CC BY
131
UDC 579.873.71.017.7 https://doi.org/10.15407/biotech9.04.077
ALPHA-AMYLASE PRODUCTION
FROM Aspergillus oryzae M BY SUBMERGED FERMENTATION
Zh. B. Suleimenova Department of Physiology and Biochemistry,
Zh. K. Saduyeva Institute of Microbiology and Virology, Almaty,
Zh. K. Rakhmetova Kazakhstan
E-mail: [email protected]
Received 28.03.2016
The main goal of present study was implementation of the Aspergillus oryzae M strain improved technology using earlier developed method of microorganism selection. The 8 pure strains of Aspergillus fungi were screened for the production of extra cellular alpha-amylase using agar medium with starch as a substrate and incubated for 72h at 30 °C. Zone of clearance was observed for screening of the amylolytic fungi (in mm). Aspergillus oryzae M has demonstrated the highest zone of clearance. Aspergillus oryzae M was cultivated for 42 days in submerged conditions of growth using new method of fungal cultivation. This method based on immobilizing enzymes producers on solid career in submerged conditions of growth gives the way to improve quality of filtrates, which remain clear, does not require additional filtering and easily separated from the mycelium. Moreover, it allows to prolong the process of fungal cultivation and to maintain high enzymatic activity for a long period of time. Presented method allowed increasing alpha-amylase production from 321 U/ml (before immobilization) to 502 U/ml (after immobilization).
Key words: a-amylase, Aspergillus oryzae, submerged fermentation, immobilization.
Amylases are among the most important industrial enzymes. This group of enzymes occupies about 25% of the whole market of produced enzymes in the world [1, 2]. Despite the fact that among microorganisms that produce amylases there are bacteria, fungi, yeast and actinomycetes, in the recent period micromycetes got wide application, particularly Aspergillus fungi type thanks to the ease of its cultivation and high productivity [3, 4]. However, great drawback of industrial strains is their low activity, despite the fact that the chief requirement to enzymes is their high catalyst activity which is directly connected with the activity of the microorganism that produces this enzyme. Scientific research of the leading biochemical laboratories of the world are directed to the solution of this problem, i.e. obtaining of a highly active strain — superproducer of enzymes. The use of such highly active strain can reduce expenses on production of the enzyme, which will positively influence the price of the final product [5].
Until recently, the main instrument in obtaining such a highly active a-amylase strain was mutagenesis [6-9]. There are available data on increasing of Aspergillus awamori strain glucoamylase productivity obtained by using recombinant-DNA technology. As a result rising in the level of starch-splitting gene activator proteinl, that was amyR gene-coded, led to 30% increasing in Aspergillus awamori strain productivity [10]. However, genetic engineering is long-lasting and requires for special complex techniques [11]. In this regards, the cells immobilization offer a multitude of advantages in enzymes production, such as high metabolic activity and strong resistance to toxic chemicals [12-15]. Immobilized microorganisms can be used for a long period of time with increased enzymatic activity [16, 17].
Traditionally used enzymatic agent processing technology applies the submerged culture method of micromycetes in periodic conditions. Under such conditions of micromycetes growth, their biomass forms
a shape of pellets, which during the culture process firms and becomes inaccessible for oxygen and nutrients. The biomass forms the maximum amount of the target product in 3-5 days only once. After that, this culture autolyzes and its enzyme activity decrease. A new method of cultivation of filamentous fungi has been developed by Bliyeva R.K., as well as devices and equipment for their cultivation [18]. Such devices and equipment prolong producers' cultivation period to 30-60 days and create the opportunity to obtain enzymes repeatedly in every 2-3 days of cultivation. This method is based on immobilization enzymes producers on solid career in submerged conditions of growth. Immobilization has a range of advantages: decreasing the price of the final product, absence of foreign substances, controlled process of enzyme-genesis, ability of various enzymes simultaneous production, etc. [19-21]. Design of proposed equipment gives the opportunity to increase the activity of immobilized cells culture filtrate comparing to free cells. In the present work a-amylase production using immobilized Aspergillus species was studied.
Materials and Methods
Screening for the alpha-amylase activity
The 8 pure strains of Aspergillus fungi (own collection) were screened for the production of extra cellular alpha-amylase using agar medium with starch using as a substrate (Aspergillus awamori 16, Aspergillus awamori 1-8, Aspergillus awamori 22, Aspergillus oryzae M, Aspergillus oryzae 3-9-15, Aspergillus foetidus, Aspergillus niger n, Aspergillus niger 355). Aspergillus fungi were maintained on Czapek medium. CZAPEK: NaNO3 — 9.0 g, sucrose — 20.0 g; KH2P04 — 1.0 g; MgSO4 — 0.5 g; KCl — 0.5 g; FeSO4 — 0.001 g; agar-agar — 20,0 g — per liter. The pure cultures were inoculated on starch agar medium and incubated for 72 h at room temperature. After obtained colonies of each plate iodine solution was layered on the agar plates and zone of clearance was observed for screening of the amylolytic fungi. A positive test is indicated by a clear, colorless zone around the growth. Only positive and better zone formed strain was taken for further study.
