Етаполан — мікробний екзополісахарид мультифункціонального призначення

Етаполан — мікробний екзополісахарид мультифункціонального призначення

Підсумовано опубліковані експериментальні дані стосовно інтенсифікації синтезу, регуляції фізико-хімічних властивостей і практичного використання мікробного екзополісахариду (ЕПС) етаполану (продуцент — Acinetobacter sp. В-7005). Наведено характеристики етаполану і штаму продуцента, аналіз розроблен...

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Datum:2006
Hauptverfasser: Пирог, Т.П., Корж, Ю.В.
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Sprache:Ukrainian
Veröffentlicht: Інститут молекулярної біології і генетики НАН України 2006
Schriftenreihe:Біополімери і клітина
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Огляди
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Zitieren:Етаполан — мікробний екзополісахарид мультифункціонального призначення / Т.П. Пирог, Ю.В. Корж // Біополімери і клітина. — 2006. — Т. 22, № 3. — С. 171-185. — Бібліогр.: 68 назв. — укр., англ.

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spelling nasplib_isofts_kiev_ua-123456789-1566002025-02-09T15:31:40Z Етаполан — мікробний екзополісахарид мультифункціонального призначення Этаполан — микробный экзополисахарид мультифункционального назначения Ethapolan–microbial exopolysaccharide multifunctional assignment Пирог, Т.П. Корж, Ю.В. Огляди Підсумовано опубліковані експериментальні дані стосовно інтенсифікації синтезу, регуляції фізико-хімічних властивостей і практичного використання мікробного екзополісахариду (ЕПС) етаполану (продуцент — Acinetobacter sp. В-7005). Наведено характеристики етаполану і штаму продуцента, аналіз розроблених технологій біосинтезу цього ЕПС на різних вуглецевих субстратах (у тому числі і на су миші ростових С₂-С₆-сполук), переваги етаполану порівняно з відомими ЕПС, а також нові підходи до регуляції синтезу і властивостей етаполану. The review summaries the data concerning the synthesis intensification, the regulation of physical and chemical properties and practical application of microbial exopolysaccharide (EPS) ethapolan (producer – Acinetobacter sp. B-7005). Ethapolan consist of the acylated (C10-C18 fatty acids) and non-acylated components, which are identical by the content of carbohydrates to pyruvic and glucuronic acids. The presence of fatty acids in this EPS composition causes the unique properties of the ethapolan solutions: ability to emulsify; to increase viscosity in presence of the cations, at low pH values and in Cu²⁺-glycin system. These properties determine practical ethapolan application in the oil, chemical, food industry and agriculture. The use of one tone of ethapolan in oil industry enables to obtain 240 tones of oil, in addition. The data were summarized concerning the technological parameters of this EPS biosynthesis on different carbon substrates (ethanol, carbohydrates, mixture of growth С₂-С₆-compounds), which allow decreasing the salts content in the producent cultivation medium 3–4 fold (to 2,95 g/l), to increase increasing the EPS quantity 2–5 fold and controlling simultaneously its composition and its physical and chemical properties necessary for a definite field of the ethapolan application. Суммированы опубликованные экспериментальные данные, касающиеся интенсификации синтеза, регуляции физико-химиче­ских свойств и практического использования микробного экзополисахарида (ЭПС) этаполана (продуцент — Acinetobacter sp. В-7005). Приведены характеристики этаполана и штамма продуцента, анализ разработанных технологий биосинтеза этого ЭПС на разных углеродных субстратах (в том числе и на смеси ростовых С₂-С₆ -соединений), преимущества этапо­лана по сравнению с известными ЭПС, а также новые подходы к регуляции синтеза и свойств этаполана. 2006 Article Етаполан — мікробний екзополісахарид мультифункціонального призначення / Т.П. Пирог, Ю.В. Корж // Біополімери і клітина. — 2006. — Т. 22, № 3. — С. 171-185. — Бібліогр.: 68 назв. — укр., англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.00072E https://nasplib.isofts.kiev.ua/handle/123456789/156600 579.222:577.114 uk Біополімери і клітина application/pdf application/pdf Інститут молекулярної біології і генетики НАН України
institution Digital Library of Periodicals of National Academy of Sciences of Ukraine
collection DSpace DC
language Ukrainian
topic Огляди
Огляди
spellingShingle Огляди
Огляди
Пирог, Т.П.
Корж, Ю.В.
Етаполан — мікробний екзополісахарид мультифункціонального призначення
Біополімери і клітина
description Підсумовано опубліковані експериментальні дані стосовно інтенсифікації синтезу, регуляції фізико-хімічних властивостей і практичного використання мікробного екзополісахариду (ЕПС) етаполану (продуцент — Acinetobacter sp. В-7005). Наведено характеристики етаполану і штаму продуцента, аналіз розроблених технологій біосинтезу цього ЕПС на різних вуглецевих субстратах (у тому числі і на су миші ростових С₂-С₆-сполук), переваги етаполану порівняно з відомими ЕПС, а також нові підходи до регуляції синтезу і властивостей етаполану.
format Article
author Пирог, Т.П.
Корж, Ю.В.
author_facet Пирог, Т.П.
Корж, Ю.В.
author_sort Пирог, Т.П.
title Етаполан — мікробний екзополісахарид мультифункціонального призначення
title_short Етаполан — мікробний екзополісахарид мультифункціонального призначення
title_full Етаполан — мікробний екзополісахарид мультифункціонального призначення
title_fullStr Етаполан — мікробний екзополісахарид мультифункціонального призначення
title_full_unstemmed Етаполан — мікробний екзополісахарид мультифункціонального призначення
title_sort етаполан — мікробний екзополісахарид мультифункціонального призначення
publisher Інститут молекулярної біології і генетики НАН України
publishDate 2006
topic_facet Огляди
url https://nasplib.isofts.kiev.ua/handle/123456789/156600
citation_txt Етаполан — мікробний екзополісахарид мультифункціонального призначення / Т.П. Пирог, Ю.В. Корж // Біополімери і клітина. — 2006. — Т. 22, № 3. — С. 171-185. — Бібліогр.: 68 назв. — укр., англ.
series Біополімери і клітина
work_keys_str_mv AT pirogtp etapolanmíkrobnijekzopolísaharidmulʹtifunkcíonalʹnogopriznačennâ
AT koržûv etapolanmíkrobnijekzopolísaharidmulʹtifunkcíonalʹnogopriznačennâ
AT pirogtp étapolanmikrobnyjékzopolisaharidmulʹtifunkcionalʹnogonaznačeniâ
AT koržûv étapolanmikrobnyjékzopolisaharidmulʹtifunkcionalʹnogonaznačeniâ
AT pirogtp ethapolanmicrobialexopolysaccharidemultifunctionalassignment
AT koržûv ethapolanmicrobialexopolysaccharidemultifunctionalassignment
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last_indexed 2025-11-27T10:36:04Z
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fulltext Ethapolan As A mi cro bial exopolysaccharide of multifunctional IN VOLVe MENT T.P. Pirog, Yu.V. Korzh The In sti tute of Mi cro bi ol ogy and Vi rol ogy, NAS of Ukraine 154 Zabolotny Str., Kyiv GSP, DO 3680, Ukraine E-mail: tapirog@usuft.kiev.ua, Korzh@serv.imv.kiev.ua The re view rep re sents the data of the study of mi cro bial exopolysaccharide (EPS) ethapolan (pro ducer – Acinetobacter sp. B-7005) con cern ing the syn the sis pro cesses ac ti va tion, the in flu ences on its phys i cal and chem i cal prop er ties as well as its prac ti cal ap pli ca tions. Ethapolan con sists of both non-acylated and acylated (con tains the C 10 -C 18 fatty ac ids) com - po nents, which are iden ti cal as for the con tent of car bo hy drates, pyruvic and glucuronic ac ids. The pres ence of fatty ac ids moi ety in this mol e cule de ter mines unique prop er ties of the EPS so lu tions such as abil ity to emul sify; to in crease vis cos ity in the pres ence of the cat ions, and also at low pH val ues and us ing Cu2+-glycin sys tem. Due to these prop er ties the ethapolan can be ex ploited in the pe tro leum pro duc tion, chem i cal, food in dus try and ag ri cul ture. The us ing of as much as one ton of EPS in petrolium in dus try en ables yield ing ad di tional 240 tons of petrolium. The re view sum ma rizes the data of the tech no log i cal pe cu liar i ties of this EPS biosynthesis on dif fer ent car bon sub strates (eth a nol, car bo hy drates, mix ture of growth C 2 -C 6 -com pounds), this al lows de creas ing the salts con cen tra tion in nu tri ent me dium by 3-4 fold (up to 2.95g/l) and el e vat ing by 2-5 fold the quan tity of EPS syn the sized by the pro duces used. Also, the EPS com po si tion of the prop er - ties needed