Cloning of Bacillus subtilis phytase gene construct in Escherichia coli
-
Mahdiyar Iravani Saadi
,
Abbas Doosti
,
Heeva Jalali
,
Ehsan Nabi Abdolyousefi
,
Mansooreh Hooshiyar
,
Reza Tabrizi
,
Esmat Noshadi
Abstract
Background and Objectives: Phytase has a hydrolysis function of phytic acid, which yields inorganic phosphate. Bacillus species can produce thermostable alkaline phytase. The aim of this study was to isolate and clone a Phytase gene (Phy) from Bacillus subtilis in Escherichia coli.
Materials and Methods: In this study, the extracellular PhyC gene was isolated from Bacillus subtilis Phytase C. After purification of the bands, DNA fragment of Phy gene was cloned by T/A cloning technique, and the clone was transformed into Escherichia coli. Afterward, the pGEM-Phy was transferred into E. coli Top-10 strain and the recombinants were plated on LB agar containing 100 µg/ml ampicillin. The colonization of 1171 bp of gene Phytase C was confirmed by PCR. The presence of gene-targeting in vector was confirmed with enzymatic digestion by XhoI and XbaI restriction enzymes.
Results: The Phytase gene was successfully cloned in E. coli. The result of cloning of 1171 bp Phytase gene was confirmed by PCR assay.
Conclusion: Our impression of this article is that several methods, such as using along with microbial, plant phytase reproduction, or low-phytic acid corn may be the better way from a single phytase.
1. Rao DE, Rao KV, Reddy VD. Cloning and expression of Bacillus phytase gene (phy) in Escherichia coli and recovery of active enzyme from the inclusion bodies. J Appl Microbiol 2008;105:1128-1137.
2. Singh N, Kuhar S, Priya K, Jaryal R, Yadav R (2018). Phytase: The feed enzyme, an overview. Advances in Animal Biotechnology and Its Applications. pp: 269-327.
3. Farhat-Khemakhem A, Farhat MB, Boukhris I, Bejar W, Bouchaala K, Kammoun R, et al. Heterologous expression and optimization using experimental designs allowed highly efficient production of the PHY US417 phytase in Bacillus subtilis 168. AMB Express 2012;2:10.
4. Bhavsar K, Gujar P, Shah P, Kumar VR, Khire JM. Combinatorial approach of statistical optimization and mutagenesis for improved production of acidic phytase by Aspergillus niger NCIM 563 under submerged fermentation condition. Appl Microbiol Biotechnol 2013;97:673-679.
5. Nielsen AV, Tetens I, Meyer AS. Potential of phytase-mediated iron release from cereal-based foods: a quantitative view. Nutrients 2013;5:3074-3098.
6. Dersjant‐Li Y, Awati A, Schulze H, Partridge G. Phytase in non‐ruminant animal nutrition: a critical review on phytase activities in the gastrointestinal tract and influencing factors. J Sci Food Agric 2015;95:878-896.
7. Morgan NK, Walk CL, Bedford MR, Scholey DV, Burton EJ. Effect of feeding broilers diets differing in susceptible phytate content. Anim Nutr 2016;2:33-39.
8. Boehm Jr JD, Walker FR, Bhandari HS, Kopsell D, Pantalone VR. Seed inorganic phosphorus stability and agronomic performance of two low‐phytate soybean lines evaluated across six southeastern US environments. Crop Sci 2017;57:2555-2563.
9. Haros M, Bielecka M, Honke J, Sanz Y. Myo-inositol hexakisphosphate degradation by Bifidobacterium infantis ATCC 15697. Int J Food Microbiol 2007;117:76-84.
10. Lan GQ, Abdullah N, Jalaludin S, Ho YW. Culture conditions influencing phytase production of Mitsuokella jalaludinii, a new bacterial species from the rumen of cattle. J Appl Microbiol 2002;93:668-674.
11. Haefner S, Knietsch A, Scholten E, Braun J, Lohscheidt M, Zelder O. Biotechnological production and applications of phytases. Appl Microbiol Biotechnol 2005;68:588-597.
12. Wang Q, Fu SJ, Sun JY, Weng XY. Characterization of a thermostable alkaline phytase from Bacillus licheniformis ZJ-6 in Pichia pastoris. World J Microbiol Biotechnol 2011;27:1247-1253.
