تولید نانوذرات اورهآز به روش حلالزدایی و مقایسه برخی خواص آنها با اورهآز آزاد
محورهای موضوعی :
بیوشیمی
راضیه سادات حسینی
1
(کارشناسی ارشد، گروه زیست، دانشکده علوم پایه، واحد قم، دانشگاه آزاد اسلامی، قم، ایران)
سید محمد حسین رضویان
2
(استادیار، گروه زیست، دانشکده علوم پایه، واحد قم، دانشگاه آزاد اسلامی، قم، ایران)
محمدعلی قاسم‌زاده
3
(دانشیار، گروه شیمی، دانشکده علوم پایه، واحد قم، دانشگاه آزاد اسلامی، قم، ایران)
تاریخ دریافت : 1401/03/07
تاریخ پذیرش : 1401/09/11
تاریخ انتشار : 1402/02/01
کلید واژه:
پایداری حرارتی,
نانو فناوری,
نانوذرات آنزیمی,
حلالزدایی,
اورهآز,
چکیده مقاله :
هدف: آنزیم‌ها به‌عنوان کاتالیزور طبیعی در واکنش‌های بیولوژیکی عمل می‌کنند. اما دارای محدودیت‌هایی مانند عدم پایداری حرارتی، طول عمر کوتاه و عدم پایداری آنها در محیط آلی نیز می‌باشند. بنابراین، دانشمندان سعی کرده‌اند عملکرد آنزیم‌ها را به روش‎‎‎‎‎‎‎‎های‎‎‎‎‎‎‎‎‎‎ مختلف ازجمله فناوری نانو بهبود ببخشند. لذا، هدف مطالعه حاضر تولید نانوذرات آنزیمی و ارزیابی برخی خواص آنها است، که به دلیل اهمیت اوره‎آز در پزشکی، کشاورزی و صنعت روی آن کار می‌شود.مواد و روش‌ها: در این پژوهش، افزایش پایداری اوره‎آز براساس تولید نانوذرات آنزیمی با روش حلال‎زدایی انجام شد. نانوذرات سنتز شده با استفاده از طیف‌سنجی مادون قرمز تبدیل فوریه (FTIR)، طیف‌سنجی مرئی- فرابنفش (UV-Vis) و میکروسکوپ الکترونی روبشی (SEM) بررسی شد. همچنین فعالیت‌های کلی و اختصاصی آنزیم‌های آزاد و نانو در دمای °C۳۷ اندازه‌گیری و باهم مقایسه شد. به‌علاوه، آنزیم‌های آزاد و نانو به مدت 10 دقیقه در دماهای بین ۳۰ تا °C۷۰ انکوبه و سپس فعالیت آنها اندازه‌گیری شد.یافته‌ها: نتایج حاصل از طیف‌سنجی و میکروسکوپ الکترونی روبشی، تشکیل نانوذرات اوره‎آز را تأیید کرد. همچنین نتایج تعیین فعالیت نشان داد که با تشکیل نانوذرات آنزیمی، علی‌رغم کاهش فعالیت کل آنزیم، فعالیت اختصاصی آن 46/43 درصد افزایش یافت.دمای مطلوب فعالیت کل اوره‎آز آزاد °C50 و نانوذرات اوره‎آز °C60 بود. پس از 10 دقیقه انکوباسیون در °C70، آنزیم‌های آزاد و نانو به ترتیب 2 و 32 درصد فعالیت خود را حفظ کردند که نشان‌دهنده افزایش پایداری حرارتی در این روش است.نتیجه‌گیری: با تهیه نانوذرات آنزیمی می توان فعالیت و کاربرد آنها را در صنعت بهبود بخشید.
