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EXPERIMENTAL ARTICLES
UDC 577. 152.32 https://doi.org/10.15407/biotech10.01.026
YEAST P-MANNANASE ACTIVITY
N. V. Borzova
L. D. Varbanets Zabolotny Institute of Microbiology and Virology
V. S. Pidgorskyi of the National Academy of Sciences of Ukraine, Kiyv
O. D. Ianieva
E-mail: nv_borzova@bigmir.net
Received 09.01.2017
The aim of the research was to determine the mannan-degrading activity of yeasts cultures isolated from various sources and select strains with high P-mannanase activity. As a result of screening of 245 yeast strains, which are the representatives of 7 genera and 14 species, the active producers of extracellular P-mannanase were identified. To increase P-mannanase activity, the cultures were grown under submerged conditions using guar gum galactomannan as a carbon source and an inducer. P-Mannanase activity was determined by dinitrosalicylic method. The most active biosynthetic species were Cryptococcus albidus, C. gastricus, C. magnus, C. terreus, C. laurentii, Saccharomyces cerevisiae, Williopsis californica, Metschnikowia pulcherrima, Pichia anomala and P. guilliermondii. The activity in culture supernatant was ranged from 0.2 to 75 U/ml. a-Galactosidase activity was found in two strains (Debaryomyces polymorphus UCM Y-152 and Debaryomyces hansenii var. fabryi UCM Y-2400). None of the tested cultures demonstrated both P-mannanase and a-galactosidase activity, that is, they are unable to attack both the main and side chains of galactomannan.
Key words: yeast, P-mannanase, a-galactosidase, galactomannan.
High demand for enzymes that hydrolyze lignohemicelluloses determines the need for the research for screening and isolation of new high active mannanases producers (1,4-P-D-mannan mannohydrolases or P-mannanases, EC 3.2.1.78). These enzymes catalyze the hydrolysis of P-mannoside bond in the main chain of hemicellulose, as well as in gluco-and galactomannans with the formation of mannooligosaccharides, mannose, glucose and galactose. Cellulose and hemicellulose, due to their chemical properties, are the substrates of great biotechnological value. On the one hand, waste from the wood, paper industries and agriculture can be environment pollution factors, and on the other hand, they have a great technological potential as a source of poly- and oligosaccharides. Because of the ability to hydrolyze hemicellulose, P-mannanase has found an application in various industries: pharmaceutical, pulp and paper, gas; in biofuel and cheap energy, prebiotic mannooligosaccharides, as well as in food and feed production [1].
Mannanases are isolated from plants, invertebrates, bacteria and fungi. The basic requirements for enzyme producers are the simplicity of isolation of enzymes resistant to high temperature, salt concentrations, and their effectiveness over a wide pH range, i.e., biocatalysts must have the physico-chemical properties necessary for technological processes. Preferred sources of enzymes are microorganisms due to rapid growth, high productivity and the specificity of the action. P-Mannanases have been found in many species of microorganisms [2]. Currently the mannan-degrading enzymes of bacteria of Acinetobacter sp. [3], Bacillus amyloliquefaciens [4], Bacillus sp. [5, 6], Cellulosimicrobium sp. [7], Klebsiella oxytoca and Klebsiella edwardsii [3, 8], Clostridium tertium [9], Scopulariopsis candida [10], Streptomyces sp. [11], of micromycetes of the genera Aspergillus, Penicillium, Trichoderma, Trichosporo-noides and many others are described [12-16].
The least studied group of P-mannanase producers are yeasts. For these microorganisms a-mannanase activity as a component of their lytic complex is more characteristic. However, even among them, the cultures with high P-mannanase activity are described, although not as numerous as among bacteria and micromycetes [1, 2]. The undoubted advantage of yeasts as enzyme producers over other microorganisms is their resistance to infections and ease of separation from the culture medium due to large cell sizes.
It is known that mannans constitute an extremely diverse group of glycopolymers, including homomannans and galacto-, gluco-, galactoglucomannans. The degradation efficiency of these polysaccharides depends on the complex of enzymes of different specificity, which is due to the nature of the raw materials used in this or that biotechnological process. Therefore, the search for producers of the enzymes of the mannan-degrading complex of definite specificity remains an actual problem.