13. Ries EF, Alves Macedo G. Improvement of phytase activity by a new Saccharomyces cerevisiae strain using statistical optimization. Enzyme Res 2011;2011: 796394.
14. Vohra A, Satyanarayana T. Phytase production by the yeast, Pichia anomala. Biotechnol Lett 2001;23:551-554.
15. Azeem M, Riaz A, Chaudhary AN, Hayat R, Hussain Q, Tahir MI, et al. Microbial phytase activity and their role in organic P mineralization. Arch Agron Soil Sci 2015;61:751-766.
16. Nakamura Y, Fukuhara H, Sano K. Secreted phytase activities of yeasts. Biosci Biotechnol Biochem 2000;64:841-844.
17. Huang H, Luo H, Yang P, Meng K, Wang Y, Yuan T, et al. A novel phytase with preferable characteristics from Yersinia intermedia. Biochem Biophys Res Commun 2006;350:884-889.
18. Bekalu ZE, Madsen CK, Dionisio G, Brinch-Pedersen H. Aspergillus ficuum phytase activity is inhibited by cereal grain components. PLoS One 2017;12(5):e0176838.
19. Han N, Miao H, Yu T, Xu B, Yang Y, Wu Q, et al. Enhancing thermal tolerance of Aspergillus niger PhyA phytase directed by structural comparison and computational simulation. BMC Biotechnol 2018;18:36.
20. Quan CS, Tian WJ, Fan SD, Kikuchi J. Purification and properties of a low-molecular-weight phytase from Cladosporium sp. FP-1. J Biosci Bioeng 2004;97:260-266.
21. Hamada A, Yamaguchi K, Ohnishi N, Harada M, Nikumaru S, Honda H. High‐level production of yeast (Schwanniomyces occidentalis) phytase in transgenic rice plants by a combination of signal sequence and codon modification of the phytase gene. Plant Biotechnol J 2005;3:43-55.
22. Tye AJ, Siu FK, Leung T, Lim B. Molecular cloning and the biochemical characterization of two novel phytases from B. subtilis 168 and B. licheniformis. Appl Microbiol Biotechnol 2002;59:190-197.
23. Priyodip P, Prakash PY, Balaji S. Phytases of probiotic bacteria: characteristics and beneficial aspects. Indian J Microbiol 2017;57:148-154.
24. Singh S, Singh S, Sharma PK, Sharma D. Isolation, identification and molecular characterization of phytase producing bacteria, Pseudomonas sp. aazad. J Pure Appl Microbiol 2017;11:1845-1850.
25. Roy MP, Mazumdar D, Dutta S, Saha SP, Ghosh S. Cloning and expression of phytase appA gene from Shigella sp. CD2 in Pichia pastoris and comparison of properties with recombinant enzyme expressed in E. coli. PLoS One 2016;11(1):e0145745.
26. Hong CY, Cheng KJ, Tseng TH, Wang CS, Liu LF, Yu SM. Production of two highly active bacterial phytases with broad pH optima in germinated transgenic rice seeds. Transgenic Res 2004;13:29-39.
27. Shamna KS, Rajamanikandan KCP, Mukesh Kumar DJ, Balakumaran MD, Kalaichelvan PT. Extracellular production of phytases by a native Bacillus subtilis strain. Ann Biol Res 2012;3:979-987.
28. Gulati HK, Chadha BS, Saini HS. Production and characterization of thermostable alkaline phytase from Bacillus laevolacticus isolated from rhizosphere soil. J Ind Microbiol Biotechnol 2007;34:91-98.
29. Barbe V, Cruveiller S, Kunst F, Lenoble P, Meurice G, Sekowska A, et al. From a consortium sequence to a unified sequence: the Bacillus subtilis 168 reference genome a decade later. Microbiology (Reading) 2009;155:1758-1775.
30. Liu J, Fang C, Jiang Y, Yan R. Characterization of a hemolysin gene ytjA from Bacillus subtilis. Curr Microbiol 2009;58:642-647.
31. Heinzmann S, Entian KD, Stein T. Engineering Bacillus subtilis ATCC 6633 for improved production of the lantibiotic subtilin. Appl Microbiol Biotechnol 2006;69:532-536.
