شناسایی مولکولی و بهینه سازی شرایط تولید آنزیم لیپاز باسیلوس تورنجینسیس L26
محورهای موضوعی : میکروب شناسی صنعتیفرزانه کریمیان 1 , محمدحسن قربانی 2 , سید حسین میردامادیان 3
1 - دانشجوی کارشناسی ارشد، دانشگاه آزاد اسلامی واحد فلاورجان، دانشکده علوم زیستی، گروه میکروبیولوژی، ایران
2 - استادیار، دانشگاه آزاد اسلامی، واحد فلاورجان، دانشکده علوم زیستی، گروه بیوشیمی، ایران
3 - مربی، دانشگاه آزاد اسلامی، واحد فلاورجان، دانشکده علوم زیستی، گروه میکروبیولوژی
کلید واژه: لیپاز, باسیلوس تورنجینسیس, آنزیم‌های هیدرولیتیک, سنجش فعالیت,
چکیده مقاله :
سابقه و هدف: لیپازها مهم‌ترین آنزیم‌های هیدرولیتیک تولید شده توسط میکروارگانیسم‌های مختلف به ویژه باکتری‌ها هستند که در صنایع غذایی و دارویی کاربردهای گسترده ای دارند. هدف از این مطالعه، جداسازی و شناسایی باکتری‌های مولد لیپاز از منابع در دسترس به منظور استفاده در صنعت است. مواد و روش‌ها: نمونه‌برداری و جداسازی باکتری‌های مولد آنزیم لیپاز از پساب و خاک تصفیه‌خانه حبیب‌آباد، پساب و لجن فیلتر شنی کارخانه روغن نباتی گلبهار، دنبه و کنجاله‌های کنجد صورت گرفت. به منظور سنجش میزان تولید و فعالیت آنزیم‌های لیپازی، از روماند به‌دست آمده از کشت باکتری در محیط کشت پایه لیپیدی استفاده گردید. سپس مقدار آنزیم موجود در روماند توسط سنجش کمی فعالیت آنزیمی با استفاده از تکنیک اسپکتروفتومتری و سوبسترای اختصاصی پارانیتروفنیل استات در دمای 28 درجه سلسیوس صورت گرفت. شناسایی باکتری توسط آنالیزهای ماکروسکوپی، میکروسکوپی و مولکولی انجام شد. یافته‌ها: بر اساس نتایج غربال‌گری سویه‌های جداسازی شده و همچنین ارزیابی‌های آنزیمی، سویه 31 به‌عنوان سویه برتر انتخاب گردید. با استفاده از آنالیز مولکولی 16S rRNA این جدایه به‌عنوان باسیلوس تورنجینسیس L26 تعیین شد. بیشترین پایداری و فعالیت آنزیمی در این سویه در دمای 48 درجه سلسیوس، 8.5 pH و به ترتیب در حضور کلرید سدیم ، کلرید کلسیم، کلرید پتاسیم ، کلرید منیزیم، سولفات روی و کلرید منگنز به دست آمد.نتیجه گیری: نتایج این پژوهش نشان داد آنزیم لیپاز باسیلوس تورنجینسیسL26 در شرایط قلیایی و در حضور کاتیون های مختلف پایداری متفاوتی دارند.
Background & Objectives: Lipases are important hydrolytic enzymes produced by various microorganisms such as bacteria. This enzyme is applied in various industries including food and pharmaceutical industries. This study aimed to isolate and identify lipase-producing bacteria from various sources to use in different industries.Materials & Methods: Sampling and screening of lipase- producing bacteria were carried out from wastewater and soils of Habib-abad refinery, wastewater and slugs of Golbahar oil factory, sheep’s tail fat, and sesame meal. To evaluate the enzymatic activity, bacterial isolates were cultured in lipid-based media, where the supernatant was used for the next assay. A quantitative assessment of enzymatic activity was performed using a spectrophotometer, in the presence of para-nitrophenol acetate at the temperature of 28°C. The identification of bacterial isolates was carried out by macroscopic, microscopic and molecular analysis.Results: Screening bacterial isolates and the results of enzymatic activity assays showed strain 31 as the superior one. Molecular analysis results identified this strain as Bacillus thuringiensis L26. The highest enzymatic activity and stability were obtained at the temperature of 48°C, pH value of 8.5, and in the presence of sodium chloride, calcium chloride, potassium chloride, magnesium chloride, zinc sulfate, and manganese chloride, respectively.Conclusion: Our results showed the stability of tested lipase enzymes under alkaline conditions and the presence of different cations. Therefore, further complementary tests are recommended o assess practical uses.
in a bioreactor from Aspergillus species. Ministry Sci Technol. 2012; 1: 1-30.
