طراحی بیوانفورماتیکی و بهینه سازی تولید آنزیم گلوکزاکسیداز نوترکیب بیان شده در مخمر یارروویا لیپولیتیکا
محورهای موضوعی : زیست فناوری میکروبی
فاطمه خدیوی درخشان
1
(دانشجوی دکتری، دانشگاه آزاد اسلامی، واحد کرج، دانشکده علوم پایه، گروه میکروب شناسی)
فرشاد درویشی
2
(دانشیار، دانشگاه مراغه، دانشکده علوم پایه، گروه میکروب شناسی)
مهروز دزفولیان
3
(استادیار، دانشگاه آزاد اسلامی، واحد کرج، دانشکده علوم پایه، گروه میکروب شناسی)
کاترین مادزاک
4
(استاد، دانشگاه پاریس، آگروپاریس تک)
کلید واژه: تاگوچی, بیوانفورماتیک, گلوکزاکسیداز, یارروویا لیپولیتیکا,
چکیده مقاله :
سابقه و هدف: آنزیم گلوکزاکسیداز کاربرد فراوانی در صنایع پزشکی، دارویی، غذایی، نساجی و محیطی دارد. بیان هترولوگ این آنزیم در میزبان های مخمری به دلیل برخی از محدودیت های تولید صنعتی این آنزیم توسط آسپرژیلوس نایجر بررسی شده است. این مطالعه با هدف بررسی ویژگی های بیوانفورماتیکی گلوکزاکسیداز در سویه نوترکیب مخمر یارروویا لیپولیتیکا Po1g-GOX و بهینه سازی شرایط تولید این آنزیم به روش تاگوچی صورت گرفت.مواد و روش ها: توالی پروتئین، ساختارهای اول، دوم، سوم و میزان گلیکوزیلاسیون آنزیم نوترکیب گلوکزاکسیداز با نرم افزارهای بیوانفورماتیکی بررسی شد. بهینه سازی تولید آنزیم نوترکیب برای گلوکز، پپتون، عصاره مخمر و pH در محیط YPD دارای تیامین در 4 سطح برای نرم افزار Qualitek-4 تعریف شد. میزان تولید آنزیم سنجش و با نرم افزار تجزیه و تحلیل شد.یافته ها: گلوکزاکسیداز نوترکیب دارای 605 اسیدآمینه بوده که ساختار دوم آن دارای 29 درصد مارپیچ آلفا، 16 درصد صفحه بتا و 54 درصد کویلاست که با ساختار دوم توالی اسیدآمینه گلوکزاکسیداز طبیعی آسپرژیلوس نایجر تشابه زیادی را نشان داد. 8 جایگاه گلیکوزیلاسیون در این پروتئین نوترکیب قرار دارد و ساختار سوم دارای یک دومین است و تشابه بالایی با گلوکزاکسیداز طبیعی دارد. بیشترین تولید آنزیم گلوکزاکسیداز نوترکیب در شرایط بهینه در محیط کشت حاوی 30 گرم در لیتر گلوکز، 5 گرم در لیتر عصاره مخمر، 15 گرم در لیتر پپتون و pH حدود 7 به دست آمد.نتیجه گیری: گلوکز اکسیداز نوترکیب یارروویا به دلیل شباهت بالا با گلوکزاکسیداز طبیعی پتانسیل مناسبی به منظور کاربرد در صنایع دارویی و غذایی دارد.
Background & Objectives: Glucose oxidase has a wide range of application in medical, pharmaceutical, food, textile, and environmental industries. Heterologous expression of the enzyme has been investigated in yeast hosts due to several industrial-scale limitations in enzyme production by Aspergillus niger. The present study aimed to investigate bioinformatics characteristics of recombinant glucose oxidase in Yarrowia lipolytica Po1g-GOX and to optimize the production conditions of the enzyme by Taguchi experimental design method. Materials & Methods: Protein sequence, primary, secondary and tertiary structures and glycosylation rate of recombinant glucose oxidase enzyme were studied via bioinformatics tools. Optimization of recombinant glucose oxidase for glucose, peptone, yeast extract and pH at four levels in YPD media with thiamine were defined to Qualitek-4 software. The level of enzyme production was measured and analyzed by the software.Results: Recombinant glucose oxidase had 605 amino acids, with 29% α-helix, 16% β sheet and 54% coil in its secondary structure showing similarity to that of native glucose oxidase of A. niger. Eight glycosylation sites have been located in this recombinant protein, and the tertiary structure had one domain showing high similarity to native glucose oxidase. The highest level of recombinant glucose oxidase production was obtained in medium containing 30 g/L glucose, 5 g/L yeast extract, 15 g/L peptone, and pH 7.Conclusion: Recombinant glucose oxidase produced in Yarrowia has a suitable potential to be used in pharmaceutical and food industries due to high similarity to native glucose oxidase.
