PT101813B - USE OF DI-MANOSIL-DI-MIO-INOSITOL AND PHOSPHATE OF 1,3-DI-MIO-INOSITOL PHOSPHATE IN THERMOESTABILIZATION, OSMOPROTECTION AND PROTECTION AGAINST DEHYDRATION OF CELL COMPONENTS AND CELLS - Google Patents
USE OF DI-MANOSIL-DI-MIO-INOSITOL AND PHOSPHATE OF 1,3-DI-MIO-INOSITOL PHOSPHATE IN THERMOESTABILIZATION, OSMOPROTECTION AND PROTECTION AGAINST DEHYDRATION OF CELL COMPONENTS AND CELLS Download PDFInfo
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- PT101813B PT101813B PT10181396A PT10181396A PT101813B PT 101813 B PT101813 B PT 101813B PT 10181396 A PT10181396 A PT 10181396A PT 10181396 A PT10181396 A PT 10181396A PT 101813 B PT101813 B PT 101813B
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- inositol phosphate
- phosphate
- mannosyl
- myo
- inositol
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- 229910019142 PO4 Inorganic materials 0.000 title claims abstract description 15
- 239000010452 phosphate Substances 0.000 title claims abstract description 14
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 title claims description 7
- 229960000367 inositol Drugs 0.000 title abstract description 11
- 210000004027 cell Anatomy 0.000 title description 12
- 230000018044 dehydration Effects 0.000 title description 5
- 238000006297 dehydration reaction Methods 0.000 title description 4
- 210000003850 cellular structure Anatomy 0.000 title description 2
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 14
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 14
- 102000004190 Enzymes Human genes 0.000 claims abstract description 11
- 108090000790 Enzymes Proteins 0.000 claims abstract description 11
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 11
- 150000001875 compounds Chemical class 0.000 claims abstract description 11
- 238000000034 method Methods 0.000 claims abstract description 11
- 230000008569 process Effects 0.000 claims abstract description 11
- 239000011942 biocatalyst Substances 0.000 claims abstract description 10
- 239000003599 detergent Substances 0.000 claims abstract description 8
- 239000012528 membrane Substances 0.000 claims abstract description 6
- 239000003960 organic solvent Substances 0.000 claims abstract description 5
- 238000004321 preservation Methods 0.000 claims abstract description 5
- 239000003814 drug Substances 0.000 claims abstract description 4
- 239000002502 liposome Substances 0.000 claims abstract description 4
- 244000005700 microbiome Species 0.000 claims abstract description 4
- 239000002537 cosmetic Substances 0.000 claims abstract description 3
- 229940079593 drug Drugs 0.000 claims abstract description 3
- 229920002521 macromolecule Polymers 0.000 claims abstract description 3
- 239000007800 oxidant agent Substances 0.000 claims abstract description 3
- 241001465754 Metazoa Species 0.000 claims abstract 2
- 230000015572 biosynthetic process Effects 0.000 claims abstract 2
- 238000004108 freeze drying Methods 0.000 claims abstract 2
- 210000000056 organ Anatomy 0.000 claims abstract 2
- 230000006641 stabilisation Effects 0.000 claims description 7
- 238000011105 stabilization Methods 0.000 claims description 7
- 150000002632 lipids Chemical class 0.000 claims description 4
- 238000012423 maintenance Methods 0.000 claims description 3
- 150000003384 small molecules Chemical class 0.000 claims description 3
- 241000204652 Thermotoga Species 0.000 claims description 2
- 241000204664 Thermotoga neapolitana Species 0.000 claims description 2
- 239000003398 denaturant Substances 0.000 claims description 2
- 238000004925 denaturation Methods 0.000 claims description 2
- 230000036425 denaturation Effects 0.000 claims description 2
- 230000000813 microbial effect Effects 0.000 claims description 2
- 230000001737 promoting effect Effects 0.000 claims description 2
- 230000001580 bacterial effect Effects 0.000 claims 1
- 238000001035 drying Methods 0.000 abstract description 7
- 230000000694 effects Effects 0.000 abstract description 7
- 238000007710 freezing Methods 0.000 abstract description 4
- 230000008014 freezing Effects 0.000 abstract description 4
- 239000004202 carbamide Substances 0.000 abstract description 3
- 230000007613 environmental effect Effects 0.000 abstract description 3
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 abstract description 3
- 230000003834 intracellular effect Effects 0.000 abstract description 3
- 230000000087 stabilizing effect Effects 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 241000894006 Bacteria Species 0.000 abstract description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 abstract description 2