Enzyme production
For inoculum preparation, 25 ml of sterile distilled water was added to the 5-day-old
culture grown on Czapek agar plate and scraped aseptically with inoculating loop. This suspension with spore concentration of 1.3107 cells/ml, was used as inoculum for the fungal cultivation. Submerged fermentation was carried out in 750 ml Erlenmeyer flask by taking 100 ml of mineral salt medium (%): NH4NO3 — 0.5; KH2P04 — 0.1; MgSO4 — 0.05; KCl — 0.05; FeSO4 — 0.001; maltose — 1.0; starch — 1.0. They were incubated at 30 °C on a rotary shaker (180 rpm) for 42 days. The growth medium was exchanged at 3-day intervals.
Alpha-amylase enzyme assay
The amylase activity was assayed by spectrohotometric measurement of a starch-iodine complex (State Standard of Russian Federation). The reaction mixture (15 ml) consisted of 10 ml of 1% (w/v) soluble starch and 0.5 ml appropriately diluted enzyme source in 25 ml of distilled water. After incubation at 30 °C temperature for 10 min the reaction was stopped by addition of iodine solution with 0.2 mol/dm3 HCl. Then the enzymatic hydrolysis of starch was determined on spectrophotometer at 670 nm. One unit of the a-amylase activity was defined as the amount of enzyme that hydrolyses 1 g of starch for 1 hour in 30 °C, pH 4.7. The experiment was carried out in triplicates. The results were expressed as mean ± standard deviation using Excel 2010.
Results and Discussion
Screening for the alpha-amylase activity
During the initial screening of Aspergillus fungi for synthesis of alpha-amylase enzyme it was found that almost all studied strains grew on the starch medium. The clear zone formation concerns the ability of colonies with confirmed starch hydrolysis (Fig. 1).
Aspergillus oryzae M showed the highest clearance zone (29.3 mm) and was used for further studies (Table). Alpha-amylase activity of free cells of Aspergillus oryzae M after 3-days liquid cultivation was 321 U/ml.
Immobilization of Aspergillus oryzae M
A novel immobilization technique was developed by using the cheapest and most easily available cotton material as an immobilizing carrier for absorption of spores of A. oryzae M, which has been for the first time used for immobilization of microorganisms. Immobilized A. oryzae M on carrier is presented in Fig. 2.
Aspergillus oryzae M Control
Fig. 1. Iodine clearing zone assay
Starch hydrolysis test by Aspergillus fungi for alpha-amylase activity
NN Strain Clear zone, mm
1 Aspergillus awamori 16 11.5 ± 1.0
2 Aspergillus awamori 1-8 20.0 ± 0.9
3 Aspergillus awamori 22 14.3 ± 0.9
4 Aspergillus oryzae М 29.3 ± 2.8
5 Aspergillus oryzae 3-9-15 16.4 ± 1.9
6 Aspergillus foetidus 11.8 ± 1.0
7 Aspergillus niger II 16.7 ± 0.7
8 Aspergillus niger 355 12.1 ± 1.2
Fig. 2. Immobilization of Aspergillus oryzae M
Cultivation period was extended to 42 days. Results show that immobilization procedure has significant effect both on growth and bioactivity of A. oryzae M when compared to periodic cultivation by free cells (Fig. 3). As shown on Fig. 3 enzymatic activity was enhanced significantly after 6 days of cultivation of immobilized cells and keeps the same value for 42 days of fungal cultivation. The immobilized system showed a significant stability of the enzyme biosynthesis. Maximum of the alpha-amylase production was obtained at 9 days interval. Enzyme activity ranged from 286 to 502 U/ml.
In contrast to immobilized cells cultivation by free cells has a limited development cycle. It forms greatest amount of alpha-amylase activity only on 3d day. After that, A. oryzae M cells autolyze and its enzyme activity decrease (Fig. 3).
Thus, our presented method extends continuously without interruption for a period of 42 days. Frequency of obtainment of the desired product is gradually increasing. If the periodic cultivation by free cells allows to obtain target product (alpha-amylase enzyme) only once (on the 3d day), during continuous cultivation by immobilized cells target product can be obtained every 2-3 days. In addition, presented immobilization procedure allows maintain high fungal enzymatic activity for a long period of time.