Biotechnol Adv. 2009; 27(4): 489-501.
2. Wong CM, Wong KH, Chen XD. Glucose oxidase: Natural occurrence, function, properties and
industrial applications. Appl Microbiol Biotechnol. 2008; 78(6): 927-938.
3. Shaikh Sumaiya A, Trived R. A review on glucose oxidase. Int J Curr Microbiol App Sci. 2015;
4: 636-642.
4. Frederick KR, Tung J, Emerick RS, Masiarz FR, Chamberlain SH, Vasavada A, Rosenberg S,
Chakraborty S, Schopfer LM. Glucose oxidase from Aspergillus niger. Cloning, gene sequence,
secretion from Saccharomyces cerevisiae and kinetic analysis of a yeast-derived enzyme. J Biol
Chem. 1990; 265(7): 3793-3802.
5. Cox H, Mead D, Sudbery P, Eland RM, Mannazzu I, Evans L. Constitutive expression of
recombinant proteins in the methylotrophic yeast Hansenula polymorpha using the PMA1
promoter. Yeast. 2000; 16(13): 1191-1203.
6. Malherbe DF, Toit Md, Cordero Otero RR, van Rensburg P, Pretorius IS. Expression of the
Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential
applications in wine production. Appl Microbiol Biotechnol. 2003;61(5-6):502-511.
7. Rocha SN, Abrahão-Neto J, Cerdán ME, González-Siso MI, Gombert AK. Heterologous
expression of glucose oxidase in the yeast Kluyveromyces marxianus. Microb Cell Fact. 2010;
9(1): 4-15.
8. Madzak C, Beckerich JM. Heterologous protein expression and secretion in Yarrowia
lipolytica. in Yarrowia lipolytica: Biotechnological applications. Edited by Barth G. Berlin,
Heidelberg, Springer Berlin Heidelberg; 2013. pp. 1-76.
9. Madzak C, Gaillardin C, Beckerich JM. Heterologous protein expression and secretion in the
non-conventional yeast Yarrowia lipolytica: A review. J Biotechnol. 2004; 109(1-2): 63-81.
10. Darvishi Harzevili F: Yarrowia lipolytica: An overview. in Biotechnological Applications of
the Yeast Yarrowia lipolytica. Cham, Springer International Publishing; 2014. pp. 1-16.
11. Madzak C. Yarrowia lipolytica: recent achievements in heterologous protein expression and
pathway engineering. Appl Microbiol Biotechnol. 2015; 99: 4559-4577.
12. Khadivi Derakshan F, Darvishi F, Dezfulian M, Madzak C. Expression and characterization of
glucose oxidase from Aspergillus niger in Yarrowia lipolytica. Mol Biotechnol. 2017; 59(8):
307-314.
13. Khuri AI, Mukhopadhyay S. Response surface methodology. Wiley Interdisciplinary Reviews:
Computational Statistics. 2010; 2: 128-149.
14. Barton RR. Response surface methodology. In: Encyclopedia of operations research and
management science. Edited by Gass SI, Fu MC. Boston, MA, Springer US; 2013. pp.
1307-1313.
15. Madzak C, Otterbein L, Chamkha M, Moukha S, Asther M, Gaillardin C, Beckerich JM.
Heterologous production of a laccase from the basidiomycete Pycnoporus cinnabarinus in the
dimorphic yeast Yarrowia lipolytica. FEMS Yeast Res. 2005; 5(6-7): 635-646.
16. Tsui KL. An overview of Taguchi method and newly developed statistical methods for
Robust design. IIE Transactions. 1992; 24: 44-57.
17. Karna SK, Sahai R. An overview on Taguchi method. Int J Eng sci. 2012;1:1-7.
18. Hodgkins M, Mead D, Ballance DJ, Goodey A, Sudbery P. Expression of the glucose oxidase
gene from Aspergillus niger in Hansenula polymorpha and its use as a reporter gene to isolate
regulatory mutations. Yeast. 1993; 9(6): 625-635.
19. Park EH, Shin YM, Lim YY, Kwon TH, Kim DH, Yang MS. Expression of glucose oxidase by
using recombinant yeast. J Biotechnol. 2000; 81: 35-44.
20. Zhanjun Q, Yuanfang G, Xiaoming B, Jianrong H, Gaoying S, Bingyin P, Wenxiang B.
Expression of Aspergillus niger glucose oxidase in yeast Pichia pastoris SMD1168. Biotechnol
Biotechnol Equip. 2016; 30(5): 998-1005.
21. Zhou YF, Zhang XE, Liu H, Zhang CG, Cass AE. Cloning and expression of Aspergillus niger
glucose oxidase gene in methylotrophic yeast. Sheng Wu Gong Cheng Xue Bao. 2001; 17(4):
400-405.