چکیده انگلیسی:
Objective: Enzymes act as natural catalysts in biological reactions. But they have limitations such as lack of thermal stability, short life span and their lack of stability in the organic environment. Therefore, scientists have tried to improve the performance of enzymes in different ways, including nanotechnology. Therefore, the aim of the current study is to produce enzyme nanoparticles and evaluate some of their properties, which are being worked on due to the importance of urease in medicine, agriculture and industry.Materials and methods: In this research, increasing the stability of urease was done based on the production of enzyme nanoparticles by desolvation method. Synthesized nanoparticles were examined using Fourier Transform Infrared Spectroscopy (FTIR), Visible-Ultraviolet Spectroscopy (UV-Vis) and Scanning Electron Microscope (SEM). Also, the general and specific activities of free and nano enzymes were measured and compared at 37°C. In addition, free and nano enzymes were incubated for 10 minutes at temperatures between 30 and 70°C and then their activity was measured.Findings: The results of spectroscopy and scanning electron microscopy confirmed the formation of urease nanoparticles. Also, the activity determination results showed that with the formation of enzyme nanoparticles, despite the decrease in the total activity of the enzyme, its specific activity increased by 43.46%. The optimal activity temperature of total free urease was 50°C and urease nanoparticles was 60°C.After 10 minutes of incubation at 70°C, the free and nano enzymes retained 2% and 32% of their activity, respectively, which indicates an increase in thermal stability in this method.Conclusion: By preparing enzyme nanoparticles, it is possible to improve their activity and application in the industry.
منابع و مأخذ:
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Lizeng G & Xiyun Y. Nanozymes: an emerging field bridging nanotechnology and biology. Science China Life Sciences. 2016; 59(4): 400-2.
Khosravi A, Vossoughi M, Sharokhian S & Alemzadeh I (eds). Synthesis and Stability evaluation of HRP Single Enzyme Nanoparticles. Proceedings of International Conference on Nanostructures (ICNS4); 2012.
Cuesta SM, Rahman SA, Furnham N & Thornton JM. The classification and evolution of enzyme function. Biophysical journal. 2015; 109(6): 1082-6.
Sheldon RA & van Pelt S. Enzyme immobilisation in biocatalysis: why, what and how. Chemical Society Reviews. 2013; 42(15): 6223-35.
Kim J, Jia H & Wang P. Challenges in biocatalysis for enzyme-based biofuel cells. Biotechnology advances. 2006; 24(3): 296-308.
Mohamad NR, Marzuki NHC, Buang NA, Huyop F & Wahab RA. An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes. Biotechnology & Biotechnological Equipment. 2015; 29(2): 205-20.
Leong MK. Green Synthesis, Characterization of Copper (II) Oxide Nanoparticles and Their Photocatalytic Activity. UTAR; 2016.
Ghosh Chaudhuri R & Paria S. Core/shell nanoparticles: classes, properties, synthesis mechanisms, characterization, and applications. Chemical reviews. 2012; 112(4): 2373-433.
Olveira S, Forster SP & Seeger S. Nanocatalysis: academic discipline and industrial realities. Journal of Nanotechnology. 2014. https://doi.org/10.1155/2014/324089
Koo OM, Rubinstein I & Onyuksel H. Role of nanotechnology in targeted drug delivery and imaging: a concise review. Nanomedicine: nanotechnology, biology and medicine. 2005; 1(3): 193-212.
NH K. Synthesis Characterization and Application of Nickel Nanoparticles. Jamshoro-PAKISTAN.: Doctoral dissertation; 2013.
Kundu N, Yadav S & Pundir C. Preparation and characterization of glucose oxidase nanoparticles and their application in dissolved oxygen metric determination of serum glucose. Journal of Nanoscience and Nanotechnology. 2013; 13(3): 1710-6.
Jakhar S & Pundir C. Preparation, characterization and application of urease nanoparticles for construction of an improved potentiometric urea biosensor. Biosensors and Bioelectronics. 2018; 100: 242-50.
Kim J, Grate JW & Wang P. Nanobiocatalysis and its potential applications. Trends in biotechnology. 2008; 26(11): 639-46.
Yadav N, Narang J, Chhillar AK & Pundir CS. Chapter Six - Preparation, characterization, and application of enzyme nanoparticles. Methods in Enzymology. 2018; 609: 171-196.
Cuenot S, Frétigny C, Demoustier-Champagne S & Nysten B. Surface tension effect on the mechanical properties of nanomaterials measured by atomic force microscopy. Physical Review B. 2004; 69(16): 165410.