This research work was funded by Ministry of Education and Sciences of the Republic of Kazakhstan.
Grant № 0347 "Biosynthesis of a-amylase enzyme by Aspergillus fungi to improve the quality of bread and baked goods".
1 ♦ • periodic cultivation (free cells) Fig. 3. Alpha-amylase production by immobilized Aspergillus oryzae M
REFERENCES
1. Rani G., Paresh G., Harapriya M., Vineet K. G., Bhavna C. Microbial a-amylases: a biotechnological perspective. Proc. Biochem. 2013, V. 38, P. 1599-1616. doi: 10.1016/ S0032-9592(03)00053-0.
2. Lili Kandra. a-Amylases of medical and industrial importance. Journal of Molecular Structure: Theochem. 2003, N 666, P. 487-498. doi: 10.1016/ j.theochem. 2003.08.073.
3. David B. Finkelstein. Improvement of enzyme production in Aspergillus. Antonie van Leeuwenhoek J. Microbiol. 1987, 53 (5), 349-352. doi: 10.1007/BF00400559.
4. Ward O. P., Qin W. M., Dhanjoon J., Ye J., Singh A. Physiology and Biotechnology of Aspergillus. Adv. Appl. Microbiol. 2005, V. 58, P. 1-75. doi: 10.1016/S0065-2164(05)58001-8.
5. Vanek Z, Hosalek Z, Spizek J. Overproduction of microbial products — Facts and ideas. Biotechnol. Adv. 1990, 8 (1), 1-27. doi: 10.1016/0734-9750(90)90003-T.
6. Sidhu G. S, Sharma P., Chakrabarti T., Gupta J. K. Strain improvement for the production of a thermostable a-amylase. Enzyme and Microbial Technology. 1997, 21 (7), 525-530. doi: 10.1016/S0141-0229(97)00055-0.
7. Clark Ford. Improving operating performance of glucoamylase by mutagenesis. Cur. Opin. Biotechnol. 1999, 10 (4), 353-357. doi: 10.1016/S0958-1669(99)80064-0.
8. Parekh S. Strain Improvement. Encyclopedia of Microbiology, 3rd edn. 2009, P. 286-298. doi:10.1016/B978-012373944-5.00169-3.
9. Rowlands R. T. Industrial strain improvement: Mutagenesis and random screening procedures. Enz. Microb. Technol. 1984, 6 (1), 3-10. doi: 0.1016/0141-0229(84)90070-X.
10. Vinetsky Y. P., Rozhkov A. M., Sereda A. S., Tsurikova N. V., NurtayevA. K, Semenov M. V., Zorov I. N., Sinicyn A. P. Increase productivity strain Aspergillus awamori glucoamylase by a combination of radiation mutagenesis and plasmid transformation. Appl. Biochem. Microbiol. 2010, 46 (6), 685-692. doi: 10.1134/S0003683810060128.
11. Sanjai Saxena. Strategies of Strain Improvement of Industrial Microbes. Appl. Microbiol. 2015, P. 155-171. doi: 10.1007/978-81-322-2259-0-10.
12. Cohen Y. Biofiltration — The Treatment of Fluids by Microorganisms Immobilized into the Filter Bedding Material: a review. Biores. Technol. Rev. 2001, V. 77, P. 257-254. doi: 10.1016/S0960-8524(00)00074-2
13. Liu H., Guo L., Liao S., Wang G. Reutilization of immobilized fungus Rhizopus sp. LG04 to reduce toxic Chromate. J. Appl. Microbiol. 2012, N 112, P. 651-659. doi: 10.1111/j.1365-2672.2012.05257.x.
14. Cai T., Chen L., Ren Q., Cai S., Zhang J. The biodegradation pathway of triethylamine and its biodegradation by immobilized Arthrobacterprotophormiae cells. J. Hazard Mater. 2001, V. 186, P. 59. doi: 10.1016/j. jhazmat.2010.10.007.
15. Wang L., Ridgway D., Gu T., Moo-Young M. Bioprocessing strategies to improve heterologous protein production in filamentous fungal fermentations. Biotech. Adv. 2005, V. 23, P. 115-129. doi: 10.1016/j. biotechadv.2004.11.001.
16. Rhee S. K., Lee G. M., Lee S. T. Influence of a supplementary carbon source on biodegradation of pyridine by freely suspended and immobilized Pimelobacter sp. J. Appl. Microbiol. Biotechnol. 1996, V. 44, P. 816-822. doi: 10.1007/BF00178624.