de pend ing on the field of its ex ploi ta tion could be prop erly reg u lated. Key words: exopolysaccharides, ethapolan, in ten si fi ca tion of syn the sis, reg u la tion of me tab o lism, phys i cal and chem i cal prop er ties, mix ture of growth sub strates, biosynthesis Both intracellular and ex og e nous mi cro bial poly - sac cha rides are of great in ter est to re search ers – mi cro - bi ol o gists, bio chem ists, mo lec u lar bi ol o gists, and bio - tech nolo gists. Prac ti cal im por tance of EPS used in small amountsis caused by their abil ity to change sig - nif i cantly rhe o log i cal char ac ter is tics of wa ter sys tems, This is im por tant for EPS us age in pe tro leum, food, chem i cal in dus try, ag ri cul ture and med i cine. The abil ity to EPS syn the sis was re vealed in many mi cro or gan isms, though the syn the sis level of these poly mers fluc tu ates in a broad range for dif fer ent EPS pro duc ers as well as for the same pro ducer at dif fer ent con di tions of its cul ti va tion. The elab o ra tion of highly ef fec tive tech nol o gies of ob tain ing me tab o lites, im por - tant for prac ti cal ap pli ca tion, is based on the pur pose ful reg u la tion of biosynthesis pro cess. This re quires deep knowl edge of phys i ol ogy, bio chem is try and ge net ics of pro duc ers. A strain of Acinetobacter sp. bac te ria which is a pro ducer of ethapolan, a com plex polysaccharide prep - a ra tion, was se lected in the In sti tute of Mi cro bi ol ogy and Vi rol ogy, NAS of Ukraine. Due to its unique phys i - cal and chem i cal prop er ties this biopolymer may be 171 ISSN 0233-7657. Biopolymers and cell. 2006. Vol. 22. ISS 3. Translated from Ukrainia. ã T.P. PIROG, YU.V. KORZH, 2006 Reviews con sid ered as a polysaccharide of multifunctional ap - pli ca tion. The char ac ter is tics of ethapolan pro ducer. Acinetobacter sp. strain was de pos ited with the Ukrai - nian mi cro or gan isms col lec tion as B-7005 num ber. The strain was iso lated from EPS-pro duc ing ac cu mu lat ing cul ture, ob tained as a re sult of sev eral sub se quent reinoculations of a sam ple of ac ti vated sludge of the bi - o log i cal pu ri fi ca tion sta tion of sew age wa ter of Nadvornyansky pe tro leum re fin ery (Ukraine) on the min eral me dium con tain ing eth a nol [1, 2]. The in ves ti ga tion of morphologic-cul ture prop er - ties of this strain showed that in the ex po nen tial phase of growth the cells are rod-like (thick short rods), and in the sta tion ary phase they are coccus-like, lo cated in pairs (diploforms) or in small chains. The cells hav ing the size of (0.95-1.5) x (1.2-2.0) mi crom e ter do not pro - duce any spores. Cells re pro duc tion oc curs by bi nary di vi sion. The cells are gram-neg a tive and not motile. On agarosed wort me dium they pro duce smooth lus - trous muculent col o nies of cream col our. The size of a three-day col ony is 4-5 mm at di am e ter. On agarosed min eral me dium con tain ing ac e tate, eth a nol, or su crose the col o nies are lus trous, mucoid, with the size of 1-2 mm; on the meat in fu sion agar they are smooth, white, pro tu ber ant, mucoid, shown the size of 3 mm at the di - am e ter. Dur ing the cul ti va tion on liq uid me dia the cell pop u la tion pro duces a ho mog e nous sus pen sion. The strain is an ob li gate aer obe, which is catalase-pos i tive and oxidase-neg a tive. It grows on com plex or ganic me - dia, and ac e tate is ac cu mu lated at the growth on min eral me dium con tain ing eth a nol lack ing growth fac tors in cul ture liq uid [2]. Ethapolan pro ducer is a nat u ral auxotroph, which needs for its growth pantothenic acid and non-iden ti - fied growth fac tor that is in yeast ex tract [2]. Tax o nomic study of ethapolan pro ducer. On the ba - sis of the in ves ti ga tion of mor pho log i cal, phys i o log i cal in ves ti ga tions, con ducted in late 80s of the pre vi ous cen tury, ethapolan pro ducer was placed among Acinetobacter sp. Ac cord ing to the 9th edi tion of Bergey’s man ual of sys tem atic bac te ri ol ogy [3], all the strains of Acinetobacter sp. bac te ria orig i nate from of one kind of Acinetobacter calcoaceticus. Due to the fact that the in ves ti gated strain dif fered from A. calcoaceticus in some prop er ties, it was iden ti fied as Acinetobacter sp. [1]. It should be men tioned that af ter the pub li ca tion of the abovementioned edi tion on sys - tem atic bac te ri ol ogy, sev eral doz ens kinds of Acinetobacter sp. were de scribed [4], but ethapolan pro ducer was dif fer ent from them as well. A great vol - ume of in for ma tion con cern ing B-7005 strain prop er - ties caused the ne ces sity of de fin ing its tax o nomic sta - tus. There fore a se ries of mo lec u lar bi o log i cal re - searches of this strain were con ducted, the anal y sis of 16S rRNA, in par tic u lar. Gene 16S rRNA se quenc ing proved that B-7005 strain is the clos est to the mem bers of Rhodobacter and Paracoccus spe cies [5]. Thus, the strain of Acinetobacter sp. B-7005 is a mem ber of a sin gle clus ter with a photosynthetic strain of Rhodobacter capsulatus ATCC 11166 (D16428) re - veal ing the sim i lar ity co ef fi cient as 95.7%. The strain of Acinetobacter sp. B7005 is also close to photosynthetic Rhodobacter massiliae and R. sphaeroides bac te ria (the sim i lar ity level achieves 94.6-94.3%) and non-photosynthetic Paracoccus bac - te ria (the sim i lar ity level – 95.2-95.0%). Con sid er ing the re sults ob tained, nu cle o tide se quences of 16S rRNA genes of sev eral strains of R. capsulatus and Paracoccus denitrificans were com pared us ing the ClastalX pro gram (1.81 ver sion). It was de ter mined that dif fer ences be tween 16S rRNA genes of these kinds are lo cated in the fol low ing po si tions: 36 non-con cur rent nu cleo tides are dis trib - uted prac ti cally equally in dif fer ent loci of 16S rRNA gene and 26 non-con cur rent nu cleo tides are con cen - trated in one of 16S rRNA gene frag ments (which cor - re sponds to the nu cle o tide po si tions from 935 to 980). In the same frag ment 16S rRNA Acinetobacter sp. B-7005 gene is prac ti cally iden ti cal to R. capsulatus. At the same time in other po si tions (where there are 36 non-con cur rent nu cleo tides in R. capsulatus and P. denitrificans) 16S rRNA gene of Acinetobacter sp. B-7005 strain is iden ti cal to P. denitrificans. In this work the strain name is given ac cord ing to data avail - able at the De pos i tary of mi cro or gan isms of the In sti - tute of Mi cro bi ol ogy and Vi rol ogy, NAS of Ukraine (Acinetobacter sp. B-7005); it could be seen that the men tioned dis crep an cies of nu cle o tide se quence in dif - fer ent 16S rRNA gene frag ments did not al low the iden - ti fi ca tion of B-7005 strain as a mem ber of Rhodobacter or Paracoccus spe cies. 172 T.P. Pirog, Yu.V. Korzh The prep a ra tion char ac ter is tics of ethapolan, a mi - cro bial exopolysaccharide. Ethapolan, a com plex polysaccharide ma te rial, con sists of neu tral and acid poly sac cha rides [2, 6-8]. A neu tral polysaccharide, con tain ing glu cose, manose, galactose (3:2:1) is a mi - nor com po nent – be cause its con tent does not ex ceed 5-6%. Ac cord ing to the data of gas-liq uid chro ma tog ra - phy, the res i dues of glu cose, manose, galactose and rhamnose are found in the com po si tion of acid EPS in the mo lar cor re la tion of 3:2:1:1. The pres ence of uronic and pyruvic ac ids is de ter mined in acid EPS. In the pro cess of al ka line pro cess ing of acid EPS the mix ture of fatty ac ids was de ter mined. The main com po nents turned out to be dodecanoic acid, hexadecane acid, hexadecenic acid, octadecanoic acid and cis-octadecenic acid in the as 10:29:12:7:20 [1, 8]. It is ap pro pri ate to men tion that the pres ence of fatty ac - ids, etherifying a car bon chain, is not char ac ter is tic for mi cro bial EPS. 40-50% of car bo hy drates were re vealed in acid EPS con tent, pro tein was not found at all. This was proved by both neg a tive col oured re ac tions of Lori and Brad ford, and the data of 13C-NMR-spec tros copy. In 13C-NMR spec tra of this EPS the sig nals with the chem - i cal shift of 55-57 m.d., char ac ter is tic of C-N-bond are ab sent [9]. Acid EPS also con tains 20-30% of min eral com po nents [8, 10]. It was es tab lished that de pend ing on the na ture of the car bon source and po tas sium cat ions con cen tra tion used in the