MD1624Z - Process for submerged cultivation of Rhizopus arrhizus CNMN FD 03 fungus strain, producer of lipases - Google Patents
Process for submerged cultivation of Rhizopus arrhizus CNMN FD 03 fungus strain, producer of lipases Download PDFInfo
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- 239000004367 Lipase Substances 0.000 title claims abstract description 23
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- 238000000034 method Methods 0.000 title claims abstract description 15
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- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 6
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- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 6
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims abstract description 6
- 238000003756 stirring Methods 0.000 claims abstract description 6
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- 235000019764 Soybean Meal Nutrition 0.000 claims description 4
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- SNQQJEJPJMXYTR-UHFFFAOYSA-N dimethyl pyridine-2,6-dicarboxylate Chemical compound COC(=O)C1=CC=CC(C(=O)OC)=N1 SNQQJEJPJMXYTR-UHFFFAOYSA-N 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 abstract description 19
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- 239000012153 distilled water Substances 0.000 description 2
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- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 2
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- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 description 1
- PXGOKWXKJXAPGV-UHFFFAOYSA-N Fluorine Chemical compound FF PXGOKWXKJXAPGV-UHFFFAOYSA-N 0.000 description 1
- 102000004157 Hydrolases Human genes 0.000 description 1
- 108090000604 Hydrolases Proteins 0.000 description 1
- 101100396546 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) tif-6 gene Proteins 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
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- 241000235527 Rhizopus Species 0.000 description 1
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 description 1
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- KJNGJIPPQOFCSK-UHFFFAOYSA-N [H][Sr][H] Chemical compound [H][Sr][H] KJNGJIPPQOFCSK-UHFFFAOYSA-N 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- SOIFLUNRINLCBN-UHFFFAOYSA-N ammonium thiocyanate Chemical compound [NH4+].[S-]C#N SOIFLUNRINLCBN-UHFFFAOYSA-N 0.000 description 1
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- 150000001869 cobalt compounds Chemical class 0.000 description 1
- FDBAIUSFRPWFTD-UHFFFAOYSA-L cobalt(2+) dithiocyanate trihydrate Chemical compound O.O.O.[Co++].[S-]C#N.[S-]C#N FDBAIUSFRPWFTD-UHFFFAOYSA-L 0.000 description 1
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- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
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- GWHOGODUVLQCEB-UHFFFAOYSA-N pyridine-2,6-dicarbonyl chloride Chemical compound ClC(=O)C1=CC=CC(C(Cl)=O)=N1 GWHOGODUVLQCEB-UHFFFAOYSA-N 0.000 description 1
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- AHBGXTDRMVNFER-UHFFFAOYSA-L strontium dichloride Chemical compound [Cl-].[Cl-].[Sr+2] AHBGXTDRMVNFER-UHFFFAOYSA-L 0.000 description 1
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- -1 titanate anion Chemical class 0.000 description 1
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Abstract
Description
Invenţia se referă la biotehnologie, în particular, la un procedeu de cultivare submersă a tulpinii de fungi Rhizopus arrhizus CNMN FD 03, producătoare de enzime lipolitice şi poate fi utilizată în industria microbiologică pentru obţinerea lipazelor cu largi aplicări în industria alimentară, de producere şi prelucrare a grăsimilor şi uleiurilor vegetale, în medicină ca mijloc terapeutic şi de diagnostică. The invention relates to biotechnology, in particular, to a process for submerged cultivation of the fungal strain Rhizopus arrhizus CNMN FD 03, producing lipolytic enzymes and can be used in the microbiological industry to obtain lipases with wide applications in the food industry, production and processing of vegetable fats and oils, in medicine as a therapeutic and diagnostic means.