17. Devi S., Sridhar P. Production of cephamycin C in repeated batch operations from immobilized Streptomyces clavuligerus. Proc. Biochem. 2000, V. 36, P. 225-231. doi:10.1016/S0032-9592(00)00194-1.
18. Blieva R. K. An apparatus for the cultivation of microorganisms with filamentous structure. Innovation Patent Kazakhstan № 27164. June 25, 2013.
19. Guisan J. M. Immobilization of Enzymes and Cells (Third Edition). 2013, P. 115-119. doi: 10.1007/978-1-62703-550-7.
20. Konstantin A., Lusta K. C., Whan S., Hee S. P., Dong S. Immobilization of fungus Aspergillus sp. by a novel cryogel technique for production of extracellular hydrolytic enzymes. Proc. Biochem. 2000, 35 (10), 1177-1182. doi: 10.1016/S0032-9592(00)00158-8.
21. Fiedurek J., Ilczuk Z., Lobarzewski J., Pleszczynska M. Optimization of pectinolytic enzymes biosynthesis by immobilized mycelium of Aspergillus niger 71. Zentralblatt f r Mikrobiologie. 1992, 147 (1-2), 15-21. doi: 10.1016/S0232-4393(11)80357-4.
ПРОДУКУВАННЯ АЛЬФА-АМ1ЛАЗИ З Aspergillus oryzae M ЗА УМОВ
ГЛИБИННОГО КУЛЬТИВУВАННЯ
Ж. Б. Сулейменова
Ж. К. Садуева Ж. К. Рахметова
1нститут мшробюлогп i вiрусологii, Алмати, Казахстан
E-mail: [email protected]
Метою дослщження було отримання висо-коактивного штаму-продуцента а-ам^ази на основi iммобiлiзацii гриба Aspergillus oryzae M в умовах глибинного культивування за розробленим рашше методом селекцп мшро-органiзмiв. Об'ектами дослiджень слугували 8 штамiв роду Aspergillus iз колекцii 1нсти-туту мшробюлогп та вiрусологii. Первинний вiдбiр активноi культури проводили якiсним методом шляхом вимiрювання дiаметра зон гiдролiзу дослiджуваними культурами субстрату (крохмалю) на третю добу iнкубацii за 30 °С (у мм). Найбьльшу активнiсть мав штам Aspergillus oryzae М. Проведено три-вале культивування ввдбраного продуцента а-амiлази Aspergillus oryzae M упродовж 42 дiб. Актившсть а-амiлази варiювала вiд 286 до 502 од/мл. Вщзначено, що куль-туральна рiдина прозора i не потребуе до-датково'1 ф^ьтрацп, легко вiддiляеться вiд мiцелiю. Пропонований метод селекцп уможливив шдвищення активност аль-фа-амiлази з 321 од/мл (до iммобiлiзацii) до 502 од/мл (шсля iммобiлiзацii), а також дав змогу зберегти актившсть культури протя-гом тривалого перiоду часу.
Ключовi слова: а-ам1лаза, Aspergillus oryzae; глибинне культивування, 1ммоб1л1защя.
ПРОДУЦИРОВАНИЕ АЛЬФА-АМИЛАЗЫ ИЗ Aspergillus oryzae M В УСЛОВИЯХ ГЛУБИННОГО КУЛЬТИВИРОВАНИЯ
Ж. Б. Сулейменова
Ж. К. Садуева Ж. К. Рахметова
Институт микробиологии и вирусологии, Алматы, Казахстан
E-mail: [email protected]
Целью исследования было получение высокоактивного штамма-продуцента а-амилазы на основе иммобилизации гриба Aspergillus oryzae M в условиях глубинного культивирования по разработанному ранее методу селекции микроорганизмов. Объектами исследований служили 8 штаммов рода Aspergillus из коллекции Института микробиологии и вирусологии. Первичный отбор активной культуры проводили качественным методом путем измерения диаметра зон гидролиза исследуемыми культурами субстрата (крахмала) на третьи сутки инкубации при 30 °С (в мм). Наибольшую активность имел штамм Aspergillus oryzae М. Проведено длительное культивирование отобранного продуцента а-амилазы Aspergillus oryzae M в течение 42 суток. Активность а-амилазы варьировала от 286 до 502 ед/мл. Отмечено, что культуральная жидкость прозрачная и не требует дополнительной фильтрации, легко отделяется от мицелия. Предлагаемый метод селекции дал возможность повысить активность альфа-амилазы с 321 ед/мл (до иммобилизации) до 502 ед/мл (после иммобилизации), а также позволил сохранить активность культуры в течение длительного периода времени.
Ключевые слова: а-амилаза, Aspergillus oryzae, глубинное культивирование, иммобилизация.