cul ti va tion me dium of Acinetobacter sp. B-7005 the con tent of PVK res i dues in EPS var ies (in % to the mass of rel a tively dry sub stance) from 3.0 to 4.3; for uronic ac ids it is 15.3 to 22.5; for fatty ac ids it is from 1.8 to 6.5 [2, 8, 11]. Our fur ther re searches showed that the acid com po - nent of ethapolan con sists both of acylated (AP) and non-acylated (NAP) poly sac cha rides [7]. For this rea - son a sim ple method to sep a rate the EPS into acylated and non-acylated com po nents was de vel oped. It was based on the emul si fy ing prop er ties of acylated EPS so - lu tions [7]. The need to de velop such method was caused by the fact that the meth ods poly sac cha rides sub di vid ing, so the one could con tain fatty ac ids, were un known. The ap proaches, avail able at that time, al - lowed sep a rat ing poly sac cha rides into com po nents of dif fer ent mo lec u lar weight (e.g. the meth ods of gel-chro ma tog ra phy [12] and gra di ent centrifugation [13]); as well as fractioning acid and neu tral poly sac - cha rides (on cel lu lose anionites – DEAE and ECTEOLA or at treat ing with bro mide cetylthreemetylamonium) [14-16]. The avail able method of the com po nents, as a hy dro pho bic part, was frac tion ation us ing the col umn with hy dro pho bic car ri - ers. How ever, its us age to sep a rate EPS in large amounts was time and la bor-con sum ing. The au thors of the work [17] used emulsification of car bo hy drate in EPS so lu tion at the iso la tion of acylated polysaccharide emulsane, syn the sized Acinetobacter calcoaceticus RAG-1. Our ex per i ments showed that this ap proach can also be suc cess fully used for the sep a ra tion of poly sac cha rides, one of which con tains res i dues of fatty ac ids. The com par a tive anal y sis of the chem i cal com po si - tion of yielded EPS, AP and NAP ma te rial, iso lated from it, did n’t dif fer in mo lar re la tion of D-glu cose, D-manose, D-galactose, L-rhamnose, D-glucuronic and pyruvic ac ids (3:2:1:1:1:1). The dif fer ences be - 173 Ethapolan As A mi cro bial exopolysaccharide tween these poly sac cha rides cover only the fact that acylated EPS con tains fatty ac ids (C10-C18) [7, 18, 19]. The same struc ture of a re peated unit of a car bo hy - drate chain of non-acylated polysaccharide and acylated EPS af ter the ex trac tion of fatty ac ids was de - ter mined on the ba sis of chem i cal EPS mod i fi ca tions, solvolysis of EPS by wa ter-free an hy drous hy dro gen flu o ride, deg ra da tion by Smith, 1H- and 13C-NMR-spec tros copy [19] (a scheme). The re sis tance of glu cose and galactose at deg ra da - tion ac cord ing to Smith al lowed sup pos ing that at least these two monosaccharide res i dues are acylated, though the ex act place of O-acylation of AP was not de - ter mined [19]. The anal y sis of rhe o log i cal prop er ties of na tive and disacylated ethapolan so lu tions showed that the so lu - tions of disacylated EPS are not struc tured by cat ions, their vis cos ity does not in crease at low shift speeds, as well as at pH de crease and in Cu2+-glycin sys tem [2, 8]. It is worth men tion ing that these char ac ter is tics of ethapolan so lu tions de ter mine its prac ti cal value [8]. Thus, rhe o log i cal prop er ties of ethapolan so lu tions are caused by the pres ence of fatty ac ids in its com po si tion. It was found that de pend ing on the cul ti va tion con - di tions the av er age mo lec u lar weight (m.w.) of ethapolan is 926.0-1441.0 kDa [20]. The anal y sis of mo lec u lar weight com po si tion of EPS showed the pres - ence of com po nents with m.w. mar gins from 13.5 to 2000 kDa, though the main com po nent of the prep a ra - tion was com posed of a frac tion hav ing m.w. of more than 2000 kDa. Af ter EPS pre cip i ta tion by eth a nol (isopropanol, ac e tone) the av er age m.w. de creased 2-3 fold and achieved 400-500 kDa [20]. In our opin ion, it can be ex plained by the de struc tion of the ethapolan so - lu tions struc ture in the pro cess of their treat ment with or ganic dissolvents. Such a phe nom e non is char ac ter is - tic of the ma jor ity of mi cro bial EPS [2]. Ethapolan syn the sis on dif fer ent car bon sub - strates. Ethapolan syn the sis on eth a nol. It was shown that in the growth pro cess of Acinetobacter sp. B-7005 in the min eral me dium with eth a nol with out growth fac tors the level of bio mass and EPS amounted to 0.3-0.5 g/l, and ac e tate com pounds were ac cu mu lated in the cul tural me dium [2, 21, 22]. The in tro duc tion of yeast ex tract into the cul ti va tion me dium was ac com pa - nied by the bio mass and EPS in crease to 2-3 g/l. As it is known yeast ex tract is a source of aminoacids, vi ta - mins, pep tides, pur ines, py rimi dines, and a se ries of other fac tors [23]. There fore we in ves ti gated the in flu - ence of these ma te ri als on the growth of Acinetobacter sp. B-7005. It was found that the strain does not need aminoacids, and as for vi ta mins we ob served the in - crease of bio mass and EPS level only in the pres ence of cal cium pantothenate [2, 21]. Nev er the less, in some sub se quent reinoculations of Acinetobacter sp. B-7005 in the me dium with eth a nol and cal cium pantothenate we ob served grad ual de - crease of bio mass, EPS and wash ing-out of the cul ture. The in tro duc tion of yeast ex tract (0.5%) into the me - dium with eth a nol ad di tion ally to B5 vi ta min re sulted in growth sta bi li za tion and high-vis cos ity EPS syn the sis (4.5-5.0 g/l). It proved that be sides cal cium pantothenate the strain also needs other growth fac tors that are avail able in yeast ex tract. The fur ther ex per i ments al lowed ex clud ing pu rine (ad e nine, gua nine) and py rim i dine (thy mine, cy to sine, ura cil) bases as well as cor re spond ing nu cleo tides (mono- and triphosphates) out of the group of the pos si - ble growth fac tors. The pro cess of ob tain ing ethapolan on eth a nol in the pres ence of C4-di car box yl ic ac ids. To in ten sify the pro cess of EPS biosynthesis on eth a nol we used the so called “met a bolic” ap proach, the sense of which was in the o ret i cal def i ni tion of pos si ble “nar row places” of cul ture me tab o lism and the search of the ways of their re mov ing [2, 24, 25]. While solv ing this task we pro ceeded from the fact that eth a nol me tab o lism in heterotrophic bac te ria may be per formed due to its ox i da tion through acetaldehyde to ac e tate. Ac e tate is in tro duced to fur ther me tab o lism due to the me di a tion of acetyl-coenzyme A (CoA) [26, 27]. At the growth on eth a nol bac te ria use acetyl-CoA mainly for the syn the sis of fatty ac ids, 2-oxoglutarate, C4-di car box yl ic ac ids, from which then C3-in ter me di - ates, car bo hy drates, aminoacids, nu cleo tides are formed. The syn the sis of C4-di car box yl ic ac ids in mi - cro or gan isms, which grow on eth a nol, ac e tate, fatty ac - ids or car bo hy drates, oc curs in glyoxylate cy cle [26, 27]. As for CoA syn the sis Acinetobacter sp. B-7005 bac te ria need ex og e nous cal cium pantothenate – the an - te ced ent to coenzyme A [2, 25], this branch of me tab o - 174 T.P. Pirog, Yu.V. Korzh lism is ev i dently lim ited by acetyl-CoA. Hence, it was sug gested to in tro duce C4-di car box yl ic ac ids into the me dium with eth a nol to in ten sify gluconeogenesis and to in crease EPS syn the sis. It was shown that while cul - ti vat ing Acinetobacter sp. B-7005 in the me dium con - tain ing eth a nol, cal cium pantothenate and C4-di car box - yl ic ac ids, the EPS out put from the sub strate used in - creased 2-fold, and the quan tity of EPS in creased by 30% [2, 24, 25]. For fur ther ex per i ments po tas sium fumarate was se lected as C4-blocks; the op ti mum con - cen tra tion of fumarate which was ap plied to the cul ti va - tion me dium, amounted to 0.2%. Add ing fumarate in the ex po nen tial phase of Acinetobacter sp. B-7005 growth at the cul ti va tion in the me dium with eth a nol and cal cium pantothenate de pressed cells growth com - pletely and in creased the EPS syn the sis by them con - sid er ably. These re searches showed that ex og e nous po tas sium fumarate is used for the EPS for ma tion i.e. fumarate ad - di tion al lows reg u lat ing the di rec tion of pro cesses of ethapolan biosynthesis of Acinetobacter sp. B-7005 [2, 24, 25]. It was found in fur ther ex per i ments that the ad di