32. Swain M, Ray R. Alpha‐amylase production by Bacillus subtilis CM3 in solid state fermentation using cassava fibrous residue. J Basic Microbiol 2007;47:417-425.
33. Chang CT, Fan MH, Kuo FC, Sung HY. Potent Fibrinolytic Enzyme from a Mutant of Bacillus s ubtilis IMR-NK1. J Agric Food Chem 2000;48:3210-3216.
34. Earl AM, Losick R, Kolter R. Ecology and genomics of Bacillus subtilis. Trends Microbiol 2008;16:269-275.
35. Tran TT, Mamo G, Mattiasson B, Hatti-Kaul R. A thermostable phytase from Bacillus sp. MD2: cloning, expression and high-level production in Escherichia coli. J Ind Microbiol Biotechnol 2010;37:279-287.
36. Sambrook J, Fritsch EF, Maniatis T (1989). Molecular cloning: a laboratory manual 2nd ed. Cold spring harbor laboratory press.
37. Zimmerman SA, Tomb JF, Ferry JG. Characterization of CamH from Methanosarcina thermophila, founding member of a subclass of the {gamma} class of carbonic anhydrases. J Bacteriol 2010;192:1353-1360.
38. Golovan S, Wang G, Zhang J, Forsberg CW. Characterization and overproduction of the Escherichia coli appA encoded bifunctional enzyme that exhibits both phytase and acid phosphatase activities. Can J Microbiol 2000;46:59-71.
39. Lei XG, Porres JM. Phytase enzymology, applications, and biotechnology. Biotechnol Lett 2003;25:1787-1794.
40. Bikker P, Spek JW, Van Emous RA, Van Krimpen MM. Precaecal phosphorus digestibility of inorganic phosphate sources in male broilers. Br Poult Sci 2016;57:810-817.
41. Selle PH, Ravindran V. Microbial phytase in poultry nutrition. Anim Feed Sci Technol 2007;135:1-41.
42. Kumar V, Yadav AN, Verma P, Sangwan P, Saxena A, Kumar K, et al. β-Propeller phytases: diversity, catalytic attributes, current developments and potential biotechnological applications. Int J Biol Macromol 2017;98:595-609.
43. Ozusaglam MA, Ozcan N. Cloning of phytase gene in probiotic bacterium Bacillus coagulans. Adv Studies Biol 2009;1:15-24.
44. Bawane R, Tantwai K, Rajput LPS, Kadam-Bedekar M, Kumar S, Gontia I, et al. Molecular analysis of phytase gene cloned from Bacillus subtilis. Adv Stud Biol 2011;3:103-110.
2. Singh N, Kuhar S, Priya K, Jaryal R, Yadav R (2018). Phytase: The feed enzyme, an overview. Advances in Animal Biotechnology and Its Applications. pp: 269-327.
3. Farhat-Khemakhem A, Farhat MB, Boukhris I, Bejar W, Bouchaala K, Kammoun R, et al. Heterologous expression and optimization using experimental designs allowed highly efficient production of the PHY US417 phytase in Bacillus subtilis 168. AMB Express 2012;2:10.
4. Bhavsar K, Gujar P, Shah P, Kumar VR, Khire JM. Combinatorial approach of statistical optimization and mutagenesis for improved production of acidic phytase by Aspergillus niger NCIM 563 under submerged fermentation condition. Appl Microbiol Biotechnol 2013;97:673-679.
5. Nielsen AV, Tetens I, Meyer AS. Potential of phytase-mediated iron release from cereal-based foods: a quantitative view. Nutrients 2013;5:3074-3098.
6. Dersjant‐Li Y, Awati A, Schulze H, Partridge G. Phytase in non‐ruminant animal nutrition: a critical review on phytase activities in the gastrointestinal tract and influencing factors. J Sci Food Agric 2015;95:878-896.
7. Morgan NK, Walk CL, Bedford MR, Scholey DV, Burton EJ. Effect of feeding broilers diets differing in susceptible phytate content. Anim Nutr 2016;2:33-39.
8. Boehm Jr JD, Walker FR, Bhandari HS, Kopsell D, Pantalone VR. Seed inorganic phosphorus stability and agronomic performance of two low‐phytate soybean lines evaluated across six southeastern US environments. Crop Sci 2017;57:2555-2563.