- 238000009825 accumulation Methods 0.000 abstract description 2
- 235000021317 phosphate Nutrition 0.000 abstract 8
- 239000002671 adjuvant Substances 0.000 abstract 1
- 230000031018 biological processes and functions Effects 0.000 abstract 1
- 238000010438 heat treatment Methods 0.000 abstract 1
- 230000007774 longterm Effects 0.000 abstract 1
- 230000002035 prolonged effect Effects 0.000 abstract 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 230000001413 cellular effect Effects 0.000 description 6
- 208000005156 Dehydration Diseases 0.000 description 4
- 230000000065 osmolyte Effects 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 230000016571 aggressive behavior Effects 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000012153 distilled water Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 230000007704 transition Effects 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000003196 chaotropic effect Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 230000003204 osmotic effect Effects 0.000 description 2
- 230000001012 protector Effects 0.000 description 2
- JHJLBTNAGRQEKS-UHFFFAOYSA-M sodium bromide Chemical compound [Na+].[Br-] JHJLBTNAGRQEKS-UHFFFAOYSA-M 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000819038 Chichester Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 1
- 239000000232 Lipid Bilayer Substances 0.000 description 1
- 101100496858 Mus musculus Colec12 gene Proteins 0.000 description 1
- 229910004616 Na2MoO4.2H2 O Inorganic materials 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 241000205160 Pyrococcus Species 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 229960002433 cysteine Drugs 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 102000034238 globular proteins Human genes 0.000 description 1
- 108091005896 globular proteins Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000009931 harmful effect Effects 0.000 description 1
- 229910001410 inorganic ion Inorganic materials 0.000 description 1
- 125000003010 ionic group Chemical group 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- DHCDFWKWKRSZHF-UHFFFAOYSA-N sulfurothioic S-acid Chemical compound OS(O)(=O)=S DHCDFWKWKRSZHF-UHFFFAOYSA-N 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 230000005074 turgor pressure Effects 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
Description
DESCRIÇÃODESCRIPTION
Utilização de fosfato de di-manosil-di-mzo-inositol e de fosfato de l,3'-di-m/o-inositol na termoestabilização, osmoprotecção e protecção contra a desidratação de componentes celulares e célulasUse of di-mannosyl-di-mzo-inositol phosphate and 1,3'-di-m / o-inositol phosphate in thermostabilization, osmoprotection and protection against dehydration of cellular components and cells
Vários solutos intracelulares de baixo peso molecular acumulam em níveis elevados em alguns microrganismos quando estes são sujeitos a condições ambientais extremas, nomeadamente de agressão osmótica. Estes solutos servem geralmente como osmólitos em organismos halotolerantes ou halofílicos (que toleram ou exigem meios com elevadas concentrações salinas, respectivamente), ou ainda como reservas de carbono e energia. Na célula, os osmólitos têm como função manter a pressão de turgescência e o volume celular, sem que a sua acumulação prejudique as estruturas e funcionamento celulares em condições de agressão osmótica. Praticamente todos os organismos halotolerantes ou halófilos até agora examinados acumulam ou iões inorgânicos (K*, Nia+, Cf) ou solutos orgânicos. Estes, também designados por solutos compatíveis, incluem açúcares, aminoácidos e substâncias derivadas de aminoácidos, polióis e oligopeptídeos [1].Several low molecular weight intracellular solutes accumulate in high levels in some microorganisms when they are subjected to extreme environmental conditions, namely osmotic aggression. These solutes generally serve as osmolytes in halotolerant or halophilic organisms (which tolerate or require media with high salt concentrations, respectively), or as carbon and energy reserves. In the cell, osmolytes have the function of maintaining the turgor pressure and the cell volume, without their accumulation harming the cellular structures and functioning in conditions of osmotic aggression. Virtually all halotolerant or halophilic organisms examined so far accumulate either inorganic ions (K *, Nia + , Cf) or organic solutes. These, also called compatible solutes, include sugars, amino acids and substances derived from amino acids, polyols and oligopeptides [1].