This work is devoted to the study of the mannan-degrading activity of yeast cultures isolated from various sources, in particular from plant material, soil, water, gastrointestinal tract (GIT) of fish and warm-blooded animals, in order to select high-activity P-mannanase producers among them.
Materials and Methods
245 strains of yeast, representatives of 7 genera and 14 species from the Ukrainian Collections of Microorganaisms maintained at the Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine were studed. Strains were isolated from various sources (Table 1).
Cultivation of yeasts was carried out in wort broth containing 1% guar gum under submerged conditions in tubes containing 10 ml of nutrient medium at 25 °C and shaker rotation speed of 220 rpm for 4-5 days.
The mannanase activity determination was performed by dinitrosalicylic method; guar gum galactomannan was used as a substrate [17]. A reaction mixture containing 0.5 ml of culture liquid (CL) and 0.5 ml of 1% galactomannan in 0.1 M phosphate-citrate buffer, pH 5.2, was incubated for 20 min at 45 °C, then 1 ml of dinitrosalicylic reagent (DSR) was added and the mixture was boiled for 10 min. The color intensity was evaluated spectrophotometrically at 540 nm. Mannose was used as the standard. One unit of enzyme activity was defined as the amount of the enzyme that releases 1 pmol of mannose per 1 min under experimental conditions.
a-Galactosidase activity was determined using p-nitrophenyl-a-D-galactopyranoside [18].
All experiments were performed in at least 3-5 repetitions. Statistical processing of
Table 1. Sources of yeast strains isolation
№№ Genus Strains amount The source of isolation
1 Candida 41 Sewage, water of the Dnieper, silage, GIT of longliver of the State Institute of Gerontology of AMS of Ukraine
2 Cryptococcus 97 Vegetables, alfalfa, trees, soil of Ukraine, Crimea, Yakutia, Israel
3 Debaryomyces 22 Israel Soil, beet, trees, sedge
4 Metschnikowia 23 Vegetables, fruits, lake water
5 Pichia 25 Vegetables, fruits, silage, gills, skin and fish GIT, oil contaminated soil
6 Saccharomyces 17 GIT of long-liver of the State Institute of Gerontology of NAMS of Ukraine, wine, soil, wort, kvass, pistachios
7 Williopsis 20 Vegetables, cereals, soil
experimental series results was carried out by standard methods using Student's t-test at 5% significance level.
Results and Discussion
Mannans are the main component of the hemicellulose of coniferous trees, and also widely distributed in the tissues of other plants: ivory nuts and coconuts, coffee beans, fenugreek, guar, caesalpinia, soy, carob, lichens. The main mannan-degrading enzymes are P-mannanase, P-mannosidase (EC 3.2.1.25) and P-glucosidase (EC 3.2.1.21). This group also includes a-galactosidase (EC 3.2.1.22) and acetylmannane esterase (EC 3.1.1.6), hydrolyzing the side chains of heteromannans with the galactose splitting off and acetyl ester bonds cleavage. The enzymatic hydrolysis of heteromannan is carried out as follows:
r Tv-r ri.tn r i- flannanass
I 4
.. .-P-Glc/?-0.>4)-£(-Harv3-l?4-Hario-Cl>4)-P-Harv?
6
c*-gal act asi das e
p-namosi dase
tt-0-Gal/?
Acetyl
Manp-1 >4 -Ma np- CI >4) -fi-Manp
acetyl nwnane esterase
p-glucosidase
Glc/J-[I>4)-P-Harv).
Various plant substrates, including agricultural waste, can contain lignin, cellulose, hemicellulose, as well as activators and inhibitors of unknown nature, which in turn makes them a rich substrate for the isolation of specific enzymes. Due to its chemical composition, such a substrate is easily colonized by microorganisms and can serve as a source of highly active enzymes involved in the degradation of biopolymers. Various vegetable substrates are used as sources of carbohydrates for the synthesis of mannanases: wheat, rice and corn bran, potato peel, cassava, pineapple, acacia seeds, palm, coconut and peanut oil cake [13, 15].