tion of fumarate in the be gin ning of the sta tion ary phase of bac te ria growth re sulted in the in crease of EPS-syn the - sis abil ity, and ex og e nous fumarate was me tab o lized in EPS stoichiometrically. The pro cess of fumarate as sim - i la tion which is trans ported into cells to gether with pro - ton, was ac com pa nied by the in crease of pH value of the me dium, which reached max i mum at com plete fumarate us age, af ter which it de creased to the pri mary level. Reach ing the pH neu tral value served as a “sig - nal” of the ne ces sity of in tro duc ing the fol low ing por - tion of C4-di car box yl ic ac ids. As at the re peated ad di - tion of fumarate there was a grad ual de crease of pH value to the pri mary level, its fol low ing por tions were in tro duced af ter the acidation of the cul ture liq uid to pH 7.0. It al lowed de creas ing the du ra tion of the cul ti va tion pro cess of Acinetobacter sp. B-7005 due to the pe ri od - ic ity re duc tion of the ad di tion of C4-di car box yl ic acid. It was found in the fol low ing ex per i ments that sep a - rate in tro duc tion of po tas sium fumarate (in por tions of 0.2%) al lowed in creas ing the num ber of syn the sized EPS 4-5 fold (to 10-15 gr/l), and EPS out put from the used sub strate (eth a nol+fumarate) amounted to 60-80% [2, 24, 25]. Enzymological re searches of the Acinetobacter sp. B-7005 strain, which were con ducted later, proved our the o ret i cal as sump tion con cern ing pos si ble ways of eth a nol me tab o lism in these bac te ria. Thus, it was de - ter mined that the “bot tle neck” of C2-me tab o lism of Acinetobacter sp. B-7005 is ac e tate as sim i la tion, which was proved by the fact that so dium ions in hibit both ac - e tate ox i da tion in in tact cells, and acetyl-CoA-synthetase ac tiv ity in cell-free ex tract, as well as the lim i ta tion of C2-me tab o lism by coenzyme A [28, 29]. At the in tro duc tion of C4-di car box yl ic ac ids (po tas sium fumarate) into the me dium with eth a nol there was 1.5-2 fold in crease of the ac tiv ity of glyoxylate cy cle en zymes, as well as fumarate hydrase, malate dehydrogenase and phosphoenolpyruvate synthetase (PEP-synthetase). In these con di tions the in - crease of PEP-carboxykinase ac tiv ity was more ev i dent (al most 7.5 fold) [30]. Thus, the in crease of level of ethapolan syn the sis dur ing pro ducer cul ti va tion on eth - a nol in the pres ence of C4-di car box yl ic ac ids is caused by gluconeogenesis en hanc ing. Ethapolan syn the sis on car bo hy drates sub strates. We found the pos si bil ity of ethapolan syn the sis dur ing the cul ti va tion of the Acinetobacter sp. B-7005 strain on car bo hy drate sub strates (mono- and disaccharides, mo las ses, starch) [31]. In the course of pro ducer growth on C6-sub strates with out cal cium pantothenate the num ber of syn the sized EPS amounted to 2-3 g/l, though pH value of the cul tural liq uid de creased to 5.5-5.7 till the end of cul ti va tion [31, 32]. To find out the rea sons, caus ing the de crease of pH level of the cul ture liq uid, we in ves ti gated the main stages of C6-com pounds me - tab o lism of Acinetobacter sp. B-7005. It was shown that glu cose ca tab o lism in the men tioned bac te ria oc - curs by the path ways of Embden-Mayerhof-Parnas and Entner-Dudorov, which is proved by high ac tiv ity of key en zymes of these path ways [31]. The pres ence of 2-oxoglutaratedehydrogenase ac tiv ity pointed out to the fact of the com plete cy cle of tricarboxylic ac ids (CTA) func tions in Acinetobacter sp. B-7005. It was found that the “bot tle neck” of glu cose me - tab o lism in ethapolan pro ducer is the re ac tion which is catalysed by pyruvate dehydrogenase, i.e. C6-me tab o - lism in these bac te ria is lim ited by CoA. The re plen ish - ment of in ter me di ates pool for con struc tive me tab o lism 175 Ethapolan As A mi cro bial exopolysaccharide re ac tions takes place with the par tic i pa tion of pyruvate carboxylase. Ir re spec tive of the source of car bon nu tri tion (glu - cose, eth a nol) high ac tiv ity of key en zymes of C2- and C6-me tab o lism was de ter mined in the Acinetobacter sp. B-7005 cells. At the bac te ria growth on glu cose the ac - tiv ity of isocytratliase amounted to 4.3 nmol•min-1•mg-1 of pro tein. It was found that C2-com pounds (eth a nol, ac e tate) are inducters of this en zyme in ethapolan pro - ducer. The in tro duc tion of C2-com pounds in low con - cen tra tions (0.01-0.1%) into the me dium with glu cose was ac com pa nied by si mul ta neous con sump tion of two sub strates, the in crease of the num ber of syn the sized EPS (5.1-6.1 gr/l) and EPS-syn the sis abil ity (4.07-4.25gr EPS to 1gr of bio mass) [31]. The re sults ob tained be came the ba sis for the elab o ra tion [32] of the tech nol ogy of ob tain ing ethapolan on car bo hy - drates, as well as on the mix ture of C2-C6 com pounds. The improvement of ethapolan syn the sis us ing the mix ture of growth com pounds. The tech nol ogy of syn - the sis in ten si fi ca tion of mi cro bial EPS ethapolan on the mix ture of en ergy-wise non-equal growth sub strates is prin ci pally new [32-36]. In the nat u ral places of ex is tence mi cro or gan isms de velop in the pres ence of sev eral car bon sub strates while in lab o ra tory con di tions monosubstrates are used as the only source of car bon and en ergy for their cul ti - va tion. At the same time it is known that a con sid er able part of some sub strates is spent on the ox i da tion to CO2 to ob tain the en ergy, nec es sary for con struc tive me tab - o lism (e.g. on glu cose – 40%). Be sides, there are works, prov ing the abil ity of mi cro or gan isms to use the mix - tures of two (or more) sub strates and in ves ti gat ing some as pects of such pro cesses reg u la tion [27, 37-40]. Nev er the less, the men tioned in ves ti ga tions con cern the us age of mixed sub strates only for the in crease of bio - mass out put. It is also known that the com bi na tion of en - ergy-wise non-equal sub strates al lows in creas ing the trans for ma tion ef fec tive ness of sub strates car bon into bio mass [39, 40]. We sup posed that such an ap proach may be used not only for the in crease of bio mass yield, but for the en hanc ing the sec ond ary me tab o lites syn the - sis (with the con sid er ation of ad di tional power in puts for their for ma tion). The iden ti fi ca tion of the ways of en er getic and con - struc tive me tab o lism of C2-C6 com pounds in ethapolan pro ducer al lowed de ter min ing which of these com - pounds are sur plus en ergy-wise, and which are def i cit en ergy-wise [28, 31]. It was found that ox i da tion of eth - a nol and acetaldehyde in these bac te ria is made by NAD+- and NADF+-de pend ent dehydrogenases [28], so this sub strate is clas si fied as sur plus en ergy-wise. Glu - cose ca tab o lism in the Acinetobacter sp. B-7005 strain (ethapolan pro ducer) oc curs ac cord ing to the method of Embden-Mayerhof-Parnas and Entner-Dudorov [31]. Ac cord ing to the en er getic sub strates clas si fi ca tion of Ba bel [39], glu cose is a def i cit sub strate en ergy-wise. As a re sult of the o ret i cal cal cu la tions of en ergy needs of EPS and bio mass yield of the Acinetobacter sp. B-7005 strain the cor re la tion of eth a nol and glu cose con cen tra tions was found which al lows avoid ing non-pro duc tive losses of car bon and en ergy, tak ing place at the us age of monosubstrates, and in creas ing the trans for ma tion ef fec tive ness of sub strates car bon into EPS [33, 35]. The in tro duc tion of eth a nol into the me - dium with glu cose in mo lar re la tion 3.1:1 al lowed in - creas ing the num ber of syn the sized EPS 1.8-1.9 fold (to 7.5-8.0 gr/l), EPS-syn the sis abil ity – 1.4-1.7 fold (to 3.8g EPS per 1g of bio mass), and EPS out put from the sub strate – 1.5-2 fold (to 62-65%) in com par i son with the pro ducer grow ing on monosubstrates. The in ves ti ga tions, the re sults of which are shown in works [32-34, 36] proved that dur ing cul ti vat ing Acinetobacter sp. B-7005 on the mix ture of eth a nol and glu cose both sub strates are as sim i lated at the same time, the max i mum spe cific growth speed in creases by 1.3-1.4 fold and the time of reach ing this re sult de - creases con sid er ably. Nev er the less, dur ing the cul ti va tion of ethapolan pro ducer on the mix ture of eth a nol and glu