Procedeele moderne de cultivare a tulpinilor fungice producătoare de enzime exocelulare se bazează pe aplicarea rezultatelor cercetărilor clasice îmbinate cu realizările tehnologice şi fizico-chimice moderne, precum studiul particularităţilor de creştere şi sinteză a enzimelor în funcţie de regimul termic, aeraţie, dinamica variaţiei pH-ului mediului, raportul optim al componentelor mediilor nutritive, cantitatea şi tipul materialului semincer, cât şi screening-ul inductorilor specifici reprezentaţi de ingrediente naturale (făina de soia, porumb, tărâţe de grâu, etc.). Componentele lipidice prezente în cantităţi mici în ingredientele din compoziţia mediului stimulează efectiv biosinteza lipazelor (Рубан Е.Л. Микробные липиды и липазы. Изд. Наука, Москва, 1977, p. 132-156; Калунянц К.А., Голгер Л.И. Микробные ферментные препараты. Москва. Пищ. Пром., 1979, p. 14-30). Modern methods of cultivating fungal strains producing exocellular enzymes are based on the application of classical research results combined with modern technological and physicochemical achievements, such as the study of the peculiarities of growth and synthesis of enzymes depending on the thermal regime, aeration, the dynamics of the pH variation of the medium, the optimal ratio of the components of the nutrient media, the amount and type of seed material, as well as the screening of specific inducers represented by natural ingredients (soybean flour, corn, wheat bran, etc.). Lipid components present in small quantities in the ingredients of the medium composition effectively stimulate the biosynthesis of lipases (Ruban E.L. Microbial lipids and lipases. Izd. Nauka, Moscow, 1977, p. 132-156; Kalunyants K.A., Golger L.I. Microbial enzyme preparations. Moscow. Pisch. Prom., 1979, p. 14-30).
Pentru cultivarea submersă clasică a tulpinii Rhizopus arrhizus CNMN FD 03 este cunoscut procedeul, în care se utilizează mediul nutritiv cu compoziţia, g: făină de soia - 35,0; (NH4)2SO4 -1,0; KH2PO4 -1,0; apă potabilă până la 1L [1]. For the classical submerged cultivation of the Rhizopus arrhizus strain CNMN FD 03, the procedure is known, in which the nutrient medium with the composition, g: soybean meal - 35.0; (NH4)2SO4 -1.0; KH2PO4 -1.0; drinking water up to 1L is used [1].
Dezavantajul constă în faptul că raportul componentelor mediului nu asigură realizarea pe deplin a potenţialului biosintetic al culturii, biosinteza lipazelor nu atinge nivelul maxim, iar maxima biosintezei lipazelor se manifestă în ziua a 2-a de cultivare. The disadvantage is that the ratio of the medium components does not ensure the full realization of the biosynthetic potential of the culture, lipase biosynthesis does not reach the maximum level, and the maximum of lipase biosynthesis occurs on the 2nd day of cultivation.
De asemenea, se cunoaşte un procedeu de cultivare a tulpinii Rhizopus arrhizus CNMN FD 03 în aceleaşi condiţii, timp de 48 ore, cu utilizarea în calitate de biostimulator a nanoparticulelor de Fe3O4 care asigură obţinerea unei activităţi lipolitice mult mai mari faţă de invenţie [2] . Also, a process for cultivating the Rhizopus arrhizus CNMN FD 03 strain under the same conditions, for 48 hours, is known, with the use of Fe3O4 nanoparticles as a biostimulator, which ensures the obtaining of a much higher lipolytic activity compared to the invention [2].
Deşi aplicarea nanoparticulelor oferă soluţii rapide şi fiabile în diverse domenii de activitate, inclusiv biotehnologii, impactul acestora asupra mediului şi sănătăţii este imprevizibil, variind semnificativ (benefic sau toxic) în funcţie de caracteristicile nanomaterialelor şi organismelor ţintă şi fiind dificil de prognozat şi controlat. Totodată, nanoparticulele pot suferi diferite transformări (inclusiv biotransformări), care modifică proprietăţile fizico-chimice ale acestora, rezultând un impact asupra mediului diferit de cel pe care îl pot provoca nanoparticulele originale, fiind necesare evaluări individualizate costisitoare (Martínez G., Merinero M., Pérez-Aranda M., Pérez-Soriano E. M., Ortiz T., Begines B., Alcudia A. Environmental Impact of Nanoparticles' Application as an Emerging Technology: A Review. Materials (Basel, Switzerland), 2020, 14(1), p. 166). Although the application of nanoparticles offers rapid and reliable solutions in various fields of activity, including biotechnology, their impact on the environment and health is unpredictable, varying significantly (beneficial or toxic) depending on the characteristics of the nanomaterials and target organisms and being difficult to forecast and control. At the same time, nanoparticles can undergo different transformations (including biotransformations), which modify their physicochemical properties, resulting in an environmental impact different from that caused by the original nanoparticles, requiring costly individualized assessments (Martínez G., Merinero M., Pérez-Aranda M., Pérez-Soriano E. M., Ortiz T., Begines B., Alcudia A. Environmental Impact of Nanoparticles' Application as an Emerging Technology: A Review. Materials (Basel, Switzerland), 2020, 14(1), p. 166).