Therefore, the aim of our work was to investigate the extracellular mannan-degrading activity of yeasts, which are mostly isolated from plant substrates, such as the surface of vegetables, fruits, herbs, and trees. This choice is due to the fact that microorganisms adapt to the utilization of a substrate that is abundant in their habitats, and therefore form the enzymes that react
with this substrate. In addition, the activity of cultures isolated from other sources, in particular from soil, silage, freshwater reservoirs, GIT of fish and mammals was studied.
Guar gum — galactomannan, whose main chain consists of 1,4-linked mannose residues, to which in the side chain single a-D-galactosyl residues are appended (in the ratio of 2:1), is added as a substrate for activating the synthesis of enzymes and for determining the mannan-degrading activity. Earlier it was shown [9] that guar gum serves as optimal carbon source for the synthesis of Clostridium tertium mannanases. The presence of a-linked galactose in the side chains of guar galactomannan gives reason to assume also the induction of a-galactosidase synthesis in the presence of this substrate. Therefore, to evaluate the effectiveness of hydrolysis of this galactomannan, both P-mannanase and a-galactosidase activity of yeast were studied in parallel.
Enzymatic activity was studied in 245 strains, belonging to 7 genera and 14 species. The most numerous group consisted of representatives of the genus Cryptococcus (97 strains) (Fig. 1). Extracellular P-mannanase activity in the CL supernatant of producers ranged from 0.2 to 75 U/ml. It should be noted that a fairly high percentage of active strains were found among the yeasts of the genus Cryptococcus. Thus, 28 % of the strains of C. albidus, 100% of C. gastricus, 11% of C. humicolus, 27% of C. magnus, 66% of C. terreus and 29% of C. laurentii showed P-mannanase activity. Although P-mannanase activity was present in CL of many strains of this group, the rate of hydrolysis of galactomannan was low (0.2-15 U/ml). It should also be noted that there is no a-galactosidase activity in all cases.
Representatives of the species Saccharomyces cerevisiae also actively hydrolyzed guar gums galactomannan. Among this group of microorganisms, 41 % of cultures showed activity. Among representatives of the species Williopsis californica and Metschnikowia pulcherrima 40% and 30% of studied strains demonstrated mannanase activity. The lowest number of active strains was found among representatives of the species Candida krusei — 14% and the genus Debaryomyces — 13.6%, and the most active group were yeasts of the Pichia anomala species — 76% of active strains (Fig. 2).
Thus, we established a higher frequency of P-mannanase activity in the yeast cultures
Fig. 1. Species diversity of Cryptococcus cultures used in the screening process
Saccharomvces
WiUiopsis
Metscltnikowia Cryptococcus
Picftia
Candida
Debaryomyces
□ Acth'f
In active
Fig. 2. The ratio of active (showing P-mannanase or a-galactosidase activity) and inactive strains among representatives of different yeast genera
studied than in other studies, where it was shown that only 7% of strains of the genus Cryptococcus and less than 3% of strains of the genus Pichia exhibited extracellular P-mannanase activity [19, 20]. This may be due to the sources of producer strains used by us, as extracellular enzymatic activity often depends on inducers and substrates from the external environment.
In contrast to micromycetes and actinobacteria, which, as established earlier [21], exhibited both a-galactosidase and
P-mannosidase activity, no such producers were found among the yeast cultures. In the CL supernatant of only two yeast cultures — Debaryomyces polymorphus UCM Y-152 and D. hansenii var. fabryi UCM Y-2400 — a-galactosidase activity was detected in the absence of P-mannanase. This indicates that all studied yeast strains are capable of attacking either the main galactomannan chain or its side chains. Probably, there is a phenomenon of antisinergism of the action of enzymes competing for the same substrate [22].
P-Mannanase activity in the CL supernatant of different yeast strains varied in a wide range and was depend upon both the strain and the species of the microorganism (Fig. 3). Thus, representatives of S. cerevisiae and various species of Cryptococcus showed low P-mannanase activity. The most active species were W. сalifornica UCM Y-25 (tomatoes), UCM Y-258 (soil) and UCM Y-250 (oats), M. pulcherrima UCM Y-357 (birch), UCM Y-355 (hornbeam), UCM Y-445 (soil contaminated with crude oil) and P. anomala UCM Y-244 (silage), UCM Y-237 and UCM Y-231 (GIT of trout and carp, respectively; Table 1).