22. Darvishi Harzevili F. Yarrowia lipolytica in biotechnological applications. In: Biotechnological
applications of the yeast Yarrowia lipolytica. Cham, Springer International Publishing; 2014; pp.
17-74.
23. Pulci V, D'Ovidio R, Petruccioli M, Federici F. The glucose oxidase of Penicillium variabile
P16: gene cloning, sequencing and expression. Lett Appl Microbiol. 2004; 38(3): 223-238.
24. Kiess M, Hecht HJ, Kalisz HM. Glucose oxidase from Penicillium amagasakiense. Primary
structure and comparison with other glucose-methanol-choline (GMC) oxidoreductases. Eur J
Biochem. 1998; 15: 90-99.
25. Kriechbaum M, Heilmann HJ, Wientjes FJ, Hahn M, Jany KD, Gassen HG, Sharif F,
Alaeddinoglu G. Cloning and DNA sequence analysis of the glucose oxidase gene from
Aspergillus niger NRRL‐3. FEBS Lett. 1989; 225(1): 62-66.
26. Hatzinikolaou DG, Hansen OC, Macris BJ, Tingey A, Kekos D, Goodenough P, Stougaard P.
A new glucose oxidase from Aspergillus niger: characterization and regulation studies of
enzyme and gene. Appl Microbiol Biotechnol. 1996; 46(4): 371-381.
27. El-Sherbeny G, Shindia A, Sheriff Y. Optimization of various factors affecting glucose
oxidase activity produced by Aspergillus niger. Int J Agr Biol Eng. 2005; 7: 953-958.
28. Hamid HM, Zia M, Asgher M. Optimization of various parameters for the production of
glucose oxidase from rice polishing using Aspergillus niger. Biotechnol (Pakistan). 2003; 2:
1-7.
29. Khurshid S, Kashmiri MA, Quershi Z, Ahmad W. Optimization of glucose oxidase production
by Aspergillus niger. Afr J Biotechnol. 2011; 10(9): 1674-1678.
30. Darvishi F, Moradi M, Madzak C, Jolivalt C. Production of laccase by recombinant Yarrowia
lipolytica from molasses: Bioprocess development using statistical modeling and increase
productivity in shake-flask and bioreactor cultures. Appl Biochem Biotechnol. 2017; 181:
1228-1239.
_||_
Biotechnol Adv. 2009; 27(4): 489-501.
2. Wong CM, Wong KH, Chen XD. Glucose oxidase: Natural occurrence, function, properties and
industrial applications. Appl Microbiol Biotechnol. 2008; 78(6): 927-938.
3. Shaikh Sumaiya A, Trived R. A review on glucose oxidase. Int J Curr Microbiol App Sci. 2015;
4: 636-642.
4. Frederick KR, Tung J, Emerick RS, Masiarz FR, Chamberlain SH, Vasavada A, Rosenberg S,
Chakraborty S, Schopfer LM. Glucose oxidase from Aspergillus niger. Cloning, gene sequence,
secretion from Saccharomyces cerevisiae and kinetic analysis of a yeast-derived enzyme. J Biol
Chem. 1990; 265(7): 3793-3802.
5. Cox H, Mead D, Sudbery P, Eland RM, Mannazzu I, Evans L. Constitutive expression of
recombinant proteins in the methylotrophic yeast Hansenula polymorpha using the PMA1
promoter. Yeast. 2000; 16(13): 1191-1203.
6. Malherbe DF, Toit Md, Cordero Otero RR, van Rensburg P, Pretorius IS. Expression of the
Aspergillus niger glucose oxidase gene in Saccharomyces cerevisiae and its potential
applications in wine production. Appl Microbiol Biotechnol. 2003;61(5-6):502-511.
7. Rocha SN, Abrahão-Neto J, Cerdán ME, González-Siso MI, Gombert AK. Heterologous
expression of glucose oxidase in the yeast Kluyveromyces marxianus. Microb Cell Fact. 2010;
9(1): 4-15.
8. Madzak C, Beckerich JM. Heterologous protein expression and secretion in Yarrowia
lipolytica. in Yarrowia lipolytica: Biotechnological applications. Edited by Barth G. Berlin,
Heidelberg, Springer Berlin Heidelberg; 2013. pp. 1-76.
9. Madzak C, Gaillardin C, Beckerich JM. Heterologous protein expression and secretion in the
non-conventional yeast Yarrowia lipolytica: A review. J Biotechnol. 2004; 109(1-2): 63-81.
10. Darvishi Harzevili F: Yarrowia lipolytica: An overview. in Biotechnological Applications of
the Yeast Yarrowia lipolytica. Cham, Springer International Publishing; 2014. pp. 1-16.