Para além de serem compatíveis com a actividade celular normal, os osmólitos sintetizados em condições de agressão ambiental exercem um efeito estabilizador acentuado sobre as macromoléculas constituintes celulares, nomeadamente nas proteínas. Tem sido constatado que os solutos compatíveis têm um efeito positivo quer na estabilização térmica das proteínas, quer na sua estabilização na presença de agentes desnaturantes, como detergentes, solventes orgânicos ou agentes caotrópicos - ureia e cloreto de guanidina. Vários estudos têm incidido sobre o mecanismo molecular envolvido na estabilização da estrutura de proteínas por diferentes solutos de baixo peso molecular, nomeadamente osmólitos [2,3,41.In addition to being compatible with normal cellular activity, osmolytes synthesized under conditions of environmental aggression have a marked stabilizing effect on cellular constituent macromolecules, namely proteins. It has been found that compatible solutes have a positive effect both on the thermal stabilization of proteins, and on their stabilization in the presence of denaturing agents, such as detergents, organic solvents or chaotropic agents - urea and guanidine chloride. Several studies have focused on the molecular mechanism involved in the stabilization of the protein structure by different low molecular weight solutes, namely osmolytes [2,3,41.
A adição de compostos de baixo pelo molecular a soluções contendo proteínas conduz a alterações das propriedades físicas da água, tendo como consequência umThe addition of low molecular compounds to solutions containing proteins leads to changes in the physical properties of water, resulting in a
aumento da sua tensão superficial e a exclusão desses compostos da proteína. Assim, a estrutura da água envolvida nas camadas de proteínas fica num estado de elevada organização, o que gera termodinamicamente desvaforável e que conduz à minimização da área de interface água-proteína. A área superficial das proteínas globulares é menor no estado nativo do que no desnaturado, deslocando-se o equilíbrio proteína nativa <=> desnaturada para a esquerda, do que resulta uma maior estabilização da estutura nativa. Esta maior estabilização das proteínas, induzida pela presença de compostos de baixo peso molecular em solução aquosa, permite que biocatalisadores funcionem e catalisem reacções específicas em condições extremas de temperatura, pressão, força iónica, pH ou na presença de solventes orgânicos e detergentes. Esta capacidade estabilizante dos compostos compatíveis reveste-se da maior importância visto que uma das prioridades da biotecnologia moderna passa pela obtenção de biocatalisadores estáveis ou de agentes que estabilizem os biocatalisadores contra a inactivação térmica e química.increased surface tension and the exclusion of these compounds from the protein. Thus, the water structure involved in the protein layers is in a state of high organization, which generates thermodynamically unfavorable and leads to the minimization of the water-protein interface area. The surface area of globular proteins is smaller in the native state than in the denatured one, with the balance of native protein <=> being shifted to the left, resulting in greater stabilization of the native structure. This greater stabilization of proteins, induced by the presence of low molecular weight compounds in aqueous solution, allows biocatalysts to function and catalyze specific reactions under extreme conditions of temperature, pressure, ionic strength, pH or in the presence of organic solvents and detergents. This stabilizing capacity of compatible compounds is of utmost importance since one of the priorities of modern biotechnology is to obtain stable biocatalysts or agents that stabilize biocatalysts against thermal and chemical inactivation.