An analysis of the results shows that the ability to synthesize secondary metabolites is primarily a strain-specific and not a species-specific trait, and the activity of different strains within the genus and species can differ by several orders of magnitude. The ability of a microorganism to hydrolyze a mannan-containing substrate and to show P-mannanase activity depends largely on the source of culture isolation. Although there is no clear correlation between the level of hydrolytic activity and the source of strain isolation, a high occurrence of mannanase-producing strains was found among yeasts isolated from soil and plants (Table 2).
I s S ? H s I U i S 5 ? H = = H Ti 5 I 5 s i s 4 H s H 3 s s a H S H i
Hie number of strains
Fig. 3. P-Mannanase activity in the culture liquid supernatant of the most active yeast strains
Table 2. Isolation sources of the most active yeast strains
№№ № of UCM-Y Species The strain origin (time, place of isolation or from where it was received)
1 2 3 4
1 1022 Cryptococcus albidus Soil, 1969
2 1037 Cryptococcus albidus Soil, Teremky, 1983
3 1047 Cryptococcus albidus Soil, Teremky, 1983
4 1083 Cryptococcus albidus Soil, Teremky, 1983
5 2277 Cryptococcus albidus Israel, 2000
6 2637 Cryptococcus albidus Soil under the juniper, Crimea, 2000
7 2655 Cryptococcus albidus Soil, Yakutia, 2002
8 2230 Cryptococcus gastricus Israel, 2000
end of table 2
№№ № of UCM-Y Species The strain origin (time, place of isolation or from where it was received)
1 2 3 4
9 2235 Cryptococcus gastricus Israel, 2001
10 2246 Cryptococcus gastricus Israel, 2002
11 2206 Cryptococcus magnus Israel, 2000
12 2418 Cryptococcus magnus Soil, Evolutionary Canyon, Israel, 1999
13 2447 Cryptococcus magnus Pistachio, Israel,1999
14 1098 Cryptococcus laurentii Lupine, rhizosphere, 1967
15 1102 Cryptococcus laurentii Tobacco, rhizosphere, 1969
16 1115 Cryptococcus laurentii Cucumbers, leaves, 1969
17 1116 Cryptococcus laurentii Cucumbers, rhizosphere, 1969
18 1118 Cryptococcus laurentii Carrot, leaves, 1969
19 340 Metschnikowia pulcherrima Cabbage, leaves, 1968
20 341 Metschnikowia pulcherrima Apple tree, leaves, 1969
21 355 Metschnikowia pulcherrima Grab, rhizosphere 1969
22 357Кат Metschnikowia pulcherrima Birch, rhizosphere, 1969
23 231 Pichia anomala GIT of carp, 1977
24 232 Кат Pichia anomala Gills of carp, 1977
25 236 Pichia anomala GIT of carp, 1977
26 237 Pichia anomala GIT of trout, 1978
27 242 Pichia anomala Gills of bream, the Gorky Reservoir, 1983
28 243 Pichia anomala Leather of bream, Balkhash, 1983
28 244 Pichia anomala Silage
30 444 Pichia guilliermondii Soil contaminated with crude oil, West Ukraine
31 445 Pichia guilliermondii Soil contaminated with crude oil, West Ukraine
32 446 Pichia guilliermondii Soil contaminated with crude oil, West Ukraine
33 1754 Saccharomyces cerevisiae Wine, received from Golovach
34 1887 Saccharomyces cerevisiae Intestine of pheasant, Turkmenistan, 1983
35 2232 Saccharomyces cerevisiae Israel, 2000
36 2298 Saccharomyces cerevisiae Institute of Hematology
37 2386 Saccharomyces cerevisiae Institute of Urology
38 2444 Saccharomyces cerevisiae Pistachio, Israel, 2000
39 2519 Saccharomyces cerevisiae Netherlands, Rotterdam, brewing (top fermenting yeast)
40 25 Williopsis californica Tomatoes, rhizosphere, 1969
41 250 Williopsis californica Oats, rhizosphere, 1967
42 255 Williopsis californica Cucumbers, 1969
43 256 Williopsis californica Tomatoes, 1969
44 258 Williopsis californica Soil, Teremky, 1983
45 259 Williopsis californica Soil, Teremky, 1983
46 2461 Williopsis californica Soil, Znamenka, Kirovograd region, 1999
Thus, as a result of the work, the data were obtained on the prevalence frequency of P-mannanases producers among yeast cultures. It is shown that the soil and sources of plant origin are the optimal medium for the isolation of active producers of the enzymes of mannan-degrading complex. For the first
time the strains-producers of ß-mannanase among representatives of W. californica and M. pulcherrima species have been identified. Yeast ß-mannanases are promising for use in various fields of biotechnology, in particular in the processing of mannan-containing raw materials.
