Directed evolution of an acid Yersinia mollaretii phytase for broadened activity at neutral pH

Abstract

Phytases are phosphohydrolases that initiate the sequential hydrolysis of phosphate from phytate, which is the main storage form of phosphorous in numerous plant seeds, especially in cereals and grains. Phytate is indigestible for most monogastric animals, such as poultry, swine, fish, and humans; therefore, microbial phytases have been widely used in plant (specially soy)-based animal feeding to improve nutrition by enhanced phosphorus, mineral, and trace element absorption, and reducing phosphorus pollution by animal waste. Most phytases used as animal feed additives have an acid pH optimum (pH 2.5 and 5.5 for Aspergillus and pH 4.5 for E. coli phytases) and show a sharp decrease in performance at neutral pH, correlating with intestinal digestion. Directed evolution of phytases has been previously reported to improve enzyme thermostability, pH, or specific activity. In this manuscript, we report a directed evolution campaign of the highly active bacterial phytase from Yersinia mollaretii (YmPh) towards a broadened pH activity spectrum. Directed evolution identified the key positions T44 and K45 for increased YmPh activity at neutral pH. Both positions are located in the active site loop of the phytase and have a synergistic effect on activity with a broadened pH spectrum. Kinetic characterization of the improved variants, YmPh-M10 and -M16, showed up to sevenfold increased specific activity and up to 2.2-fold reduced Khalf at pH 6.6 under screening conditions compared to Yersinia mollaretii phytase wild type (YmPhWT).

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References

  1. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17):3389–3402

    CAS  Article  Google Scholar 

  2. Ariza A, Moroz OV, Blagova EV, Turkenburg JP, Waterman J, Roberts SM, Vind J, Sjøholm C, Lassen SF, De Maria L, Glitsoe V, Skov LK, Wilson KS (2013) Degradation of phytate by the 6-phytase from Hafnia alvei: a combined structural and solution study. PLoS One 8(5):e65062. https://doi.org/10.1371/journal.pone.0065062

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. Arnold FH (2018) Directed evolution: bringing new chemistry to life. Angew Chem Int Ed Engl 57(16):4143–4148. https://doi.org/10.1002/anie.201708408

    CAS  Article  PubMed  Google Scholar 

  4. Billington DC (1993) The inositols phosphates: chemical synthesis and biological significance. Weinheim, VCH Verlagsgesellschaft

    Google Scholar 

  5. Blanusa M, Schenk A, Sadeghi H, Marienhagen J, Schwaneberg U (2010) Phosphorothioate-based ligase-independent gene cloning (PLICing): an enzyme-free and sequence-independent cloning method. Anal Biochem 406(2):141–146. https://doi.org/10.1016/j.ab.2010.07.011

    CAS  Article  PubMed  Google Scholar 

  6. Bohn L, Meyer AS, Rasmussen SK (2008) Phytate: impact on environment and human nutrition. A challenge for molecular breeding. J Zhejiang Univ Sci B 9(3):165–191. https://doi.org/10.1631/jzus.B0710640

    Article  PubMed  PubMed Central  Google Scholar 

  7. Cadwell RC, Joyce GF (1994) Mutagenic PCR. PCR Methods Appl 3(6):S136–S140. https://doi.org/10.1101/gr.3.6.S136

    CAS  Article  PubMed  Google Scholar 

  8. Cao L, Wang W, Yang C, Yang Y, Diana J, Yakupitiyage A, Luo Z, Li D (2007) Application of microbial phytase in fish feed. Enzym Microb Technol 40(4):497–507. https://doi.org/10.1016/j.enzmictec.2007.01.007

    CAS  Article  Google Scholar 

  9. Chen W, Ye L, Guo F, Lv Y, Yu H (2015) Enhanced activity of an alkaline phytase from Bacillus subtilis 168 in acidic and neutral environments by directed evolution. Biochem Eng J 98:137–143. https://doi.org/10.1016/j.bej.2015.02.021

    CAS  Article  Google Scholar 

  10. Cheng C, Lim BL (2006) Beta-propeller phytases in the aquatic environment. Arch Microbiol 185(1):1–13. https://doi.org/10.1007/s00203-005-0080-6

