu oslobođenu pri različitim uvjetima uzgoja. Informacije dobivene ovim istraživanjem mogu se upotrijebiti za izradu prijedloga metaboličkih modela koji opisuju proizvodnju inulinaze s pomoću ovoga kvasca.Kluyveromyces marxianus ATCC 16045 was cultivated in a batch on minimal medium to overproduce inulinase. The fermented broth was purified by fast protein liquid chromatography and ultrafiltration, and a pure enzyme fraction was obtained. SDS-PAGE electrophoresis allowed calculating molecular mass of 59 kDa, while nitrogen determination by the micro-Kjeldahl method allowed evaluating intrinsic inulinase activity of 879 IU/mg. These results were then used to perform material balances of the fermentation process, which suggested that no more than 0.1 % either of carbon or nitrogen initially present in the medium were incorporated in the extracellular inulinase released under different cultivation conditions. The information obtained in this study can be used for future proposal of metabolic models describing inulinase overproduction by this yeast

Kinetička i termička svojstva sirove i pročišćene β-galaktozidaze, upotrijebljenih za proizvodnju galaktooligosaharida

β-Galactosidase is an enzyme that catalyzes the hydrolysis of lactose. It has potential importance due to various applications in the food and dairy industries, involving lactose-reduced ingredients. The properties of two β-galactosidase enzymes, crude and purified, from different sources, Kluyveromyces marxianus CCT 7082 and Kluyveromyces marxianus ATCC 16045, were analyzed. The pH and temperature optima, deactivation energy, thermal stability and kinetic and thermodynamic parameters were determined, as well as the ability to hydrolyze lactose and produce galactooligosaccharides. Purification process improved the properties of the enzymes, and the results showed that purified enzymes from both strains had a higher optimum temperature, and lower values of Km, thus showing greater affinity for o-nitrophenyl-β-D-galactopiranoside than the crude enzymes. The production of galactooligosaccharides was also greater when using purified enzymes, increasing the synthesis by more than 30 % by both strains.β-Galaktozidaza je enzim koji katalizira hidrolizu laktoze. Primjenjuje se u prehrambenoj i mliječnoj industriji za dobivanje proizvoda sa smanjenim udjelom laktoze. U ovom su radu ispitana svojstva dvaju enzima, sirove i pročišćene β-galaktozidaze, dobivenih iz sojeva kvasca Kluyveromyces marxianus CCT 7082 i Kluyveromyces marxianus ATCC 16045. Određeni su sljedeći parametri: optimalna pH-vrijednost i temperatura, energija deaktivacije, termička stabilnost, kinetički i termodinamički parametri, te sposobnost enzima da hidrolizira laktozu i proizvede galaktooligosaharide. Pročišćavanjem su poboljšana svojstva enzima, što je vidljivo iz rezultata; pročišćeni enzimi iz oba soja imali su višu optimalnu temperaturu i niže vrijednosti Km, te veću specifičnost prema o-nitrofenil-β-D-galaktopiranozidu od sirovih enzima. Također je povećana proizvodnja galaktooligosaharida, i to za 30 % pomoću oba soja

Pharmacoinvasive strategy in elderly up to 75 years or non-elderly: analysis of biochemical and cardiac magnetic resonance imaging parameters

Background: Pharmacoinvasive strategy is an alternative when primary percutaneous coronary intervention (PCI) is not feasible. Objectives: This study aimed to evaluate the effects of early pharmacoinvasive strategy on the infarct size and left ventricular ejection fraction in elderly and non-elderly patients. The role of inflammatory markers was also examined. Methods: Patients (n=223) with ST segment elevation myocardial infarction (STEMI) were prospectively included and submitted to pharmacological thrombolysis in the first six hours, and underwent coronary angiogram and PCI when necessary, in the first 24 hours. Blood samples were collected in the first day (D1) and after 30 days (D30). Cardiac magnetic resonance imaging (cMRI) was performed at D30. Significance was set at p<0.05. Results: Elderly and non-elderly patients showed similar percentage of infarcted mass (13.7 [6.9-17.0] vs. 14.0 [7.3-26.0], respectively, p=0.13) (median [interquartile range]). However, elderly patients had better left ventricular ejection fraction (53 [45-62] vs. 49 [39-58], p=0.025). Titers of interleukin (IL)1beta, IL-4, IL-6, and IL-10 did not differ between D1 and D30, but elderly patients had higher titers for IL-18 at D1 and D30. Absolute numbers of B and T lymphocytes were similar in both groups at D1 and D30, but elderly patients had higher neutrophil/lymphocyte ratio at D30. Multivariate linear regression analysis of cMRI outcomes in the whole population showed that the independent predictors were not different between elderly and non-elderly patients. Conclusion: Pharmacoinvasive strategy in elderly patients was associated with small differences in inflammatory parameters, similar infarct size and better left ventricular function than non-elderly patients