cose, not only EPS but also bio mass was of ten ob served. There fore, in fur ther in ves ti ga tions the con di tions of bac te ria cul ti va tion were de ter mined, which pro vide com plete con ver sion of car bon of both sub strates into EPS it self to the max i mum [32, 36]. It was de ter mined that while grow ing ethapolan pro ducer on the mix ture of eth a nol and glu cose the high est trans for ma tion ef fec - tive ness of sub strates car bon into EPS was ob served in the fol low ing con di tions: 1) the us age of sow ing ma te - rial, grown on eth a nol or the mix ture of eth a nol and glu - 176 T.P. Pirog, Yu.V. Korzh cose; 2) the con cen tra tion de crease of ni tro gen nu tri tion source (am mo nium ni trate) in the me dium to 0.3-0.45 g/l; 3) the ab sence of so dium cat ions in the cul ti va tion me dium. The study of the key en zymes ac tiv i ties of C2-me - tab o lism (al co hol- and acetaldehyde dehydrogenases, ac e tate kinase, and acetyl-CoA-synthetase) and C6-me - tab o lism (phosphofructokinase and phosphogluconate dehydratase) at the ESP pro ducer grow ing on the mix - ture of eth a nol and glu cose showed si mul ta neously that their val ues were some what lower in com par i son to grow ing on cor re spond ing monosubstrates [36, 41]. The de crease of isocytratliase and malate synthase ac - tiv ity while grow ing on mixed sub strate was more sig - nif i cant (in com par i son with the cul ti va tion on eth a - nol). Thus, we sup posed that acetyl-CoA, which is formed from eth a nol in ac e tate kinase and acetyl-CoA-synthetase re ac tions, is in tro duced to fur - ther me tab o lism mostly through CTA. It was shown that dur ing grow ing bac te ria on the mix ture of eth a nol and glu cose the CTA en zymes ac tiv - ity was higher than on eth a nol only (es pe cially the ac - tiv ity of isocytratdehydrogenase). Prob a bly, the role of glyoxylate cy cle in ethapolan pro ducer me tab o lism on the mixed sub strate is not so im por tant as that on eth a - nol. More over, the ac tiv ity in crease of pyruvate carboxylase, which is an en zyme of anaplerotic re ac - tion, was ob served in the con di tions of mixotrophic bac te ria growth which pro vides re plen ish ment of C4-di - car box yl ic ac ids pool while grow ing on car bo hy drates. At the same time si mul ta neous func tion ing of two anaplerotic routes (glyoxylate cy cle and pyruvate carboxylase re ac tion) may be ac counted for the re in - force ment of gluconeogenesis in the con di tions of mixotrophic pro ducer growth. Ac tu ally, the ac tiv ity of PEP-synthetase, which is a key en zyme of glu co neo - gen et ic branch of me tab o lism on mixed sub strate, was 3 and 1.5-fold higher than on glu cose and eth a nol re spec - tively. Thus, the re sults of enzymologic re searches could be ex plained by the change of biosynthesis pro cesses di rec tion on the mix ture of growth C2-C6 com pounds to wards EPS for ma tion in com par i son with bac te ria grow ing on monosubstrates. The data ob tained are the ba sis for cre at ing fun da - men tally new tech nol o gies prep a ra tion of valu able sec - ond ary me tab o lites on the mix ture of en ergy-wise non-equal growth sub strates. C2-me tab o lism reg u la tion in Acinetobacter sp. B-7005 and im prov ing ethapolan biosynthesis tech nol - ogy on eth a nol. The draw back of the tech nol ogy of ob - tain ing ethapolan on eth a nol, which was elab o rated pre - vi ously, was the ne ces sity of keep ing neu tral pH value in the pro cess of pro ducer cul ti va tion, which was ac - com plished by the in tro duc tion of salts in high con cen - tra tions (11 g/l) into the liq uid to cre ate suf fi cient high-ca pac ity phos phate buffer (0.05 M). While grow - ing Acinetobacter sp. B-7005 on the me dium with eth a - nol, which does not con tain buffer, pH value low ered to 4-5 in hi bi tions of bac te ria growth and EPS in creas ing syn the sis [2, 28, 29]. Study of pe cu liar i ties of en er getic and con struc tive me tab o lism in ethapolan pro ducer, al lowed the ap - proaches to reg u late C2-me tab o lism and to find the ways of reg u la tion of EPS biosynthesis pro cess. It is known that ox i da tion of eth a nol and acetaldehyde is car ried out by NAD+- and NADF+-de - pend ent dehydrogenases, ac e tate is in cluded into the me tab o lism due to acetyl-CoA-synthetase ac tiv ity [28]. Anaplerotic re ac tion se quence, which re plen ishes C4-di car box yl ic ac ids pool in Acinetobacter sp. B-7005 cells, is glyoxylate cy cle, and CTA has a biosynthetic role mainly. Phosphoenolpyruvate syn the sis is pro - vided by two key gluconeogenesis en zymes namely PEP-carboxynase and PEP-synthetase [30]. It was found that the “bot tle neck” of eth a nol me - tab o lism of Acinetobacter sp. B-7005 is ac e tate as sim i - la tion. This re ac tion is cat a lyzed by acetyl-CoA-synthetase and lim ited in speed-lim ited [28, 29]. Thus, in the cells of Acinetobacter sp. B-7005 bac te ria, which grow on eth a nol, ac e tate is formed at a higher speed than it is be ing in cluded into fur ther me - tab o lism. The fol low ing in ves ti ga tions were di rected to the search of fac tors which pro mote the same speed of for - ma tion and fur ther me tab o lism of ac e tate in Acinetobacter sp. B-7005 cells, grown on eth a nol. It was found that the in hib i tors of acetyl-CoA-synthetase are so dium ions as well as prod ucts of eth a nol and acetaldehyde ox i da tion – NADN and NADFN. En zyme ac ti va tors are pantothenic acid, cat ions of po tas sium and mag ne sium [29]. The de crease of the pri mary eth a - 177 Ethapolan As A mi cro bial exopolysaccharide nol con cen tra tion from 1.0 to 0.5% (with sub se quent in - tro duc tion of 0.5% eth a nol in the mid dle of ex po nen tial growth phase), the ab sence of so dium ions and the pres - ence of 100mM K+ in the me dium of ethapolan pro - ducer cul ti va tion con di tioned prac ti cally the same speed of eth a nol, acetaldehyde and ac e tate ox i da tion in in tact bac te ria cells, and the ac tiv ity of acetyl-CoA-synthetase in cell-free ex tract in creased 3 fold. It al lowed im ple ment ing the ethapolan syn the sis pro cess in the me dium, the molarity of which is 2 fold de creased. Ob tain ing an anal o gous re sult at the pri mary eth a nol con cen tra tion of 1.0% was pro vided (on the back ground of the ab sence of so dium salts and pres ence of 100mM K+) at the in crease of pantothenic acid and Mg2+ con cen tra tion in the me dium to 0.0009-0.0012% and 5mM re spec tively. Nev er the less, at fur ther de - crease of salts con cen tra tion and buffer ca pac ity of the me dium there was a drop of ethapolan syn the sis level and ac e tate ac cu mu la tion in the cul tural liq uid. In such con di tions of ethapolan pro ducer cul ti va tion the ac tiv - ity of acetyl-CoA-synthetase was 2-fold lower [29]. The lit er a ture data tes tify to the fact that acetyl-CoA-synthetase, which func tions in cells of many pro- and eukaryotes, is an in duc ible en zyme [42, 43], and ac e tate is an in ducer of this syn the sis [44]. Our ex per i ments showed that the in tro duc tion of ex og e nous ac e tate into the me dium with eth a nol in creases the ac - tiv ity of acetyl-CoA-synthetase in Acinetobacter sp. B-7005 cells [45]. On the ba sis of the in ves ti ga tion of C2-me tab o lism reg u la tion the method of ob tain ing ethapolan was de - vel oped. The key el e ments of the method are the ab - sence of so dium cat ions in the cul ti va tion me dium, the in crease of cal cium pantothenate con cen tra tion in it to 0.0009%, as well as the pres ence of 0.1% of po tas sium ac e tate dur ing cell subculturing and polysaccharide biosynthesis. It al lowed per form ing the pro cess of ethapolan ac cu mu la tion in the me dium (with out de - crease of syn the sis in di ca tors), in which the salts con - tent was de creased 4 fold (from 11 to 2.95 g/l). Reg u la tion prin ci ples of phys i cal and chem i cal prop er ties of ethapolan. It is known that pro ducer cul - ti va tion con di tions in flu ence not only EPS syn the sis, but also phys i cal and chem i cal prop er ties of the fi nal prod uct [46-48]. In dif fer ent cul ti va tion con di tions the chem i cal com po si tion of EPS, their mo lec u lar weight, cor re la tion of sev eral poly sac cha rides may change, which in flu ences EPS rhe o log i cal prop er ties, de ter min - ing the prac ti cal value of