2. Stoytcheva M, Montero G, Zlatev RA, Leon J, Gochev V. Analytical methods for lipases activity
determination: A review. Curr Anal Chem. 2012; 8(3): 400-407.
3. Balashev K, Jensen TR, Kjaer K, Bjornholm T. Novel methods for studying lipids and lipases and
their mutual interaction at interfaces. Part I. Atomic force microscopy. Biochimie. 2001; 83:
387-397.
4. Gao W, Wu K, Chen L, Fan H, Zhao Z, Gao B, Wang H, Wei D. A novel esterase from a marine
mud metagenomic library for biocatalytic synthesis of short‑chain flavor esters. Microb Cell Fact.
2016; 15: 41.
5. Kiran S, Arshad Z, Nosheen S, Kamal S, Gulzar T, Majeed MS, Jannat M, Rafique MA.
Microbial lipases: Production and applications: A review. J Biochem. 2016; 1(2): 7-20.
6. Gandolfi R, Marinelli F, Lazzarini A, Molinari F. Cell-bound and extracellular carboxylesterases
from Sterptomyces: hydrolytic and synthetic activities. J Appl Microbiol. 2000; 89(5): 870-875.
7. Pandey A, Benjamin S, Soccol CR, Nigam P, Krieger N, Soccol VT. The realm of microbial
lipases in biotechnology. Biotechnol Appl Biochem. 1999; 29: 119-131.
8. Jaeger KE, Ransac S, Dijkstra BW, Colson C, van Heuvel M, Misset O. Bacterial lipases.
FEMS Microbiol Rev. 1994; 15: 29-63.
9. Mobarak Qamsari E, Kasra Kermanshahi R, Moosavi nejad Z. Isolation and identification of a
novel, lipase-producing bacterium, Pseudomnas aeruginosa KM110. Iran J Microbiol. 2011; 3(2):
92-98.
10. Adami Ghamsari F. Isolation of lipolytic bacteria from environmental resources for
biodegradation polysorbate in industrial wastewater. Bimonthly J Sabzevar Univ Med Sci. 2015;
22(4): 685-693.
11. Torres S, Baigorí MD, Swathy SL, Pandey A, Castro GR. Enzymatic synthesis of banana flavour
(isoamyl acetate) by Bacillus licheniformis S-86 esterase. Food Res Int. 2009; 42: 454-460.
12. Moradi S, Razavi SH, Mousavi SM. Isolation of lipase producing bacteria from olive and the
various parameter effect on enzyme production in solid state fermentation.2015; 46(3): 315-325.
[In Persian].
13. Xavier AREO, Lima ER, Oliveira AME, Cardoso L and Santos J. Genetic diversity
of Bacillus sp. producers of amylase isolated from the soil. Genet. Mol. Res. 2017; 16(3).
10.4238/gmr16039771
14. Badoei-Dalfard A, Karami Z, Ramezani-Pour N. Bench scale production of nicotinic acid using a
newly isolated Stenotrophomonas maltophilia AC- 21 producing highly-inducible and versatile
nitrilase. J Mol Catal B Enzym. 2016; 133: 552-559.
15. Afrisham S, Badoei-Dalfard A, Namaki-Shoushtari A, Karami Z, Malekabadi S. Isolation and
identification of Bacillus producing thermophilic alpha amylase: production optimization and
investigation of the activity and stability of enzyme. Nova Biologica Reperta. 2018; 4(4):
288-298. [In Persian].
16. Sharma MR, Chisti Y, Banerjee UC. Production, purification, characterization, and applications
of lipases. Biotechnol Adv. 2001; 1: 627-662.
17. Stergiou PY, Foukis A, Filippou M, Koukouritaki M, Parapouli M, Theodorou Lg. Advances in
lipase-catalyzed esterification reactions. Biotechnol Adv. 2013; 31: 1846-1859.