9. Haros M, Bielecka M, Honke J, Sanz Y. Myo-inositol hexakisphosphate degradation by Bifidobacterium infantis ATCC 15697. Int J Food Microbiol 2007;117:76-84.
10. Lan GQ, Abdullah N, Jalaludin S, Ho YW. Culture conditions influencing phytase production of Mitsuokella jalaludinii, a new bacterial species from the rumen of cattle. J Appl Microbiol 2002;93:668-674.
11. Haefner S, Knietsch A, Scholten E, Braun J, Lohscheidt M, Zelder O. Biotechnological production and applications of phytases. Appl Microbiol Biotechnol 2005;68:588-597.
12. Wang Q, Fu SJ, Sun JY, Weng XY. Characterization of a thermostable alkaline phytase from Bacillus licheniformis ZJ-6 in Pichia pastoris. World J Microbiol Biotechnol 2011;27:1247-1253.
13. Ries EF, Alves Macedo G. Improvement of phytase activity by a new Saccharomyces cerevisiae strain using statistical optimization. Enzyme Res 2011;2011: 796394.
14. Vohra A, Satyanarayana T. Phytase production by the yeast, Pichia anomala. Biotechnol Lett 2001;23:551-554.
15. Azeem M, Riaz A, Chaudhary AN, Hayat R, Hussain Q, Tahir MI, et al. Microbial phytase activity and their role in organic P mineralization. Arch Agron Soil Sci 2015;61:751-766.
16. Nakamura Y, Fukuhara H, Sano K. Secreted phytase activities of yeasts. Biosci Biotechnol Biochem 2000;64:841-844.
17. Huang H, Luo H, Yang P, Meng K, Wang Y, Yuan T, et al. A novel phytase with preferable characteristics from Yersinia intermedia. Biochem Biophys Res Commun 2006;350:884-889.
18. Bekalu ZE, Madsen CK, Dionisio G, Brinch-Pedersen H. Aspergillus ficuum phytase activity is inhibited by cereal grain components. PLoS One 2017;12(5):e0176838.
19. Han N, Miao H, Yu T, Xu B, Yang Y, Wu Q, et al. Enhancing thermal tolerance of Aspergillus niger PhyA phytase directed by structural comparison and computational simulation. BMC Biotechnol 2018;18:36.
20. Quan CS, Tian WJ, Fan SD, Kikuchi J. Purification and properties of a low-molecular-weight phytase from Cladosporium sp. FP-1. J Biosci Bioeng 2004;97:260-266.
21. Hamada A, Yamaguchi K, Ohnishi N, Harada M, Nikumaru S, Honda H. High‐level production of yeast (Schwanniomyces occidentalis) phytase in transgenic rice plants by a combination of signal sequence and codon modification of the phytase gene. Plant Biotechnol J 2005;3:43-55.
22. Tye AJ, Siu FK, Leung T, Lim B. Molecular cloning and the biochemical characterization of two novel phytases from B. subtilis 168 and B. licheniformis. Appl Microbiol Biotechnol 2002;59:190-197.
23. Priyodip P, Prakash PY, Balaji S. Phytases of probiotic bacteria: characteristics and beneficial aspects. Indian J Microbiol 2017;57:148-154.
24. Singh S, Singh S, Sharma PK, Sharma D. Isolation, identification and molecular characterization of phytase producing bacteria, Pseudomonas sp. aazad. J Pure Appl Microbiol 2017;11:1845-1850.
25. Roy MP, Mazumdar D, Dutta S, Saha SP, Ghosh S. Cloning and expression of phytase appA gene from Shigella sp. CD2 in Pichia pastoris and comparison of properties with recombinant enzyme expressed in E. coli. PLoS One 2016;11(1):e0145745.
26. Hong CY, Cheng KJ, Tseng TH, Wang CS, Liu LF, Yu SM. Production of two highly active bacterial phytases with broad pH optima in germinated transgenic rice seeds. Transgenic Res 2004;13:29-39.
27. Shamna KS, Rajamanikandan KCP, Mukesh Kumar DJ, Balakumaran MD, Kalaichelvan PT. Extracellular production of phytases by a native Bacillus subtilis strain. Ann Biol Res 2012;3:979-987.