E também de salientar que os solutos compatíveis contrariam os efeitos prejudiciais provocados pela desidratação em proteínas [5], membranas celulares [6] e células (7,8). Apesar da importância dos processos de secagem e congelação na conservação e armazenagem de amostras biológicas ocorrem sempre efeitos indesejáveis nestas operações, tais como a desnaturação de proteínas sensíveis ou o decréscimo na viabilidade de muitos tipos de células. A preservação de componentes celulares quando desidratados e/ou congelados tem grande importância em aplicações em medicina (conservação de tecidos e outros produtos de origem biológica), na indústria farmacêutica (microencapsulação de fármacos), cosmética (lipossomas), na indústria alimentar (conservação de alimentos) e em investigação científica (manutenção de colecções de culturas de microrganismos). Nos processos de desidratação de membranas celulares e células a remoção da água ligada aos grupos iónicos polares das bicamadas lipídicas, conduz a uma transição de fase dessas estruturas lipídicas. Após re-hidratação, as membranas secas sofrem uma transição de fase inversa, ocorrendo geralmente, defeitos que provocam rupturas em aleumas regiões das membranas. A proteccção exercida pelos solutos de baixo peso molecular nas membranas celulares e células relaciona-se com o tacto destes compostos substituírem a água (removida na secagem) entre as cabeças polares dos lípides, promovendo a manutenção da mesma fase lipídica. mesmo quando estas estruturas seIt should also be noted that the compatible solutes counteract the harmful effects caused by dehydration in proteins [5], cell membranes [6] and cells (7,8). Despite the importance of drying and freezing processes in the conservation and storage of biological samples, there are always undesirable effects in these operations, such as the denaturation of sensitive proteins or the decrease in viability of many types of cells. The preservation of cellular components when dehydrated and / or frozen is of great importance in applications in medicine (preservation of tissues and other products of biological origin), in the pharmaceutical industry (drug microencapsulation), cosmetics (liposomes), in the food industry (preservation of food) and scientific research (maintaining collections of microorganism cultures). In the dehydration processes of cell membranes and cells, the removal of water linked to the polar ionic groups of the lipid bilayers, leads to a phase transition of these lipid structures. After rehydration, the dry membranes undergo an inverse phase transition, with defects generally occurring that cause ruptures in some regions of the membranes. The protection exercised by low molecular weight solutes in cell membranes and cells is related to the feel of these compounds to replace water (removed during drying) between the polar heads of the lipids, promoting the maintenance of the same lipid phase. even when these structures are
encontram desidratadas. Nos processos de desidratação de proteínas os compostos de baixo peso molecular exercem uma protecção através de um mecanismo de que resulta uma maior estabilização da estrutura nativa, análogo ao efeito de protecção exercido por solutos em proteínas, quando sujeitas a temperaturas elevadas ou à presença de agentes desnaturantes em solução aquosa. Assim, a integridade destas estruturas é mantida e a sua actividade normal recuperável numa maior extensão.are dehydrated. In protein dehydration processes, low molecular weight compounds exert a protection through a mechanism that results in greater stabilization of the native structure, analogous to the protective effect exerted by solutes in proteins, when subjected to high temperatures or the presence of agents denaturants in aqueous solution. Thus, the integrity of these structures is maintained and their normal activity recoverable to a greater extent.
Foram identificados alguns solutos em bactérias e arquebacténas hipertermófilas (temperatura miníma de crescimento de 70°C) que revelaram exercer funções de termoproteccção. Esta conclusão baseia-se essencialmente no facto de estes solutos se acumularem preferencialmente durante o crescimento a temperaturas supra-óptimas. Estão nestas condições os solutos fosfato de 1 .F-di-wío-inositol em Pyrococcus furiosas e 2,3'-ciclo-difosfoglicerato em Metíianotliermus fervidas [9.10].Some solutes have been identified in bacteria and hyperthermophilic archebacteria (minimum growth temperature of 70 ° C) that have been shown to exercise thermoprotection functions. This conclusion is essentially based on the fact that these solutes preferentially accumulate during growth at supra-optimal temperatures. These are the phosphate solutes of 1 .F-di-wio-inositol in boiled Pyrococcus and 2,3'-cyclo-diphosphoglycerate in boiled Methylotliermus [9.10].