Deep Eutectic Solvents: The Organic Reaction Medium of the Century

This microreview summarizes the use of deep eutectic solvents (DESs) and related melts in organic synthesis. Solvents of this type combine the great advantages of other proposed environmentally benign alternative solvents, such as low toxicity, high availability, low inflammability, high recyclability, low volatility, and low price, avoiding many disadvantages of the more modern media. The fact that many of the components of these mixtures come directly from nature assures their biodegradability and renewability. The classification and distribution of the reactions into different sections in this microreview, as well as the emphasis paid to their scope, easily allow a general reader to understand the actual state of the art and the great opportunities opened, not only for academic purposes but also for industry.This work was supported by the University of Alicante, Spain (VIGROB-173 and UAUSTI13-09)

Role of B lymphocytes in the infarcted mass in patients with acute myocardial infarction

Despite early reperfusion, patients with ST segment elevation myocardial infarction (STEMI) may present large myocardial necrosis and significant impairment of ventricular function. The present study aimed to evaluate the role of subtypes of B lymphocytes and related cytokines in the infarcted mass and left ventricular ejection fraction obtained by cardiac magnetic resonance imaging performed after 30 days of STEMI. This prospective study included 120 subjects with STEMI submitted to pharmacoinvasive strategy. Blood samples were collected in subjects in the first (D1) and 30th (D30) days post STEMI. The amount of CD11b+ B1 lymphocytes (cells/ml) at D1 were related to the infarcted mass (rho = 0.43; P=0.033), measured by cardiac MRI at D30. These B1 cells were associated with CD4+ T lymphocytes at D1 and D30, while B2 classic lymphocytes at day 30 were related to left ventricular ejection fraction (LVEF). Higher titers of circulating IL-4 and IL-10 were observed at D30 versus D1 (P=0.013 and P<0.001, respectively). Titers of IL-6 at D1 were associated with infarcted mass (rho = 0.41, P<0.001) and inversely related to LVEF (rho = -0.38, P<0.001). After multiple linear regression analysis, high-sensitivity troponin T and IL-6 collected at day 1 were independent predictors of infarcted mass and, at day 30, only HDL-C. Regarding LVEF, high-sensitivity troponin T and high-sensitivity C-reactive protein were independent predictors at day 1, and B2 classic lymphocytes, at day 30. In subjects with STEMI, despite early reperfusion, the amount of infarcted mass and ventricular performance were related to inflammatory responses triggered by circulating B lymphocytes

Atherosclerosis severity in patients with familial hypercholesterolemia: the role of T and B lymphocytes

Background and aims Familial hypercholesterolemia (FH) is characterized by lifelong exposure to high LDL-c concentrations and premature atherosclerotic cardiovascular disease; nevertheless, disease severity can be heterogeneous. We aimed at evaluating if the immune-inflammatory system could modulate atherosclerosis burden in FH. Methods From a cohort of subjects with confirmed FH (Dutch Lipid Clinic Network and genotype), 92 patients receiving high-intensity lipid-lowering therapy (statin ± ezetimibe) were included. The extension and severity of coronary atherosclerosis was assessed by standardized reporting systems (CAD-RADS) for coronary computed tomography angiography (CCTA) and coronary artery calcium (CAC) scores. Lipids, apolipoproteins, anti-oxLDL and anti-apolipoprotein B-D peptide (anti-ApoB-D) autoantibodies (IgM and IgG), lymphocytes subtypes, platelet, monocyte and endothelial microparticles (MP), IgM levels (circulating or produced by B1 cells) and cytokines in the supernatant of cultured cells were determined. Multiple linear regression models evaluated associations of these biomarkers with CAC and CAD-RADS scores. Results In univariate analysis CAC correlated with age, systolic blood pressure, TCD4+ cells, and titers of IgM anti-ApoB-D. In multiple linear regression [ANOVA F = 2.976; p = 0.024; R2 = 0.082), CD4+T lymphocytes (B = 35.289; beta = 0.277; p = 0.010; 95%CI for B 8.727 to 61.851), was independently associated with CAC. CAD-RADS correlated with age, systolic blood pressure, titers of IgM anti-ApoB-D, and endothelial MP in univariate analysis. In multiple linear regression, [ANOVA F = 2.790; p = 0.032; R2 = 0.119), only age (B = 0.027; beta = 0.234; p = 0.049; 95% CI for B 0.000 to 0.053) was independent predictor. Conclusions In subjects with FH, under high-intensity lipid-lowering therapy, age and CD4+T cells were associated to atherosclerosis burden