11. Madzak C. Yarrowia lipolytica: recent achievements in heterologous protein expression and
pathway engineering. Appl Microbiol Biotechnol. 2015; 99: 4559-4577.
12. Khadivi Derakshan F, Darvishi F, Dezfulian M, Madzak C. Expression and characterization of
glucose oxidase from Aspergillus niger in Yarrowia lipolytica. Mol Biotechnol. 2017; 59(8):
307-314.
13. Khuri AI, Mukhopadhyay S. Response surface methodology. Wiley Interdisciplinary Reviews:
Computational Statistics. 2010; 2: 128-149.
14. Barton RR. Response surface methodology. In: Encyclopedia of operations research and
management science. Edited by Gass SI, Fu MC. Boston, MA, Springer US; 2013. pp.
1307-1313.
15. Madzak C, Otterbein L, Chamkha M, Moukha S, Asther M, Gaillardin C, Beckerich JM.
Heterologous production of a laccase from the basidiomycete Pycnoporus cinnabarinus in the
dimorphic yeast Yarrowia lipolytica. FEMS Yeast Res. 2005; 5(6-7): 635-646.
16. Tsui KL. An overview of Taguchi method and newly developed statistical methods for
Robust design. IIE Transactions. 1992; 24: 44-57.
17. Karna SK, Sahai R. An overview on Taguchi method. Int J Eng sci. 2012;1:1-7.
18. Hodgkins M, Mead D, Ballance DJ, Goodey A, Sudbery P. Expression of the glucose oxidase
gene from Aspergillus niger in Hansenula polymorpha and its use as a reporter gene to isolate
regulatory mutations. Yeast. 1993; 9(6): 625-635.
19. Park EH, Shin YM, Lim YY, Kwon TH, Kim DH, Yang MS. Expression of glucose oxidase by
using recombinant yeast. J Biotechnol. 2000; 81: 35-44.
20. Zhanjun Q, Yuanfang G, Xiaoming B, Jianrong H, Gaoying S, Bingyin P, Wenxiang B.
Expression of Aspergillus niger glucose oxidase in yeast Pichia pastoris SMD1168. Biotechnol
Biotechnol Equip. 2016; 30(5): 998-1005.
21. Zhou YF, Zhang XE, Liu H, Zhang CG, Cass AE. Cloning and expression of Aspergillus niger
glucose oxidase gene in methylotrophic yeast. Sheng Wu Gong Cheng Xue Bao. 2001; 17(4):
400-405.
22. Darvishi Harzevili F. Yarrowia lipolytica in biotechnological applications. In: Biotechnological
applications of the yeast Yarrowia lipolytica. Cham, Springer International Publishing; 2014; pp.
17-74.
23. Pulci V, D'Ovidio R, Petruccioli M, Federici F. The glucose oxidase of Penicillium variabile
P16: gene cloning, sequencing and expression. Lett Appl Microbiol. 2004; 38(3): 223-238.
24. Kiess M, Hecht HJ, Kalisz HM. Glucose oxidase from Penicillium amagasakiense. Primary
structure and comparison with other glucose-methanol-choline (GMC) oxidoreductases. Eur J
Biochem. 1998; 15: 90-99.
25. Kriechbaum M, Heilmann HJ, Wientjes FJ, Hahn M, Jany KD, Gassen HG, Sharif F,
Alaeddinoglu G. Cloning and DNA sequence analysis of the glucose oxidase gene from
Aspergillus niger NRRL‐3. FEBS Lett. 1989; 225(1): 62-66.
26. Hatzinikolaou DG, Hansen OC, Macris BJ, Tingey A, Kekos D, Goodenough P, Stougaard P.
A new glucose oxidase from Aspergillus niger: characterization and regulation studies of
enzyme and gene. Appl Microbiol Biotechnol. 1996; 46(4): 371-381.
27. El-Sherbeny G, Shindia A, Sheriff Y. Optimization of various factors affecting glucose
oxidase activity produced by Aspergillus niger. Int J Agr Biol Eng. 2005; 7: 953-958.
28. Hamid HM, Zia M, Asgher M. Optimization of various parameters for the production of
glucose oxidase from rice polishing using Aspergillus niger. Biotechnol (Pakistan). 2003; 2:
1-7.
29. Khurshid S, Kashmiri MA, Quershi Z, Ahmad W. Optimization of glucose oxidase production
by Aspergillus niger. Afr J Biotechnol. 2011; 10(9): 1674-1678.
30. Darvishi F, Moradi M, Madzak C, Jolivalt C. Production of laccase by recombinant Yarrowia
lipolytica from molasses: Bioprocess development using statistical modeling and increase
productivity in shake-flask and bioreactor cultures. Appl Biochem Biotechnol. 2017; 181:
1228-1239.