Os compostos, objecto da presente invenção, fosfato de di-manosil-di-mminositol e fosfato de l,3'-di-/?uo-inositol podem ser obtidos por fermentação de duas bactérias hipertermófilas e halófilas Thermotoga marítima e Thermotoga neapolitana, à temperatura de 88°C (sendo a temperatura óptima de 80cC), utilizando um meio de crescimento [11] que contém em 1 litro de água destilada: 5g de amido, 0,5g de KH2PO4, 20g de NaCl, 0,5g de extracto de levedura, 0,5g de cisteína-HCl, 2mg de NiCl2.6H2O, 2mg de biotina, 20mM de tiossulfato, 15 ml de uma solução de oligoelementos (que contém em 1 litro de água destilada: l,5g de ácido nitriloacético, 3g de MgSO4.2H2O, 0,5g MnSO4.2H2O, lg NaCl, O.lg de FeSO4.7H2O, 0,l8g de CoSO4.7H2O, O.lg de CaCl2.2H2O, 0.18g de ZnSO4.7H2O, O.Olg CuSO4.5H2O, 0,02g de KAI(SO4)2.12H2O. 0.01g de H-.BO3. O.Olg de Na2MoO4.2H2O, 0,025g de NiCl2.6H2O e 0,3g de Na2SeO3.5H2O). 250 ml de uma solução de água do mar (que contêm em l litro de água destilada: 27,7g de NaCl. 7c de MgSO4.2H2O, 5,5g de MgCl2.6H2O, O.65g de KC1, O.lg de NaBr (KBr). 30 mg de H3BO3, 15 mg de SrCl2.6H2O, 10 mg de ácido cítrico, 0,05 mg de Kl e 2,25 mc, de CaCl2.2H2O). O meio foi acertado com uma solução de H2SO4 a pH 6.5. A recuperação deste soluto é feita por extracçao das células a 100JC com uma mistura de etanokágua a 80% [9],The compounds, object of the present invention, di-mannosyl-di-minositol phosphate and 1,3'-di - / - uo-inositol phosphate can be obtained by fermentation of two hyperthermophilic and halophilic bacteria Thermotoga maritime and Thermotoga neapolitana, at temperature 88 ° C (the optimal temperature being 80 C. c) using a growth medium [11] containing in 1 liter of distilled water: 5 g of starch, 0.5 g KH 2 PO 4, 20 g NaCl, 0.5g of yeast extract, 0.5g of cysteine-HCl, 2mg of NiCl 2 .6H 2 O, 2mg of biotin, 20mM of thiosulfate, 15 ml of a trace element solution (which contains in 1 liter of distilled water: 1.5g nitrileacetic acid, 3g MgSO 4 .2H 2 O, 0.5g MnSO 4 .2H 2 O, 1g NaCl, O.lg FeSO 4 .7H 2 O, 0.18g CoSO 4 .7H 2 O , O.lg of CaCl 2 .2H 2 O, 0.18g of ZnSO 4 .7H 2 O, O.Olg CuSO 4 .5H 2 O, 0.02g of KAI (SO 4 ) 2 .12H 2 O. 0.01g of H-.BO 3. O.Olg of Na 2 MoO 4 .2H 2 O, 0.025g of NiCl 2 .6H 2 O and 0.3g of Na 2 SeO 3 .5H 2 O). 250 ml of a solution of sea water (containing in 1 liter of distilled water: 27.7 g of NaCl. 7c of MgSO 4 .2H 2 O, 5.5g of MgCl 2 .6H 2 O, O.65g of KC1 , Na.lg of NaBr (KBr). 30 mg of H 3 BO 3 , 15 mg of SrCl 2 .6H 2 O, 10 mg of citric acid, 0.05 mg of Kl and 2.25 mc of CaCl 2 . 2H 2 O). The medium was adjusted with a solution of H 2 SO 4 at pH 6.5. The recovery of this solute is done by extracting the cells at 100 J C with a mixture of 80% ethanokagua [9],
O facto dos solutos apresentados nesta invenção, fosfato de di-manosil-di-mioinositol e fosfato de 1,3'-di-wío-inositol, serem sintetizados por organismosThe fact that the solutes presented in this invention, di-mannosyl-di-myoinositol phosphate and 1,3'-di-wo-inositol phosphate, are synthesized by organisms
hipertemiófilos, e da sua concentração intracelular aumentar drasticamente a temperaturas de crescimento supraóptimas, toma estes novos solutos excelentes candidatos a acentes termoestabilizadores.hyperemiophiles, and their intracellular concentration increases dramatically at supra-optimal growth temperatures, makes these new solutes excellent candidates for thermostabilizing effects.