these poly mers. There fore, an im por tant and nec es sary con di tion of de vel op ing mi - cro bial EPS biotechnologies is the sta bil ity of phys i cal and chem i cal prop er ties of poly sac cha rides as well as the pos si bil ity of reg u lat ing poly mers prop er ties de - pend ing on the fields of their prac ti cal use. It was found that Acinetobacter sp. B-7005 cells subcultured pe ri od - i cally cul ti va tion of the com po si tion of ethapolan of AP/NAP change [6, 49, 50]. The syn the sis of both poly - sac cha rides started in the first hours of bac te ria growth and oc curs parallelly. At the mo ment of reach ing max i - mum vis cos ity of 0.02% so lu tions of ethapolan, the AP con tent in its com po si tion was the high est and amounted to 65-68%. The con tent of NAP in creased to the end of the pe ri odic pro cess in EPS com po si tion (to 60%) which was ac com pa nied by vis cos ity de crease of its so lu tions [6]. Be sides, vis cos ity did not in crease in EPS so lu tions with high NAP con tent in the pres ence of cat ions, in the area of low shift speeds, at pH level de - crease, as well as in Cu2+-glycin sys tem, i.e. they did not show rhe o log i cal prop er ties which de ter mine prac ti cal value of ethapolan [49]. Thus, the prop er ties of ethapolan so lu tions de pend on the cor re la tion of AP and NAP in its com po si tion [50]. To ob tain EPS, the so - lu tions of which are char ac ter ized by nec es sary rhe o - log i cal prop er ties, the pro ducer cul ti va tion pro cess should be stopped be fore the sta tion ary growth phase, which leads to the de crease of syn the sized EPS quantity. The fol low ing in ves ti ga tions showed that at the 2 fold in crease (from 0.025 to 0.05M) of K+ con cen tra - tion in the me dium, there was grad ual vis cos ity in crease of syn the sized EPS so lu tions in the course of all the cul - ti va tion pro cess. In these con di tions AP con tent did not change much and amounted to 70-75%, and its for ma - tion oc curred in the sta tion ary growth phase as well [51]. Anal o gous pat terns were found while grow ing the pro ducer in the me dium, con tain ing 0,10M K+. In this case AP con tent in EPS amounted to 90-95%. Ob tained data showed that while cul ti vat ing Acinetobacter sp. B-7005 in the me dium with in creased K+ con tent it is pos si ble to ob tain EPS with nec es sary rhe o log i cal prop - er ties (ir re spec tive of pro cess du ra tion). The anal y sis of EPS chem i cal com po si tion, syn the sized in the me dia 178 T.P. Pirog, Yu.V. Korzh with dif fer ent K+ con cen tra tion (0.025-0.1M) showed that they dif fer in AP con tent as well as in fatty ac ids con tent in AP [50]. The so lu tions of in ves ti gated EPS were char ac ter ized by dif fer ent vis cos ity in the pres - ence of cat ions, at low pH val ues, as well as in Cu2+-glycin sys tem. Thus, rhe o log i cal prop er ties of ethapolan so lu tions are de fined not only by the AP and NAP re la tion in its com po si tion, but also by the con tent of fatty ac ids in AP [6, 49-51]. It was found that the syn - the sis of acylated EPS de pended on the con tent of monovalent cat ions (K+ and Na+) in the me dium of pro ducer cul ti va tion. To form the highly acylated AP, con tain ing 12-16% of fatty ac ids, the con cen tra tion of monovalent cat ions (K+ and Na+) in the me dium should be not less than 0.09M [49, 52]. To make ethapolan a com mer cial prod uct, it was nec es sary to de velop a con trolled tech nol ogy of its ob - tain ing which would pro vide high EPS out put pos sess - ing cer tain pre de ter mined prop er ties. Pre vi ously [2, 24, 25] it was found that there is a pos si bil ity of EPS syn - the sis in creas ing of Acinetobacter sp. B-7005 due to the in tro duc tion into the me dium of C4-di car box yl ic ac ids that are gluconeogenesis an te ced ents. It is known that AP or NAP is syn the sized de pend - ing on the con cen tra tion of monovalent cat ions in the me dium with po tas sium fumarate [49]. Thus, to gether with the in crease of monovalent cat ions con cen tra tion in the me dium, AP con tent in the com po si tion of syn - the sized EPS in creased which re sulted in the im prove - ment of rhe o log i cal prop er ties of their so lu tions. Nev er - the less, in the course of the pro cess the con tent of fatty ac ids de creased from 7.2% (af ter the in tro duc tion of the 1st fumarate por tion) to 3.5% (af ter the ad di tion of the 4th por tion). It was ac com pa nied by vis cos ity de crease of EPS so lu tions, syn the sized at the end of the pro cess in the pres ence of cat ions in H+-form and in Cu2+-glycin sys tem [49]. The fol low ing re searches showed that the de crease of fatty ac ids con tent in AP, syn the sized af ter the in tro - duc tion of the last fumarate por tion added, is caused by in suf fi cient quan tity of uni va lent cat ions in the bac te ria cul ti va tion me dium. At the in crease of the pri mary con - cen tra tion of monovalent cat ions in the me dium to 0.14M AP with high con tent of fatty ac ids was syn the - sized from po tas sium fumarate (7.5-8.5%) which was con stant in the course of the whole cul ti va tion pro cess. At the same time the prop er ties of EPS so lu tions, syn - the sized af ter the in tro duc tion of each of four fumarate por tions, did not change and were anal o gous to EPS prop er ties, formed in the me dium lack ing fumarate [8, 49]. The fol low ing ex per i ments showed that the syn the - sis of highly acylated ethapolan is also pos si ble in the course of grow ing the pro ducer in the me dia with eth a - nol, which con tain only 20-40mM K+. Thus, as a re sult of in ves ti ga tion of C2-me tab o lism reg u la tion in Acinetobacter sp. B-7005 it was found that bac te ria cul - ti va tion in the con di tions lack ing the met a bolic lim i ta - tions, con nected with the for ma tion of acetyl-CoA (fatty ac ids an te ced ent), is ac com pa nied by the syn the - sis of mostly acylated EPS with high m.w. (1.5 mln Da) which does not de crease in the pro cess of iso lat ing and pu ri fy ing the prep a ra tion [45]. It was found that ethapolan pro tects pro ducer cells from un fa vour able en vi ron men tal fac tors namely, the ac tion of toxic ox y gen de riv a tives, heavy met als (Cu2+, Pb2+, Cr3+, Zn2+), biocide (form al de hyde), de ter gent (so dium dodecyl sul fate), high and low pH val ues, heat - ing, dry ing, and freez ing [53-56]. It was shown that ethapolan im ple ments pro tec tive func tions not only con cern ing pro ducer cells, but also con cern ing mi cro - or gan isms cells which are in trophic re la tions with Acinetobacter sp. B-7005 [55]. The re searches, the re sults of which are stated in the works [8, 57], showed that at non-op ti mal cul ti va tion con di tions of Acinetobacter sp. B-7005 (at the change of tem per a ture, pH of en vi ron ment, dis solved ox y gen) EPS quan tity de creased, but EPS-syn the sis abil ity re - mained sta ble and amounted to 1g EPS per 1g of bio - mass. EPS so lu tions, syn the sized at non-op ti mal con di - tions, were char ac ter ized by dif fer ent vis cos ity in the pres ence of cat ions, in H+-form and in Cu2+-glycin sys - tem. Ob tained re sults al lowed sup pos ing that the ef fec - tive ness of pro tec tive func tions of such EPS is dif fer - ent, it cor re lates with rhe o log i cal prop er ties of ethapolan and is im ple mented un der non-op ti mal con - di tions of Acinetobacter sp. B-7005 cul ti va tion the most. Nev er the less, at bac te ria cul ti va tion in non-op ti - mal con di tions the quan tity of syn the sized EPS de - creased. In this con nec tion we made a sup po si tion that it is pos si ble to in crease EPS con cen tra tion and im - prove ethapolan so lu tions prop er ties at the same time 179 Ethapolan As A mi cro bial exopolysaccharide due to grow ing the pro ducer in a two-stage pro cess. On the first stage of the bac te ria growth con di tions are used op ti mal for the EPS growth and syn the sis, while on the sec ond stage non-op ti mal con di tions are cre ated (that is tem per a ture de crease to 24°C, pH in crease to 8.0, ad di - tion of form al de hyde into the me dium – 30 mkg/ml). It was found that while cul ti vat ing Acinetobacter sp. B-7005 in two-stage mode, the quan tity of EPS syn the - sized reached the level, cor re spond ing to that achieved at op ti mal con di tions of growth [20]. EPS so lu tions were