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P-МАНАНАЗНА АКТИВН1СТЬ ДР1ЖДЖ1В
Н. В. Борзова Л. Д. Варбанець В. С. Пiдгорський О. Д. Янева
1нститут мшробмлогп i вiрусологii iM. Д. К. Заболотного НАН Украши, Кшв
E-mail: nv_borzova@bigmir.net
Метою роботи було визначення манан-деградувальноi активност дрiжджових культур, iзольованих i3 рiзних джерел, для выбору серед них високоактивних проду-ценив P-мананаз. У результат скриншгу серед 245 штамiв дрiжджiв, представникiв 7 родiв, 14 видiв, виявлено активнi проду-центи позаклiтинноi P-мананази. Для ощ-нювання активност культури вирощували в глибинних умовах, як джерело вуглецю та iндуктор використовували галактома-нан камедi гуару. P-Мананазну активнiсть визначали динiтросалiциловим методом. Найб^ьш активними бiосинтетиками ви-явились представники видiв Cryptococcus albidus, С. gastricus, C. magnus, C. terreus, C. laurentii, Saccharomyces cerevisiae, Williopsis calif ornica, Metschnikowia pulcherrima, Pichia anomala та Р. guilliermondii. Актившсть у супернатан-т культуральноi рщини становила в^ 0,2 до 75 од/мл. У двох штамiв Debaryomyces polymorphus УКМ Y-152 i Debaryomyces hansenii var. fabryi УКМ Y-2400 виявлено а-галактозидазну активнiсть. Жодна з досль джених культур не виявляла одночасно P-ма-наназноi та а-галактозидазноi активносм, що свiдчить про нездатнiсть iх атакувати як ос-новний, так i бiчнi ланцюги галактоманану.
Ключовi слова: дрiжджi, P-мананаза, а-галак-то зидаза, галактоманан.
Р-МАННАНАЗНАЯ АКТИВНОСТЬ ДРОЖЖЕЙ
Н. В. Борзова Л. Д. Варбанец В. С. Подгорский О. Д. Янева
Институт микробиологии и вирусологии им. Д. К. Заболотного НАН Украины, Киев
E-mail: nv_borzova@bigmir.net
Целью работы было определение ман-нандеградирующей активности дрожжевых культур, выделенных из различных источников, для отбора высокоактивных продуцентов Р-маннаназ. В результате скрининга среди 245 штаммов дрожжей, представителей 7 родов, 14 видов, выявлены активные продуценты внеклеточной Р-маннаназы. Для оценки активности культуры выращивали в глубинных условиях, в качестве источника углерода и индуктора использовали галакто-маннан камеди гуара. Р-Маннаназную активность определяли динитросалициловым методом. Наиболее активными биосинтетиками оказались представители видов Cryptococcus albidus, С. gastricus, C. magnus, C. terreus, C. laurentii, Saccharomyces cerevisiae, Williopsis calif ornica, Metschnikowia pulcherrima, Pichia anomala и Р. guillier-mondii. Активность в супернатанте куль-туральной жидкости составила от 0,2 до 75 Е/мл. У двух штаммов Debaryomyces polymorphus УКМ Y-152 и Debaryomyces hansenii var. fabryi УКМ Y-2400 выявлена а-галактозидазная активность. Ни одна из изученных культур не проявляла одновременно Р-маннаназную и а-галактозидазную активность, что свидетельствует о неспособности их атаковать как основную, так и боковые цепи галактоманнана.
Ключевые слова: дрожжи, Р-маннаназа, а-га-лак тозидаза, галактоманнан.