18. Hasan F, Hasan AA, Hameed A. Industrial applications of microbial lipases. Enzyme Microb
Technol. 2006; 39: 235-251.
19. Leung DYC, WU X, Leung MKH. A review on biodiesel production using catalyzed
transesterification. Appl Energy. 2010; 87: 1083-1095.
20. Baltz RH, Davies JE, Demain AL. Mannual of industrial microbiology and biotechnology. Third
ed. Washington DC: ASM press. 2010. pp: 745.
21. Wanatab N, Ota Y, Minoda Y, Yamada K. Isolation and identification pf alkaline lipase
producing microorganisms, cultural conditions and some properties of crude enzyme. Agric Biol
Chem. 2016; 41(8): 1353-1358.
22. Mobarak Qamsari E, Kasra Kermanshahi R, Moosavi nejad Z. Isolation and identification of a
novel, lipase-producing bacterium, Pseudomnas aeruginosa KM110. Iran J Microbiol. 2011;
3(2): 92-98.
23. Kumar D, Kumar L, Nagar S, Raina C, Parshad R. Screening, isolation and production of lipase/
esterase producing Baclillus sp. Strain DVL2 and evaluation in esterification and resolution
reactions. Arch Appl Sci Res. 2012; 4(4): 1763-1770.
24. Gupta R, Gupta N, Rathi P. Bacterial lipases: An overview of production, purification and
biochemical properties. Appl Microbiol Biotechnol. 2004; 64: 763-781.
25. Tripathi R, Singh J, kumar Bharti R, Thakur IS. Isolation, purification and characterization of
lipase from Microbacterium sp. and its application in biodiesel production. Energy Procedia.
2014; 54: 518-529.
26. Padmapriya B, Rajeswari T, Noushida E, Sethupalan DG, Venil CK. Production of lipase
enzyme from Lactobacillus spp. and its application in the degradation of meat. World Appl Sci J.
2011; 12(10): 1798-1802.
27. Sharma P, Sharma N, Pathania S, Handa S. Purification and characterization of lipase by
Bacillus methylotrophicus PS3 under submerged fermentation and its application in detergent
industry. Genet Eng Biotechnol J. 2017; 15(2): 369-377.
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in a bioreactor from Aspergillus species. Ministry Sci Technol. 2012; 1: 1-30.
2. Stoytcheva M, Montero G, Zlatev RA, Leon J, Gochev V. Analytical methods for lipases activity
determination: A review. Curr Anal Chem. 2012; 8(3): 400-407.
3. Balashev K, Jensen TR, Kjaer K, Bjornholm T. Novel methods for studying lipids and lipases and
their mutual interaction at interfaces. Part I. Atomic force microscopy. Biochimie. 2001; 83:
387-397.
4. Gao W, Wu K, Chen L, Fan H, Zhao Z, Gao B, Wang H, Wei D. A novel esterase from a marine
mud metagenomic library for biocatalytic synthesis of short‑chain flavor esters. Microb Cell Fact.
2016; 15: 41.
5. Kiran S, Arshad Z, Nosheen S, Kamal S, Gulzar T, Majeed MS, Jannat M, Rafique MA.
Microbial lipases: Production and applications: A review. J Biochem. 2016; 1(2): 7-20.
6. Gandolfi R, Marinelli F, Lazzarini A, Molinari F. Cell-bound and extracellular carboxylesterases
from Sterptomyces: hydrolytic and synthetic activities. J Appl Microbiol. 2000; 89(5): 870-875.
7. Pandey A, Benjamin S, Soccol CR, Nigam P, Krieger N, Soccol VT. The realm of microbial
lipases in biotechnology. Biotechnol Appl Biochem. 1999; 29: 119-131.
8. Jaeger KE, Ransac S, Dijkstra BW, Colson C, van Heuvel M, Misset O. Bacterial lipases.
FEMS Microbiol Rev. 1994; 15: 29-63.
9. Mobarak Qamsari E, Kasra Kermanshahi R, Moosavi nejad Z. Isolation and identification of a
novel, lipase-producing bacterium, Pseudomnas aeruginosa KM110. Iran J Microbiol. 2011; 3(2):
92-98.