char ac ter ized by a higher vis cos ity in the pres ence of cat ions in H+-form and in Cu2+-glycin sys tem, than poly sac cha rides, syn the sized in op ti mal con di tions. Prac ti cal ap pli ca tion of ethapolan. Ethapolan is EPS which can be used in dif fer ent spheres of in dus try [8, 58-62] and ag ri cul ture as plant pro tec tive prep a ra - tions [63]. Pe tro leum in dus try. Such phys i cal and chem i cal prop er ties of ethapolan so lu tions as the abil ity to emul - sify, the vis cos ity in crease in the pres ence of cat ions, at low shift speeds, at pH de crease and in Cu2+-glycin sys - tem at tracted at ten tion of pe tro leum in dus try spe cial ists to this EPS as to an pe tro leum-sweep ing agent. It was shown that the us age of ethapolan con tain ing 70-95% AP with the acylation de gree of 5-12% is rea - son able for the in ten si fi ca tion of pe tro leum pro duc tion. At pres ent such mi cro bial EPS as xanthane (pro - ducer Xanthomonas campestris), scleroglucane (pro - ducer Sclerotium glucanicum, S. rolfsii), emulsane (pro ducer Acinetobacter calcoaceticus) are con sid ered the most per spec tive to be used in pe tro leum pro duc tion in the world. Ethapolan is con sid er ably dif fer ent from these poly sac cha rides by sig nif i cant in crease of so lu - tion vis cos ity in min er al ized me dia. An im por tant pe cu - liar ity of ethapolan so lu tions is the in crease of their vis - cos ity at ox i da tion which al lows us ing this EPS to pro - long acid treat ment of bottomhole for ma tion zones. Ethapolan so lu tions are more thermostable that xanthane and scleroglucane so lu tions which al lows us - ing them in the fields with high strata tem per a tures [59]. Due to the pres ence of a lipophilic moi ety in ethapolan mol e cule it can sta bi lize emul sions of wa ter and pe tro leum or other car bo hy drates. Ethapolan so lu - tions are char ac ter ized by higher abil ity (in com par i son with known biopolymers) of in creas ing vis cos ity in the ar eas of low shift speeds (0.1-1.0 c-1). Ethapolan is able to form gel-like sys tems while in ter act ing with metal ions and other cross-link ing agents. At the same time the vis cos ity in crease of mod i fied ethapolan so lu tions is ob served more than 10 fold and there is sta bi li za tion of ob tained so lu tion at long-term keep ing [2, 59]. It is pos si ble to use ethapolan in pe tro leum in dus try as a cul tural liq uid which ex cludes the ne ces sity of iso - lat ing and pu ri fy ing the prep a ra tion pre vi ously. The ap - pli ca tion of such com mer cial form of ethapolan has an ad di tional ad van tage in the fact that in it poly sac cha - rides are sta bi lized by which are of the com po nents of cul tural liq uid com po si tion. Be sides, it does not have microgel clots. This ex plains light dissolubility of ethapolan even in min er al ized me dia and its com pat i - bil ity with other chem i cal re agents while pre par ing ap - pro pri ate for mu la tions. The com po si tions of ethapolan and polyacrylamide, as well as ethapolan and so dium sil i - cate, which were de vel oped in the co op er a tion with the spe cial ists of “Soyuznefteotdacha” In sti tute (Rus sia) and “VNIIneft’” (Rus sia), were used in 1988-1990 for the im prove ment of pe tro leum out put on ex ploited pe - tro leum wells of VO “Bashneft’” (Bashkortostan). The pro duc tion of ex per i men tal lots of ethapolan was also per formed on Bashkyrsky bio chem i cal plant and on VO “En zyme” (Ukraine). The method of iso lat ing strata wa ter in put us ing gel-form ing com po si tions on the ba sis of ethapolan, non-or ganic cop per salt and glycin, pro vid ing highly ef fec tive iso la tion of per me able seams, ac com pa nied by the in crease of pe tro leum yield and sig nif i cant de - crease of pe tro leum flood ing, was de vel oped in the co - op er a tion with the work ers of “VNIIneft’” and ENTO “ITIN” In sti tute (Rus sia) [64]. The de vel oped method of strata wa ter in put iso la tion was used in 1991-1992 for hydroisolation of ex ploited pe tro leum wells in Prylutsky pe tro leum field (Chernigiv oblast). Pro duc - tion lots of ethapolan were made on ex per i men tal mo - bile plant, de vel oped to gether with ENTO “ITIN” in the con di tions of Trypilsky bio chem i cal plant VAT “Stirolbiotech” (Ukraine). The use of much as 1 tone of ethapolan in pe tro leum in dus try en ables to yield 240 pe tro leum tones in ad di - tion. 180 T.P. Pirog, Yu.V. Korzh House hold chem i cal goods and cos me tol ogy. Ethapolan prep a ra tions, the so lu tions of which are char - ac ter ized by high emul si fy ing ac tiv ity, are used in house hold chem i cal goods and cos me tol ogy [8]. Ethapolan, con tain ing 70% AP with the acylation de - gree of 12-16%, meets these re quire ments. A tech ni cal de ter gent BIMS-1 was de vel oped on ethapolan ba sis, the sale vol ume of which amounted to 1000 tones of the prod ucts in 1989-1990. A tech nol ogy of pro duc ing cos - metic creams with the gen eral name “Ekol” was elab o - rated on the ba sis of ethapolan [8]. Ethapolan with high con tent of non-acylated polysaccharide may be used as a thick en ing agent in al - ka line me dia (e.g. while pro duc ing some de ter gents, wash ing pastes etc). In such con di tions the use of ethapolan with high con tent of fatty ac ids is not rea son - able as al ka line me dium (with pH 10 and higher) may cause par tial disacylation of EPS which re sults in vis - cos ity de crease of its so lu tions, and there fore, in ef fec - tive ness de crease of EPS use [8]. Food in dus try. The re searches, per formed in the Ukrai nian State Uni ver sity of Food Tech nol o gies (UDUHT, since 2002 – the Na tional Uni ver sity of Food Tech nol o gies) un der the lead er ship of Cor re spond ing Mem ber of UAAS V.I. Drobot, proved the pos si bil ity and ap pro pri ate ness of us ing ethapolan in bread and bread prod ucts pro duc tion [60-62]. Tech no log i cal re gimes and pa ram e ters of pro duc - ing “Zhytomyrsky” and “Polisky” bread were de vel - oped, pro vid ing sta ble rhythm of dough for ma tion and high qual ity of ready prod ucts. The tech no log i cal dose of ethapolan for bread bak ing was de fined, it amounts to 0.3-0.5% of flour weight. Eco nomic ef fect of ethapolan use is in the pos si bil ity of us ing flour of the 2nd and 3rd sort (flour with low con tent of glu ten), flour econ omy on the con di tion of ethapolan ad di tion to it and in slow ing down hard en ing of bread prod ucts. Com plex ad di tives K-1, K-2 and K-3 were worked out in UDUHT on ethapolan ba sis for the use in bread pro duc tion from wheat flour with low con tent of glu ten. Pro duc tion test ing in bread pro duc tion with com plex ad di tives K-, K-2, and K-3 was con ducted in a bread-bak ing plant #2 (Kyiv). The vol ume of ex per i - men tal lots of bread prod ucts, pro duced on the ba sis of K-1 ad di tives, amounted to 21.4 tones; K-2 – 24.5 tones; K-3 – 21.7 tones. The re searches, con ducted in Sci ence Re search In - sti tute of Nu tri tion Hy giene of the Min is try of Health of Ukraine, showed the ab sence of mutagenicity, embryotoxicity, tera to ge nici ty and allergenicity of ethapolan, and de ter mined the com plex-form ing abil ity of ethapolan con cern ing salts of heavy met als (on the ex am ple of lead). Due to the abil ity of ethapolan to ab - sorb salts of heavy met als and take them out of the or - gan ism, bread prod ucts with it use may be rec om - mended for pre ven tive nu tri tion. Ethapolan with low con tent of fatty ac ids (40-50% AP with acylation de gree of 3-5%) may be used in food in dus try, un like to pe tro leum in dus try and cos me tol - ogy. Pro tec tion of plants from vi ral and in fec tious dis - eases. It is known that mi cro bial poly sac cha rides of dif - fer ent struc ture have an ti vi ral prop er ties [64]. Heteropolysaccharides at tract the great est at ten tion as a sig nif i cant quan tity of uronic ac ids is found in their com po si tion to gether with neu tral monosaccharides [8, 32]. Such poly mers have polyanion struc ture and due to this fact they are able to in duce or gan ism re sis tance to vi ral in fec tions de novo. Tak ing into con sid er ation the abovementioned we aimed at in ves ti gat ing the an ti vi ral ac tiv ity of these prep a ra tions on phytoviruses mod els in par tic u lar. The test ing of prep a ra tions in con tact ex per