Alt dezavantaj constă în faptul că deşi activitatea lipolitică este destul de înaltă, maximul de activitate al tulpinii de fungi miceliali Rhizopus arrhizus CNMN FD 03 se atinge în a doua zi de cultivare. Another disadvantage is that although the lipolytic activity is quite high, the maximum activity of the mycelial fungal strain Rhizopus arrhizus CNMN FD 03 is reached on the second day of cultivation.
La unele microorganisme o parte considerabilă de lipaze exocelulare sunt legate de peretele celular, ce poate inhiba secreţia lipazelor în mediul de cultură şi micşorarea randamentului lipazelor exocelulare. Includerea în mediul nutritiv a substanţelor cu abilităţi de stimulare a eliberării lipazelor legate de peretele celular, spre exemplu, surplusul de ioni ai unor metale, accelerează secreţia lipazelor în mediul de cultură şi favorizează procesul de sinteză a lipazelor exocelulare (Fogarty W.M. Микробные ферменты и биотехнология. Москва: Агропромиздат. 1986, p. 189-190). In some microorganisms, a considerable part of exocellular lipases are bound to the cell wall, which can inhibit the secretion of lipases into the culture medium and reduce the yield of exocellular lipases. The inclusion in the nutrient medium of substances with the ability to stimulate the release of lipases bound to the cell wall, for example, the surplus of metal ions, accelerates the secretion of lipases into the culture medium and favors the process of synthesis of exocellular lipases (Fogarty W.M. Microbial enzymes and biotechnology. Moscow: Agropromizdat. 1986, p. 189-190).
În acest context un efect de stimulare a biosintezei lipazelor exocelulare poate asigura şi includerea în mediul nutritiv a anumitor compuşi coordinativi ai unor metale, în special ai metalelor cu rol de microelemente. In this context, a stimulating effect on the biosynthesis of exocellular lipases can also ensure the inclusion in the nutrient medium of certain coordination compounds of some metals, especially metals acting as microelements.
În ultimele decenii în scopul sporirii biosintezei enzimelor la micromicete intensiv se studiază compuşii coordinativi ai elementelor 3d, din care unii s-au manifestat promiţător în calitate de biostimulatori ai biosintezei lipazelor. De exemplu, este cunoscută utilizarea în calitate de biostimulator al sintezei enzimelor lipolitice la tulpina Rhizopus arrhizus CNMN FD 03 a compusului de cobalt: bis(trietanolamin)-cobalt(II)diizobutirat). Dezavantajul acestui complex constă în faptul că sinteza necesită utilizarea unui compus auxiliar mai puţin accesibil ca 3,6-di-2-piridil-1,3,4,5,-tetrazină şi efectul biostimulator se manifestă mai mult în a doua şi a treia zi de cultivare [3]. In recent decades, in order to increase the biosynthesis of enzymes in micromycetes, coordination compounds of 3d elements have been intensively studied, some of which have shown promise as biostimulators of lipase biosynthesis. For example, the use of the cobalt compound bis(triethanolamine)-cobalt(II)diisobutyrate as a biostimulator of the synthesis of lipolytic enzymes in the Rhizopus arrhizus strain CNMN FD 03 is known. The disadvantage of this complex is that the synthesis requires the use of a less accessible auxiliary compound such as 3,6-di-2-pyridyl-1,3,4,5,-tetrazine and the biostimulatory effect is more pronounced on the second and third day of cultivation [3].
De asemenea se cunoaşte că utilizarea în mediul nutritiv a compuşilor coordinativi ai cobaltului(III) - [Co(DH)2(An)2]2[ZnF6]·2H2O şi [Co(DH)2(An)2]2[TiF6] în calitate de biostimulatori asigură creşterea capacităţii biosintetice a micromicetei Rhizopus arrhizus CNMN FD 03 producătoare de lipaze cu efect biostimulator din prima zi de cultivare [4]. It is also known that the use of cobalt(III) coordination compounds - [Co(DH)2(An)2]2[ZnF6]·2H2O and [Co(DH)2(An)2]2[TiF6] in the nutrient medium as biostimulators ensures an increase in the biosynthetic capacity of the micromycete Rhizopus arrhizus CNMN FD 03, which produces lipases with a biostimulatory effect from the first day of cultivation [4].