Karim MN. Modulating the NanoZyme activity for antibacterial and sensing applications. RMIT University; 2019.
Scheffel U, Rhodes BA, Natarajan T & Wagner HN. Albumin microspheres for study of the reticuloendothelial system. Journal of Nuclear Medicine. 1972; 13(7): 498-503.
Pundir CS. Enzyme nanoparticles: preparation, characterisation, properties and applications. William Andrew; 2015.
Sailaja AK, Amareshwar P & Chakravarty P. Different techniques used for the preparation of nanoparticles using natural polymers and their application. Int J Pharm Pharm Sci. 2011; 3(2): 45-50.
Liu G, Lin Y, Ostatná V & Wang J. Enzyme nanoparticles-based electronic biosensor. Chemical communications. 2005; 27: 3481-3.
Sharma S, Shrivastav A, Gupta N & Srivastava S (eds). Amperometric biosensor: increased sensitivity using enzyme nanoparticles. 2010 international conference on nanotechnology and biosensors, IPCBEE; 2011: Citeseer.
Chauhan N, Kumar A & Pundir C. Construction of an uricase nanoparticles modified Au electroe for amperometric determination of uric acid. Applied biochemistry and biotechnology. 2014; 174(4): 1683-94.
Kappaun K, Piovesan AR, Carlini CR & Ligabue-Braun R. Ureases: Historical aspects, catalytic, and non-catalytic properties–A review. Journal of advanced research. 2018; 13: 3-17.
Benini S, Rypniewski W, Wilson K, Ciurli S & Mangani S. The complex of Bacillus pasteurii urease with β-mercaptoethanol from X-ray data at 1.65-Å resolution. JBIC Journal of Biological Inorganic Chemistry. 1998; 3(3): 268-73.
Fong YH, Wong HC, Yuen MH, Lau PH, Chen YW & Wong K-B. Structure of UreG/UreF/UreH complex reveals how urease accessory proteins facilitate maturation of Helicobacter pylori urease. PLoS biology. 2013; 11(10): e1001678.
Khan M, Javed MM, Zahoor S & HAQ U. Kinetics and thermodynamic study of urease extracted from soybeans. Biologia. 2013; 59(1): 7-14.
Bzura J & Koncki R. A mechanized urease activity assay. Enzyme and microbial technology. 2019; 123: 1-7.
Boer JL & Hausinger RP. Klebsiella aerogenes UreF: identification of the UreG binding site and role in enhancing the fidelity of urease activation. Biochemistry. 2012; 51(11): 2298-308.
Chawla S, Rawal R & Pundir C. Preparation of cholesterol oxidase nanoparticles and their application in amperometric determination of cholesterol. Journal of nanoparticle research. 2013; 15(9): 1-9.
DeSantis G & Jones JB. Chemical modification of enzymes for enhanced functionality. Current Opinion in Biotechnology. 1999; 10(4): 324-30.
Kim J, Grate JW & Wang P. Nanostructures for enzyme stabilization. Chemical engineering science. 2006; 61(3): 1017-26.
Ding S, Cargill AA, Medintz IL & Claussen JC. Increasing the activity of immobilized enzymes with nanoparticle conjugation. Current opinion in biotechnology. 2015; 34:
242-50.
Ansari SA & Husain Q. Potential applications of enzymes immobilized on/in nano materials: A review. Biotechnology advances. 2012; 30(3): 512-23.
Palmer T & Bonner PL. Enzymes: biochemistry, biotechnology, clinical chemistry. Elsevier; 2007.
Pithawala K, Mishra N & Bahadur A. Immobilization of urease in alginate, paraffin and lac. Journal of the Serbian Chemical Society. 2010; 75(2): 175-83.
Brady D & Jordaan J. Advances in enzyme immobilisation. Biotechnology letters. 2009; 31(11): 1639-50.
Mirza Babaei S. Stabilization of urease enzyme on chitosan/polyvinyl alcohol nanofiber prepared by electrospinning method. Master Thesis. University of Tehran, 2012. [in persian]
Shen Y, Zhang Y, Zhang X, Zhou X, Teng X, Yan M & et al. Horseradish peroxidase-immobilized magnetic mesoporous silica nanoparticles as a potential candidate to eliminate intracellular reactive oxygen species. 2015; 7(7): 2941-50.