i ments in vi tro showed that ir re spec tive of car bon source in the me dium of pro ducer cul ti va tion (eth a nol, glu cose, eth a - nol+glu cose) EPS de pressed the in fec tion of the vi rus of to bacco mo saic and X-vi rus of po ta toes in the plants of stramonium (Datura stramonium) and gomphrena (Gomphrena globosa L.) in a wide con cen tra tion range. It is in ter est ing to men tion that both na tive and disacylated ethapolan prep a ra tions had the same ac tiv - ity. It showed that at least fatty ac ids were not the main fac tors of antiphytoviral prop er ties of this EPS [64]. It was found that ethapolan prep a ra tions at the con - cen tra tion of 500-2000 mkg/ml are able to in duce re sis - tance of super-sen si tive to bacco plants of Im mune sort 580, Nicotiana sanderae and stramonium to VTM-in - fec tion. Thus, a great va ri ety of sub strates (eth a nol, C4-di - car box yl ic ac ids, car bo hy drates – mono- and disaccharides, starch, mo las ses) can be used for ethapolan syn the sis. Such a prop erty dis tin guishes 181 Ethapolan As A mi cro bial exopolysaccharide ethapolan pro ducer fa vour ably among known mi cro - bial syn the siz ers which mainly syn the size EPS only while grow ing on car bo hy drates (Zanthomonas campestris, Azotobacter vinelandii, Sclerotium glucanicum, Aureobasidium pullulans etc). It should be men tioned that re searches, con ducted in the 70-80s of the 20th cen tury, dem on strated the pos si bil ity of ex tend - ing the source of raw ma te ri als for mi cro bi o log i cal EPS pro duc tion due to the us age of non-nu tri tive sub strates – meth ane, lower al co hols, eth yl ene gly col, car bo hy - drates and pe tro leum prod ucts [48]. The rep re sen ta tive of a new gen er a tion of mi cro bial EPS is emulsane (pro - ducer Acinetobacter calcoaceticus), ob tained in pro - duc tion scale on eth a nol ba sis [17]. Nev er the less, there are no lit er a ture data on mi cro or gan isms, able to syn - the size EPS ac tively on both car bo hy drate and non-car - bo hy drate sub strates. The abil ity of Acinetobacter sp. B-7005 to form EPS on C2-C6 com pounds al lowed usto de velop a com - plex of dif fer ent tech nol o gies on ob tain ing ethapolan on the ba sis of a wide range of car bon sub strates. Each of tech nol ogy could be used de pend ing on eco nomic ap pro pri ate ness, pres ence and avail abil ity of a cer tain sub strate, and the ne ces sity of ob tain ing EPS with cer - tain phys i cal and chem i cal prop er ties. Ethapolan syn the sis was in creased us ing the fol - low ing ap proaches: - the de ter mi na tion of to tal ity of op ti mal ex - ter nal fac tors (the na ture and con cen tra tion of growth fac tors, sources of car bon and ni tro gen nu tri tion, C/N cor re la tion, the way of sub strate giv ing etc); - the in tro duc tion of pre cur sors of poly sac - cha rides biosynthesis an te ced ents into the cul ti va tion me dium; - the use of the mix ture of en ergy-wise non-equal growth sub strates; - re veal ing “bot tle neck” of me tab o lism and the de vel op ment of ap proaches of their re moval. Phys i cal and chem i cal prop er ties of ethapolan were reg u lated, us ing the fol low ing meth ods: - re veal ing func tional groups in the EPS com - po si tion, de ter min ing their rhe o log i cal prop er ties, as well as the search of fac tors, pro vid ing EPS syn the sis with nec es sary func tional groups; - the in ves ti ga tion of EPS com po si tion and prop er ties change in the pro ducer cul ti va tion pro cess and the de ter mi na tion of the growth phase in which EPS syn the sis with nec es sary phys i cal and chem i cal prop er ties oc curs; - the study of in ter play be tween rhe o log i cal EPS prop er ties and their pro tec tive func tions as well as the de ter mi na tion of pro ducer cul ti va tion con di tions, nec es sary to re veal pro tec tive func tions. Some of these ap proaches con cern ing EPS syn the - sis in crease and their phys i cal and chem i cal prop er ties reg u la tion were used by us for the first time. Thus, the lit er a ture data con cern ing the use of mixed sub strates by mi cro or gan isms, known at pres ent, show only to the in crease of bio mass syn the sis level on the mix ture of growth and non-growth sub strates. We were the first to show the pos si bil ity of en hanc ing of the sec ond ary me tab o lites syn the sis (on the ex am ple of mi - cro bial exopolysaccharide ethapolan) on the mix ture of en ergy-wise non-equal growth sub strates. On the ba sis of ex per i men tal data the tech nol ogy of ob tain ing ethapolan was de vel oped, en abling to in crease al most 2-fold the max i mum speed of pro ducer growth, the quan tity of syn the sized EPS, their out put re lat ing to bio mass and EPS out put from the sub strate, as well as to shorten the cul ti va tion du ra tion and im prove rhe o - log i cal prop er ties of EPS so lu tions, de fin ing its prac ti - cal value. There are no lit er a ture data con cern ing rhe o log i cal prop er ties reg u la tion of mi cro bial EPS due to the ef fec - tive ness change of their pro tec tive func tions. At pres ent the re searches, con cerned with the study of phys i o log i - cal func tions of mi cro bial EPS, are dis re garded by bio - tech nolo gists. In our opin ion find ing out rea sons, caus - ing the ne ces sity of EPS syn the sis for the pro ducer it - self, will al low to view the se ries of prob lems of mi cro bial poly sac cha rides bio tech nol ogy from new an - gles, in clud ing EPS ob tain ing with pre de ter mined prop er ties. It is worth men tion ing that the meth ods of ob tain ing mi cro bial EPS in two-stage pro cess are known [65, 66], but they are char ac ter ized by the fact that on the first stage there are op ti mal con di tions for pro ducer growth, and on the sec ond – op ti mal con di tions for EPS syn the - sis. The dis tin guish ing fea ture of our ap proach is the fact that on the first stage of the pro cess the pro ducer is grown at con di tions that are op ti mal for both growth and EPS syn the sis, and on the sec ond stage – there are 182 T.P. Pirog, Yu.V. Korzh non-op ti mal (or even stress) con di tions, cre ated for the pro ducer, in which pro tec tive func tions of mi cro bial EPS are re vealed at the most. In re sponse to non-fa - vour able en vi ron ment poly sac cha rides are syn the sized with changed rhe o log i cal char ac ter is tics. The lit er a ture data high light the stim u la tion ac tion of or ganic ac ids (pyruvate, ci trate, succinate, ?-ketoglutarate) on xanthane for ma tion by Xanthomonas campestris bac te - ria [67]. The au thors think that the in flu ence of or ganic ac ids is con nected with the de ter mi na tion of pH value, fa vour able for xanthane syn the sis. The an a log i cal ef - fect takes place at the in tro duc tion of fumaric acid into the cul ti va tion me dium of X. campestris [68]. In this case there is an in crease of xanthane out put from 6.0 to 9.1 g/l. The mech a nism of ex og e nous fumarate ac tion at the growth of ethapolan pro ducer on C2-com pounds is dif fer ent, as in this case C4-di car box yl ic ac ids are in - ter me di ates of eth a nol me tab o lism, which are di rectly in cluded to gluconeogenesis. The ap proaches men tioned to syn the sis in creas ing and ethapolan prop er ties reg u la tion may be used while elab o rat ing tech nol o gies of not only mi cro bial poly sac - cha rides, but also any prac ti cally valu able sec ond ary me tab o lites. Unique rhe o log i cal prop er ties of ethapolan so lu - tions (the abil ity to emul sify, vis cos ity in crease in the pres ence of mono- and bi va lent cat ions, at pH de crease, in the area of low shift speeds, in Cu2+-glycin sys tem), the to tal ity of which is not char ac ter is tic of any of al - ready known mi cro bial EPS, al low con sid er ing it as polysaccharide of multifunctional as sign ment, which may be used in pe tro leum, house hold, chem i cal, per - fum ery-cos metic, as well as in food industry and agriculture. Ò. Ï. Ïèðîã, Þ. Â. Êîðæ Ýòàïîëàí­ìèêðîáíûé ýêçîïîëèñàõàðèä ìóëüòèôóíêöèîíàëüíîãî íàçíà÷åíèÿ Ðåçþìå Ñóììèðîâàíû îïóáëèêîâàííûå ýêñïåðèìåíòàëüíûå äàííûå, êàñàþùèåñÿ èíòåíñèôèêàöèè ñèíòåçà, ðåãóëÿöèè ôèçèêî-õèìè÷åñêèõ ñâîéñòâ è ïðàêòè÷åñêîãî èñïîëüçîâàíèÿ ìèêðîáíîãî ýêçîïîëèñàõàðèäà (ÝÏÑ) ýòàïîëàíà (ïðîäóöåíò–Acinetobacter sp. Â-7005). 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