Deşi biostimulatorul este activ, el nu este stabil la păstrare şi coroziv în raport cu sticla datorită prezenţei fluorului în anionul de hexafluorzirconat/titanat. Although the biostimulator is active, it is not stable during storage and is corrosive to glass due to the presence of fluorine in the hexafluorozirconate/titanate anion.
Cea mai apropiată soluţie de obiectul revendicat este procedeul de cultivare submersă a tulpinii Rhizopus arrhizus CNMN FD 03, în care suspensia de spori, obţinută prin spălare cu apă distilată sterilă a culturii crescute timp de 30 de zile pe suprafeţe înclinate de malţ-agar, se inoculează într-un mediu nutritiv apos cu compoziţia, g/L: făină de soia - 35,0, (NH4)2SO4 - 1,0, KH2PO4 - 5,0, compusul coordinativ CuGly2·H2O - 0,010, utilizat în calitate de biostimulator al biosintezei lipazelor. Cultivarea se realizează în condiţii de agitare continuă (200 rot/min), timp de 48 ore, la temperatura de 28ºC [5]. The closest solution to the claimed object is the submerged cultivation process of the Rhizopus arrhizus strain CNMN FD 03, in which the spore suspension, obtained by washing with sterile distilled water the culture grown for 30 days on inclined malt-agar surfaces, is inoculated into an aqueous nutrient medium with the composition, g/L: soybean meal - 35.0, (NH4)2SO4 - 1.0, KH2PO4 - 5.0, the coordination compound CuGly2·H2O - 0.010, used as a biostimulator of lipase biosynthesis. The cultivation is carried out under conditions of continuous stirring (200 rpm), for 48 hours, at a temperature of 28ºC [5].
Dezavantajul acestei soluţii constă în faptul că concentraţia maximă admisibilă de aplicare a biostimulatorului CuGly2·H2O nu asigură realizarea maximă a potenţialului de biosinteză a lipazelor, totodată maximul de conţinut de lipaze în lichidul cultural se atinge în a doua zi de cultivare. The disadvantage of this solution is that the maximum admissible application concentration of the biostimulator CuGly2·H2O does not ensure the maximum achievement of the biosynthesis potential of lipases, at the same time the maximum content of lipases in the culture liquid is reached on the second day of cultivation.
Problema tehnică pe care o rezolvă invenţia constă în elaborarea unui procedeu de cultivare submersă a tulpinii de fungi Rhizopus arrhizus CNMN FD 03 cu aplicarea unui compus coordinativ în calitate de biostimulator al biosintezei lipazelor, care asigură sporirea capacităţii biosintetice a tulpinii şi reducerea duratei de cultivare submersă cu 24 de ore. The technical problem solved by the invention consists in developing a process for submerged cultivation of the fungal strain Rhizopus arrhizus CNMN FD 03 with the application of a coordinating compound as a biostimulator of lipase biosynthesis, which ensures an increase in the biosynthetic capacity of the strain and a reduction in the duration of submerged cultivation by 24 hours.
Problema se rezolvă prin procedeul de cultivare submersă a tulpinii Rhizopus arrhizus CNMN FD 03, producătoare de lipaze, care prevede obţinerea suspensiei de spori a tulpinii crescute timp de 30 de zile pe un mediu de malţ-agar înclinat, inocularea suspensiei în cantitate de 5% v/v într-un mediu nutritiv apos ce conţine, g/L: făină de soia - 35,0, (NH4)2SO4 - 1,0, KH2PO4 - 5,0, cu adăugarea concomitentă a 0,005 g/L de [SrL3][Co(NCS)4, unde L este dimetilpiridin-2,6-dicarboxilat, şi cultivarea la agitare continuă de 180-200 rot/min în decurs de 24 de ore la temperatura de 28-30°C. The problem is solved by the submerged cultivation process of the lipase-producing Rhizopus arrhizus CNMN FD 03 strain, which provides for obtaining the spore suspension of the strain grown for 30 days on a slanted malt-agar medium, inoculating the suspension in an amount of 5% v/v in an aqueous nutrient medium containing, g/L: soybean meal - 35.0, (NH4)2SO4 - 1.0, KH2PO4 - 5.0, with the simultaneous addition of 0.005 g/L of [SrL3][Co(NCS)4, where L is dimethylpyridine-2,6-dicarboxylate, and cultivating with continuous stirring of 180-200 rpm for 24 hours at a temperature of 28-30°C.