Men D, Zhang T-T, Hou L-W, Zhou J, Zhang Z-P, Shi Y-Y & et al. Self-assembly of ferritin nanoparticles into an enzyme nanocomposite with tunable size for ultrasensitive immunoassay. Acs Nano. 2015; 9(11): 10852-60.
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Robinson PK. Enzymes: principles and biotechnological applications. Essays in biochemistry. 2015; 59: 1.
Lizeng G & Xiyun Y. Nanozymes: an emerging field bridging nanotechnology and biology. Science China Life Sciences. 2016; 59(4): 400-2.
Khosravi A, Vossoughi M, Sharokhian S & Alemzadeh I (eds). Synthesis and Stability evaluation of HRP Single Enzyme Nanoparticles. Proceedings of International Conference on Nanostructures (ICNS4); 2012.
Cuesta SM, Rahman SA, Furnham N & Thornton JM. The classification and evolution of enzyme function. Biophysical journal. 2015; 109(6): 1082-6.
Sheldon RA & van Pelt S. Enzyme immobilisation in biocatalysis: why, what and how. Chemical Society Reviews. 2013; 42(15): 6223-35.
Kim J, Jia H & Wang P. Challenges in biocatalysis for enzyme-based biofuel cells. Biotechnology advances. 2006; 24(3): 296-308.
Mohamad NR, Marzuki NHC, Buang NA, Huyop F & Wahab RA. An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes. Biotechnology & Biotechnological Equipment. 2015; 29(2): 205-20.
Leong MK. Green Synthesis, Characterization of Copper (II) Oxide Nanoparticles and Their Photocatalytic Activity. UTAR; 2016.
Ghosh Chaudhuri R & Paria S. Core/shell nanoparticles: classes, properties, synthesis mechanisms, characterization, and applications. Chemical reviews. 2012; 112(4): 2373-433.
Olveira S, Forster SP & Seeger S. Nanocatalysis: academic discipline and industrial realities. Journal of Nanotechnology. 2014. https://doi.org/10.1155/2014/324089
Koo OM, Rubinstein I & Onyuksel H. Role of nanotechnology in targeted drug delivery and imaging: a concise review. Nanomedicine: nanotechnology, biology and medicine. 2005; 1(3): 193-212.
NH K. Synthesis Characterization and Application of Nickel Nanoparticles. Jamshoro-PAKISTAN.: Doctoral dissertation; 2013.
Kundu N, Yadav S & Pundir C. Preparation and characterization of glucose oxidase nanoparticles and their application in dissolved oxygen metric determination of serum glucose. Journal of Nanoscience and Nanotechnology. 2013; 13(3): 1710-6.
Jakhar S & Pundir C. Preparation, characterization and application of urease nanoparticles for construction of an improved potentiometric urea biosensor. Biosensors and Bioelectronics. 2018; 100: 242-50.
Kim J, Grate JW & Wang P. Nanobiocatalysis and its potential applications. Trends in biotechnology. 2008; 26(11): 639-46.
Yadav N, Narang J, Chhillar AK & Pundir CS. Chapter Six - Preparation, characterization, and application of enzyme nanoparticles. Methods in Enzymology. 2018; 609: 171-196.
Cuenot S, Frétigny C, Demoustier-Champagne S & Nysten B. Surface tension effect on the mechanical properties of nanomaterials measured by atomic force microscopy. Physical Review B. 2004; 69(16): 165410.
Karim MN. Modulating the NanoZyme activity for antibacterial and sensing applications. RMIT University; 2019.
Scheffel U, Rhodes BA, Natarajan T & Wagner HN. Albumin microspheres for study of the reticuloendothelial system. Journal of Nuclear Medicine. 1972; 13(7): 498-503.
Pundir CS. Enzyme nanoparticles: preparation, characterisation, properties and applications. William Andrew; 2015.