Os compostos da presente invenção podem ser utilizados na termostabilização de vários biocatalisadores com interesse industrial afectados por inactivação, diminuição de eficiência, a temperaturas de operação elevadas, tal como acontece na indústria dos detergentes, tratamento de pasta de papel, etapas nos processos de engenharia genética, etc,.The compounds of the present invention can be used in the thermostability of various biocatalysts with industrial interest affected by inactivation, decreased efficiency, at high operating temperatures, as in the detergent industry, pulp treatment, steps in genetic engineering processes , etc,.
Os compostos da presente invenção podem ser utilizados como protectores de biocatalisadores na presença de agentes desnaturamos como solventes orgânicos, detergentes, agentes oxidantes ou agentes caotrópicos, tais corno ureia e cloreto de guanidina.The compounds of the present invention can be used as protectors of biocatalysts in the presence of denaturing agents such as organic solvents, detergents, oxidizing agents or chaotropic agents, such as urea and guanidine chloride.
Estas utilizações podem ser confirmadas pela determinação de actividades enzimáticas de um determinado biocatalisador em função da temperatura e da concentração de agentes desnaturames, na presença e na ausência destes solutos.These uses can be confirmed by the determination of enzymatic activities of a given biocatalyst depending on the temperature and the concentration of denaturing agents, in the presence and absence of these solutes.
Os compostos da presente invenção podem ser utilizados como protectores contra os efeitos negativos durante a secagem e/ou congelamento de componentes celulares (como proteínas e biomembranas), lipossomas, células ou mesmo tecidos.The compounds of the present invention can be used as protectors against negative effects during the drying and / or freezing of cellular components (such as proteins and biomembranes), liposomes, cells or even tissues.
Esta utilização pode ser confirmada adicionando estes solutos aos componentes celulares ou células ames do processo de secagem e/ou congelação. Após re-hidratação e/ou descongelamento, avalia-se o grau de manutenção da conformação molecular (quando se trata de biocatalisadores ou biomembranas) ou da viabilidade celular (quando se trata da conservação de células).This use can be confirmed by adding these solutes to the cellular components or ames cells of the drying and / or freezing process. After rehydration and / or thawing, the degree of maintenance of the molecular conformation (when it comes to biocatalysts or biomembranes) or cell viability (when it comes to cell conservation) is evaluated.
REFERÊNCIAS:REFERENCES:
[1] Brown, A.D. (1990). Microbial WaterStress Physiology - Principies and Perspectives. John Wiley & Sons, Chichester, New York, Brisbane, Toronto, Singapore.[1] Brown, A.D. (1990). Microbial WaterStress Physiology - Principies and Perspectives. John Wiley & Sons, Chichester, New York, Brisbane, Toronto, Singapore.
[2] Arakawa, T. and S.N. Timasheff (1985). The stabilizadon of proteins by osmolytes. Biophys. J. , 47: 411-414.[2] Arakawa, T. and S.N. Timasheff (1985). The stabilizadon of proteins by osmolytes. Biophys. J., 47: 411-414.
[3] Arakawa, T. and S.N. Timasheff (1983). Preferential interactions of proteins with solvent components in aqueous amino acid solutions. Arch. Biochem. Biophys.. 224: 169-177.[3] Arakawa, T. and S.N.Timasheff (1983). Preferential interactions of proteins with solvent components in aqueous amino acid solutions. Arch. Biochem. Biophys .. 224: 169-177.
[4] Arakawa, T. and S.N. Timasheff (19S2). The stabilizadon of protein structure by sugars. Biochem., 21: 6536-6544.[4] Arakawa, T. and S.N.Timasheff (19S2). The stabilized protein structure by sugars. Biochem., 21: 6536-6544.