Rezultatul tehnic al invenţiei constă în sporirea biosintezei enzimelor lipolitice faţă de martor şi reducerea duratei de cultivare cu 24 de ore. The technical result of the invention consists in increasing the biosynthesis of lipolytic enzymes compared to the control and reducing the cultivation time by 24 hours.
Rezultatele demonstrează că pe mediul cu biostimulatorul tetra(izotiocianat)cobaltat(II) de tris(dimetilpiridin-2,6-dicarboxilat)stronţiu [SrL3][Co(NCS)4], utilizat în concentraţie de 0,005, 0,010 şi 0,015 g/L, sporirea activităţii lipolitice constituie 13,1-79,5% faţă de martor (maximă de biosinteză a lipazelor la cultivare în condiţii clasice - 34167 U/mL faţă de 28413-61321 U/mL în variantele experimentale), concentraţia optimă fiind de 0,005 g/L. S-a relevat inclusiv faptul că activitatea variantelor experimentale în prima zi de cultivare prezintă valori ale activităţii lipolitice superioare nivelului maximal al probei de referinţă (a 2-a zi). Cea mai favorabilă concentraţie a compusului coordinativ este cea de 0,005 g/L, care asigură în prima zi de cultivare un spor al activităţii lipolitice cu 94,6% faţă de martorul din aceeaşi zi şi 79,5% faţă de valoarea maximă relevată la proba martor în ziua a 2-a de cultivare, depăşind analogul proxim cu 5,6% (61321 U/mL faţă de 58068 U/mL). The results demonstrate that on the medium with the biostimulator tris(dimethylpyridine-2,6-dicarboxylate)strontium tetra(isothiocyanate)cobaltate(II) [SrL3][Co(NCS)4], used in concentrations of 0.005, 0.010 and 0.015 g/L, the increase in lipolytic activity is 13.1-79.5% compared to the control (maximum lipase biosynthesis when cultivated under classical conditions - 34167 U/mL compared to 28413-61321 U/mL in the experimental variants), the optimal concentration being 0.005 g/L. It was also revealed that the activity of the experimental variants on the first day of cultivation presents lipolytic activity values higher than the maximum level of the reference sample (2nd day). The most favorable concentration of the coordinating compound is 0.005 g/L, which ensures on the first day of cultivation an increase in lipolytic activity by 94.6% compared to the control on the same day and 79.5% compared to the maximum value revealed in the control sample on the 2nd day of cultivation, exceeding the closest analogue by 5.6% (61321 U/mL compared to 58068 U/mL).
Exemplu de realizare a invenţiei Example of embodiment of the invention
Procedeul de sinteză a biostimulatorului [SrL3][Co(NCS)4] Synthesis process of the biostimulator [SrL3][Co(NCS)4]
Clorura de stronţiu cu masa de 0,16 g (0,001 mol), tiocianatul de cobalt trihidrat cu masa de 0,23 g (0,001 mol) şi tiocianatul de amoniu cu masa de 0,16 g (0,002 mol) au fost dizolvate în 10 mL metanol (soluţia 1). 2,6-piridindicarbonildiclorură cu masa de 0,61 g (0,003 moli) s-a dizolvat în 12 mL metanol (soluţia 2). Soluţia 1, la o agitare permanentă, se adaugă la soluţia 2, după care, soluţia obţinută de culoare albastră-violetă a fost refluxată timp de 3 ore. După refluxare, soluţia obţinută a fost filtrată şi lăsată la temperatura camerei pentru cristalizare. Peste 24 de ore s-au format cristale de culoare albastră. S-au obţinut 0,11 g de produs. Randamentul constituie 44%. Strontium chloride with a mass of 0.16 g (0.001 mol), cobalt thiocyanate trihydrate with a mass of 0.23 g (0.001 mol) and ammonium thiocyanate with a mass of 0.16 g (0.002 mol) were dissolved in 10 mL methanol (solution 1). 2,6-pyridinedicarbonyldichloride with a mass of 0.61 g (0.003 mol) was dissolved in 12 mL methanol (solution 2). Solution 1, with constant stirring, was added to solution 2, after which the blue-violet solution obtained was refluxed for 3 hours. After refluxing, the solution obtained was filtered and left at room temperature for crystallization. After 24 hours, blue crystals formed. 0.11 g of product was obtained. The yield is 44%.