Sailaja AK, Amareshwar P & Chakravarty P. Different techniques used for the preparation of nanoparticles using natural polymers and their application. Int J Pharm Pharm Sci. 2011; 3(2): 45-50.
Liu G, Lin Y, Ostatná V & Wang J. Enzyme nanoparticles-based electronic biosensor. Chemical communications. 2005; 27: 3481-3.
Sharma S, Shrivastav A, Gupta N & Srivastava S (eds). Amperometric biosensor: increased sensitivity using enzyme nanoparticles. 2010 international conference on nanotechnology and biosensors, IPCBEE; 2011: Citeseer.
Chauhan N, Kumar A & Pundir C. Construction of an uricase nanoparticles modified Au electroe for amperometric determination of uric acid. Applied biochemistry and biotechnology. 2014; 174(4): 1683-94.
Kappaun K, Piovesan AR, Carlini CR & Ligabue-Braun R. Ureases: Historical aspects, catalytic, and non-catalytic properties–A review. Journal of advanced research. 2018; 13: 3-17.
Benini S, Rypniewski W, Wilson K, Ciurli S & Mangani S. The complex of Bacillus pasteurii urease with β-mercaptoethanol from X-ray data at 1.65-Å resolution. JBIC Journal of Biological Inorganic Chemistry. 1998; 3(3): 268-73.
Fong YH, Wong HC, Yuen MH, Lau PH, Chen YW & Wong K-B. Structure of UreG/UreF/UreH complex reveals how urease accessory proteins facilitate maturation of Helicobacter pylori urease. PLoS biology. 2013; 11(10): e1001678.
Khan M, Javed MM, Zahoor S & HAQ U. Kinetics and thermodynamic study of urease extracted from soybeans. Biologia. 2013; 59(1): 7-14.
Bzura J & Koncki R. A mechanized urease activity assay. Enzyme and microbial technology. 2019; 123: 1-7.
Boer JL & Hausinger RP. Klebsiella aerogenes UreF: identification of the UreG binding site and role in enhancing the fidelity of urease activation. Biochemistry. 2012; 51(11): 2298-308.
Chawla S, Rawal R & Pundir C. Preparation of cholesterol oxidase nanoparticles and their application in amperometric determination of cholesterol. Journal of nanoparticle research. 2013; 15(9): 1-9.
DeSantis G & Jones JB. Chemical modification of enzymes for enhanced functionality. Current Opinion in Biotechnology. 1999; 10(4): 324-30.
Kim J, Grate JW & Wang P. Nanostructures for enzyme stabilization. Chemical engineering science. 2006; 61(3): 1017-26.
Ding S, Cargill AA, Medintz IL & Claussen JC. Increasing the activity of immobilized enzymes with nanoparticle conjugation. Current opinion in biotechnology. 2015; 34:
242-50.
Ansari SA & Husain Q. Potential applications of enzymes immobilized on/in nano materials: A review. Biotechnology advances. 2012; 30(3): 512-23.
Palmer T & Bonner PL. Enzymes: biochemistry, biotechnology, clinical chemistry. Elsevier; 2007.
Pithawala K, Mishra N & Bahadur A. Immobilization of urease in alginate, paraffin and lac. Journal of the Serbian Chemical Society. 2010; 75(2): 175-83.
Brady D & Jordaan J. Advances in enzyme immobilisation. Biotechnology letters. 2009; 31(11): 1639-50.
Mirza Babaei S. Stabilization of urease enzyme on chitosan/polyvinyl alcohol nanofiber prepared by electrospinning method. Master Thesis. University of Tehran, 2012. [in persian]
Shen Y, Zhang Y, Zhang X, Zhou X, Teng X, Yan M & et al. Horseradish peroxidase-immobilized magnetic mesoporous silica nanoparticles as a potential candidate to eliminate intracellular reactive oxygen species. 2015; 7(7): 2941-50.
Men D, Zhang T-T, Hou L-W, Zhou J, Zhang Z-P, Shi Y-Y & et al. Self-assembly of ferritin nanoparticles into an enzyme nanocomposite with tunable size for ultrasensitive immunoassay. Acs Nano. 2015; 9(11): 10852-60.