[5] Carpenter, J.F., Crowe, J.H. and T. Arakawa (1990). Comparision of solute-induced protein stabilizadon in aqueous solution and in the frozen and dried states. J. Dairy Sei., 73: 3627-3636.[5] Carpenter, J.F., Crowe, J.H. and T. Arakawa (1990). Comparision of solute-induced protein stabilized in aqueous solution and in the frozen and dried states. J. Dairy Sci., 73: 3627-3636.
[6] Rudolph, A.S., Crowe, J.H. and L.M. Crowe (1986). Effects of three stabilizing agents proline, betaine, and trehalose - on membrane phospholipids. Arch. Biochem. Biophys., 245: 134-143.[6] Rudolph, A.S., Crowe, J.H. and L.M. Crowe (1986). Effects of three stabilizing agents proline, betaine, and trehalose - on membrane phospholipids. Arch. Biochem. Biophys., 245: 134-143.
[7] Louis, P., Truper, H.G. and E.A. Galinski (1994). Survival of Escherichia coli during drying and storage in the presence of compauble solutes. Appl. Microbiol. Biotechnol. 41: 684-688.[7] Louis, P., Truper, H.G. and E.A. Galinski (1994). Survival of Escherichia coli during drying and storage in the presence of compauble solutes. Appl. Microbiol. Biotechnol. 41: 684-688.
[8] Leslie, S.B., Teter, S.A., Crowe, L.M. and J.H. Crowe (1994). Trehalose lowers membrane phase transitions in dry yeast cells. Biochm. Biophys. Acra. 1192: 7-13.[8] Leslie, S.B., Teter, S.A., Crowe, L.M. and J.H. Crowe (1994). Trehalose lowers membrane phase transitions in dry yeast cells. Biochm. Biophys. Accra. 1192: 7-13.
[9] Martins, L.O. and H Santos (1995) Accumulation of mannosylglycerate and di-mwinositol-phosphate by Pyrococcus furiosus in response to salinity and temperature. Appi. Environ. Microbiol. 61: 3299-3303.[9] Martins, L.O. and H Santos (1995) Accumulation of mannosylglycerate and di-mwinositol-phosphate by Pyrococcus furiosus in response to salinity and temperature. Appi. Environ. Microbiol. 61: 3299-3303.
[10] Hensel, R. and H. Konig (1988). Thermoadaptation of methanogenic bactéria by intracellular ion concentration. FEMS Microbiol. Lett.,49: 75-79.[10] Hensel, R. and H. Konig (1988). Thermoadaptation of methanogenic bacteria by intracellular ion concentration. FEMS Microbiol. Lett., 49: 75-79.
[11] Huber, R, Langworthy, T.A., Konig, H., Thomm, M., Woese, C.R., Sleytr, U.B. and K.O. Stetter (1986). Thermotoga marítima sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90 °C. Arch. Microbiol. 144: 324-333.[11] Huber, R, Langworthy, T.A., Konig, H., Thomm, M., Woese, C.R., Sleytr, U.B. and K.O. Stetter (1986). Thermotoga maritime sp. nov. represents a new genus of unique extremely thermophilic eubacteria growing up to 90 ° C. Arch. Microbiol. 144: 324-333.
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| PT10181396A PT101813B (en) | 1996-01-11 | 1996-01-11 | USE OF DI-MANOSIL-DI-MIO-INOSITOL AND PHOSPHATE OF 1,3-DI-MIO-INOSITOL PHOSPHATE IN THERMOESTABILIZATION, OSMOPROTECTION AND PROTECTION AGAINST DEHYDRATION OF CELL COMPONENTS AND CELLS |
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| PT10181396A PT101813B (en) | 1996-01-11 | 1996-01-11 | USE OF DI-MANOSIL-DI-MIO-INOSITOL AND PHOSPHATE OF 1,3-DI-MIO-INOSITOL PHOSPHATE IN THERMOESTABILIZATION, OSMOPROTECTION AND PROTECTION AGAINST DEHYDRATION OF CELL COMPONENTS AND CELLS |
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