28. Gulati HK, Chadha BS, Saini HS. Production and characterization of thermostable alkaline phytase from Bacillus laevolacticus isolated from rhizosphere soil. J Ind Microbiol Biotechnol 2007;34:91-98.
29. Barbe V, Cruveiller S, Kunst F, Lenoble P, Meurice G, Sekowska A, et al. From a consortium sequence to a unified sequence: the Bacillus subtilis 168 reference genome a decade later. Microbiology (Reading) 2009;155:1758-1775.
30. Liu J, Fang C, Jiang Y, Yan R. Characterization of a hemolysin gene ytjA from Bacillus subtilis. Curr Microbiol 2009;58:642-647.
31. Heinzmann S, Entian KD, Stein T. Engineering Bacillus subtilis ATCC 6633 for improved production of the lantibiotic subtilin. Appl Microbiol Biotechnol 2006;69:532-536.
32. Swain M, Ray R. Alpha‐amylase production by Bacillus subtilis CM3 in solid state fermentation using cassava fibrous residue. J Basic Microbiol 2007;47:417-425.
33. Chang CT, Fan MH, Kuo FC, Sung HY. Potent Fibrinolytic Enzyme from a Mutant of Bacillus s ubtilis IMR-NK1. J Agric Food Chem 2000;48:3210-3216.
34. Earl AM, Losick R, Kolter R. Ecology and genomics of Bacillus subtilis. Trends Microbiol 2008;16:269-275.
35. Tran TT, Mamo G, Mattiasson B, Hatti-Kaul R. A thermostable phytase from Bacillus sp. MD2: cloning, expression and high-level production in Escherichia coli. J Ind Microbiol Biotechnol 2010;37:279-287.
36. Sambrook J, Fritsch EF, Maniatis T (1989). Molecular cloning: a laboratory manual 2nd ed. Cold spring harbor laboratory press.
37. Zimmerman SA, Tomb JF, Ferry JG. Characterization of CamH from Methanosarcina thermophila, founding member of a subclass of the {gamma} class of carbonic anhydrases. J Bacteriol 2010;192:1353-1360.
38. Golovan S, Wang G, Zhang J, Forsberg CW. Characterization and overproduction of the Escherichia coli appA encoded bifunctional enzyme that exhibits both phytase and acid phosphatase activities. Can J Microbiol 2000;46:59-71.
39. Lei XG, Porres JM. Phytase enzymology, applications, and biotechnology. Biotechnol Lett 2003;25:1787-1794.
40. Bikker P, Spek JW, Van Emous RA, Van Krimpen MM. Precaecal phosphorus digestibility of inorganic phosphate sources in male broilers. Br Poult Sci 2016;57:810-817.
41. Selle PH, Ravindran V. Microbial phytase in poultry nutrition. Anim Feed Sci Technol 2007;135:1-41.
42. Kumar V, Yadav AN, Verma P, Sangwan P, Saxena A, Kumar K, et al. β-Propeller phytases: diversity, catalytic attributes, current developments and potential biotechnological applications. Int J Biol Macromol 2017;98:595-609.
43. Ozusaglam MA, Ozcan N. Cloning of phytase gene in probiotic bacterium Bacillus coagulans. Adv Studies Biol 2009;1:15-24.
44. Bawane R, Tantwai K, Rajput LPS, Kadam-Bedekar M, Kumar S, Gontia I, et al. Molecular analysis of phytase gene cloned from Bacillus subtilis. Adv Stud Biol 2011;3:103-110.
| Files | ||
| Issue | Vol 13 No 5 (2021) | |
| Section | Original Article(s) | |
| DOI | https://doi.org/10.18502/ijm.v13i5.7433 | |
| Keywords | ||
| Bacillus subtilis; Cloning; Escherichia coli; Phytase; Probiotics | ||
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How to Cite
1.
Iravani Saadi M, Doosti A, Jalali H, Nabi Abdolyousefi E, Hooshiyar M, Tabrizi R, Noshadi E. Cloning of Bacillus subtilis phytase gene construct in Escherichia coli. Iran J Microbiol. 2021;13(5):664-670.