Găsit, %: C 38,68; H 2,89: Sr 9,13: Co 6,19: N 10,23. Found, %: C 38.68; H 2.89: Sr 9.13: Co 6.19: N 10.23.
Pentru C31H27SrCoN7O12S4 For C31H27SrCoN7O12S4
calculat, %: C 38,60; H 2,82; Sr 9,08; Co 6,11; N 10,17. calculated, %: C 38.60; H 2.82; Sr 9.08; Co 6.11; N 10.17.
Procedeul de cultivare submersă a tulpinii Rhizopus arrhizus CNMN FD 03 Submerged cultivation process of Rhizopus arrhizus strain CNMN FD 03
În prealabil se obţine suspensia de spori a tulpinii de fungi prin spălare cu apă distilată sterilă a culturii de 30 zile, crescute pe suprafeţe înclinate de malţ-agar. Mediul nutritiv se prepară prin dizolvare într-un volum nu prea mare de apă potabilă a cantităţilor precântărite de săruri, urmată de dispersarea minuţioasă a făinii de soia, în raportul masic, g/L: făină de soia - 35,0; KH2PO4 - 5,0; (NH4)2SO4 - 1,0. După care volumul de apă potabilă se aduce până la 1,0 L, pH-ul iniţial al mediului - 8,0. Tulpina de fungi miceliali Rhizopus arrhizus CNMN FD 03 se cultivă în baloane Erlenmeyer cu capacitatea de 0,75 L, care conţin 0,2 L de mediu nutritiv. Mediul nutritiv se inoculează cu suspensie de spori şi miceliu în cantitate de 5% v/v. Soluţia apoasă de biostimulator [SrL3][Co(NCS)4] cu o concentraţie bine determinată, reieşind din intervalul 0,0025-0,015 g/L, nemijlocit înainte de utilizare, se agită discret timp de 2-5 minute pe baie de apă cu ultrasunet de tip DA-968 DADI, după care se adaugă la mediul nutritiv concomitent cu materialul semincer. Cultivarea se realizează în condiţii de agitare continuă (200 rot/min), timp de 24 ore, la temperatura de 28ºC. First, the spore suspension of the fungal strain is obtained by washing with sterile distilled water the 30-day culture, grown on inclined surfaces of malt-agar. The nutrient medium is prepared by dissolving in a not too large volume of drinking water the pre-weighed quantities of salts, followed by the thorough dispersion of soybean flour, in the mass ratio, g/L: soybean flour - 35.0; KH2PO4 - 5.0; (NH4)2SO4 - 1.0. After which the volume of drinking water is brought up to 1.0 L, the initial pH of the medium - 8.0. The mycelial fungal strain Rhizopus arrhizus CNMN FD 03 is cultivated in Erlenmeyer flasks with a capacity of 0.75 L, which contain 0.2 L of nutrient medium. The nutrient medium is inoculated with a spore and mycelium suspension in an amount of 5% v/v. The aqueous solution of biostimulator [SrL3][Co(NCS)4] with a well-determined concentration, ranging from 0.0025-0.015 g/L, immediately before use, is shaken discreetly for 2-5 minutes in a DA-968 DADI ultrasonic water bath, after which it is added to the nutrient medium simultaneously with the seed material. Cultivation is carried out under conditions of continuous stirring (200 rpm), for 24 hours, at a temperature of 28ºC.