10. Adami Ghamsari F. Isolation of lipolytic bacteria from environmental resources for
biodegradation polysorbate in industrial wastewater. Bimonthly J Sabzevar Univ Med Sci. 2015;
22(4): 685-693.
11. Torres S, Baigorí MD, Swathy SL, Pandey A, Castro GR. Enzymatic synthesis of banana flavour
(isoamyl acetate) by Bacillus licheniformis S-86 esterase. Food Res Int. 2009; 42: 454-460.
12. Moradi S, Razavi SH, Mousavi SM. Isolation of lipase producing bacteria from olive and the
various parameter effect on enzyme production in solid state fermentation.2015; 46(3): 315-325.
[In Persian].
13. Xavier AREO, Lima ER, Oliveira AME, Cardoso L and Santos J. Genetic diversity
of Bacillus sp. producers of amylase isolated from the soil. Genet. Mol. Res. 2017; 16(3).
10.4238/gmr16039771
14. Badoei-Dalfard A, Karami Z, Ramezani-Pour N. Bench scale production of nicotinic acid using a
newly isolated Stenotrophomonas maltophilia AC- 21 producing highly-inducible and versatile
nitrilase. J Mol Catal B Enzym. 2016; 133: 552-559.
15. Afrisham S, Badoei-Dalfard A, Namaki-Shoushtari A, Karami Z, Malekabadi S. Isolation and
identification of Bacillus producing thermophilic alpha amylase: production optimization and
investigation of the activity and stability of enzyme. Nova Biologica Reperta. 2018; 4(4):
288-298. [In Persian].
16. Sharma MR, Chisti Y, Banerjee UC. Production, purification, characterization, and applications
of lipases. Biotechnol Adv. 2001; 1: 627-662.
17. Stergiou PY, Foukis A, Filippou M, Koukouritaki M, Parapouli M, Theodorou Lg. Advances in
lipase-catalyzed esterification reactions. Biotechnol Adv. 2013; 31: 1846-1859.
18. Hasan F, Hasan AA, Hameed A. Industrial applications of microbial lipases. Enzyme Microb
Technol. 2006; 39: 235-251.
19. Leung DYC, WU X, Leung MKH. A review on biodiesel production using catalyzed
transesterification. Appl Energy. 2010; 87: 1083-1095.
20. Baltz RH, Davies JE, Demain AL. Mannual of industrial microbiology and biotechnology. Third
ed. Washington DC: ASM press. 2010. pp: 745.
21. Wanatab N, Ota Y, Minoda Y, Yamada K. Isolation and identification pf alkaline lipase
producing microorganisms, cultural conditions and some properties of crude enzyme. Agric Biol
Chem. 2016; 41(8): 1353-1358.
22. Mobarak Qamsari E, Kasra Kermanshahi R, Moosavi nejad Z. Isolation and identification of a
novel, lipase-producing bacterium, Pseudomnas aeruginosa KM110. Iran J Microbiol. 2011;
3(2): 92-98.
23. Kumar D, Kumar L, Nagar S, Raina C, Parshad R. Screening, isolation and production of lipase/
esterase producing Baclillus sp. Strain DVL2 and evaluation in esterification and resolution
reactions. Arch Appl Sci Res. 2012; 4(4): 1763-1770.
24. Gupta R, Gupta N, Rathi P. Bacterial lipases: An overview of production, purification and
biochemical properties. Appl Microbiol Biotechnol. 2004; 64: 763-781.
25. Tripathi R, Singh J, kumar Bharti R, Thakur IS. Isolation, purification and characterization of
lipase from Microbacterium sp. and its application in biodiesel production. Energy Procedia.
2014; 54: 518-529.
26. Padmapriya B, Rajeswari T, Noushida E, Sethupalan DG, Venil CK. Production of lipase
enzyme from Lactobacillus spp. and its application in the degradation of meat. World Appl Sci J.
2011; 12(10): 1798-1802.
27. Sharma P, Sharma N, Pathania S, Handa S. Purification and characterization of lipase by
Bacillus methylotrophicus PS3 under submerged fermentation and its application in detergent
industry. Genet Eng Biotechnol J. 2017; 15(2): 369-377.