Activitatea lipolitică, determinată după gradul de hidroliză în alcool polivinilic a suspensiei de ulei de măsline până la acid oleic după metoda titrimetrică Otto-Iamad (Graceva I.M., şi al., 1982), în variantele experimentale cu aplicarea compusului coordinativ [SrL3][Co(NCS)4] a constituit 39233 U/mL la concentraţia de 0,0025 g/L, 61321 U/mL la concentraţia de 0,005 g/L, 38634 U/mL la concentraţia de 0,010 g/L, şi 28413 U/mL la concentraţia de 0,015 g/L. Maxima biosintezei se manifestă în varianta cu concentraţia metalocomplexului de 0,005 g/L şi constituie 61321 U/mL (Tabel). The lipolytic activity, determined by the degree of hydrolysis in polyvinyl alcohol of the olive oil suspension to oleic acid according to the Otto-Iamad titrimetric method (Graceva I.M., et al., 1982), in the experimental variants with the application of the coordination compound [SrL3][Co(NCS)4] constituted 39233 U/mL at a concentration of 0.0025 g/L, 61321 U/mL at a concentration of 0.005 g/L, 38634 U/mL at a concentration of 0.010 g/L, and 28413 U/mL at a concentration of 0.015 g/L. The maximum of biosynthesis is manifested in the variant with a metallocomplex concentration of 0.005 g/L and constitutes 61321 U/mL (Table).
Tabel Table
Influenţa diferitor concentraţii de stimulator ([SrL3][Co(NCS)4]) asupra The influence of different concentrations of stimulator ([SrL3][Co(NCS)4]) on
activităţii lipolitice a tulpinii Rhizopus arrhizus CNMN FD 03 la cultivarea submersă lipolytic activity of the Rhizopus arrhizus strain CNMN FD 03 in submerged cultivation
Procedeul de cultivare Conc. de stimulator, g/L 1-a zi a 2-a zi Activitatea, U/mL %, de referinţă* Activitatea, U/mL %, faţă de martor Revendicat 0,0025 39233 124,5/114,8/ 40678 119,1 0,005 61321 194,6/179,5/105,6 35397 103,6 0,010 38634 122,6/113,1/ 33723 98,7 0,015 28413 90,2 27334 80,0 Martor (control) - 31500 100,0 34167 100,0 Analogul proxim 58068 100,0;*194,6/179,5/105,6 - faţă de martorul zilei/faţă de valoarea maximă a martorului (ziua a 2-a) /faţă de analogul proxim. Cultivation procedure Stimulator conc., g/L 1st day 2nd day Activity, U/mL %, reference* Activity, U/mL %, compared to control Claimed 0.0025 39233 124.5/114.8/ 40678 119.1 0.005 61321 194.6/179.5/105.6 35397 103.6 0.010 38634 122.6/113.1/ 33723 98.7 0.015 28413 90.2 27334 80.0 Control - 31500 100.0 34167 100.0 Close analog 58068 100.0;*194.6/179.5/105.6 - compared to the control of the day/compared to the maximum value of the control (day 2)/compared to the closest analogue.
Cercetările au fost efectuate în cadrul Programei de Stat 2020-2023 a Rep. Moldova prin proiectele 20.80009.5007.28 şi 20.80009.5007.15 cu finanţarea de către ANCD. The research was conducted within the framework of the 2020-2023 State Program of the Republic of Moldova through projects 20.80009.5007.28 and 20.80009.5007.15 with funding from ANCD.
1. MD 2458 F1 2004.05.31 1. MD 2458 F1 2004.05.31
2. MD 4532 B1 2017.11.30 2. MD 4532 B1 2017.11.30
3. MD a2020 0002 A2 2020.08.31 3. MD a2020 0002 A2 2020.08.31
4. Десятник А., Тюрина Ж., Клапко С., Стратан M., Лаблюк С., Болога О., Коропчану Э., Рижа A., Булхак И. Некоторые аспекты биосинтеза внеклеточных гидролаз микромицетов из родов Rhizopus и Aspergillus в присутствии комплексных соединений кобальта(III) c фторсодержащими анионами. Buletinul Academiei de Ştiinţe a Moldovei. Ştiinţele vieţii, 2010, nr. 1(310), p. 121-128 4. Десятник А., Тюрина Ж., Клапко С., Стратан M., Лаблюк С., Болога О., Коропчану Э., Рижа A., Булхак И. Some aspects of the biosynthesis of extracellular hydrolases of micromycetes from the genera Rhizopus and Aspergillus in the presence of complex compounds of cobalt(III) with fluoride-containing anions. Bulletin of the Academy of Sciences of Moldova. Life Sciences, 2010, no. 1(310), p. 121-128
5. MD 2709 F1 2005.02.28 5. MD 2709 F1 2005.02.28
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