    CAS  Article  PubMed  Google Scholar 

  11. Cheng F, Zhu L, Schwaneberg U (2015) Directed evolution 2.0: improving and deciphering enzyme properties. Chem Commun 51(48):9760–9772. https://doi.org/10.1039/C5CC01594D

    CAS  Article  Google Scholar 

  12. Choi YM, Suh HJ, Kim JM (2001) Purification and properties of extracellular phytase from Bacillus sp. KHU-10. J Protein Chem 20(4):287–292. https://doi.org/10.1023/A:1010945416862

    CAS  Article  PubMed  Google Scholar 

  13. Dvorakova J, Kopecky J, Havlicek V, Kren V (2000) Formation of myo-inositol phosphates by Aspergillus niger 3-phytase. Folia Microbiol 45(2):128–132. https://doi.org/10.1007/BF02817410

    CAS  Article  Google Scholar 

  14. Elkhalil EAI, Männer K, Borriss R, Simon O (2007) In vitro and in vivo characteristics of bacterial phytases and their efficacy in broiler chickens. Br Poult Sci 48(1):64–70. https://doi.org/10.1080/00071660601148195

    CAS  Article  PubMed  Google Scholar 

  15. Fu D, Huang H, Meng K, Wang Y, Luo H, Yang P, Yuan T, Yao B (2009) Improvement of Yersinia frederiksenii phytase performance by a single amino acid substitution. Biotechnol Bioeng 103(5):857–864. https://doi.org/10.1002/bit.22315

    CAS  Article  PubMed  Google Scholar 

  16. Fujita J, Fukuda H, Y-i Y, Kizaki Y, Shigeta S, Ono K, Suzuki O, Wakabayashi S (2001) Critical importance of phytase for yeast growth and alcohol fermentation in japanese sake brewing. Biotechnol Lett 23(11):867–871. https://doi.org/10.1023/a:1010599307395

    CAS  Article  Google Scholar 

  17. Garrett JB, Kretz KA, O’Donoghue E, Kerovuo J, Kim W, Barton NR, Hazlewood GP, Short JM, Robertson DE, Gray KA (2004) Enhancing the thermal tolerance and gastric performance of a microbial phytase for use as a phosphate-mobilizing monogastric-feed supplement. Appl Environ Microbiol 70(5):3041–3046. https://doi.org/10.1128/aem.70.5.3041-3046.2004

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. Gonzalez-Perez D, Mateljak I, Garcia-Ruiz E, Ruiz-Duenas FJ, Martinez AT, Alcalde M (2016) Alkaline versatile peroxidase by directed evolution. Catal Sci Technol 6(17):6625–6636. https://doi.org/10.1039/C6CY01044J

    CAS  Article  Google Scholar 

  19. Greiner R, Konietzny U (2006) Phytase for food application. Food Technol Biotechnol 44(2):125–140

    CAS  Google Scholar 

  20. Greiner R, Lim BL, Cheng C, Carlsson N-G (2007) Pathway of phytate dephosphorylation by β-propeller phytases of different origins. Can J Microbiol 53(4):488–495. https://doi.org/10.1139/w07-015

    CAS  Article  PubMed  Google Scholar 

  21. Haefner S, Knietsch A, Scholten E, Braun J, Lohscheidt M, Zelder O (2005) Biotechnological production and applications of phytases. Appl Microbiol Biotechnol 68(5):588–597. https://doi.org/10.1007/s00253-005-0005-y

    CAS  Article  PubMed  Google Scholar 

  22. Harland B, Oberleas D (1999) Phytase in animal nutrition and waste management. BASF Reference Manual 45:237–240

    Google Scholar 

  23. Holford ICR (1997) Soil phosphorus: its measurement, and its uptake by plants. Soil Res 35(2):227–240. https://doi.org/10.1071/S96047

    CAS  Article  Google Scholar 

  24. Huang H, Luo H, Wang Y, Fu D, Shao N, Wang G, Yang P, Yao B (2008) A novel phytase from Yersinia rohdei with high phytate hydrolysis activity under low pH and strong pepsin conditions. Appl Microbiol Biotechnol 80(3):417–426. https://doi.org/10.1007/s00253-008-1556-5

    CAS  Article  PubMed  Google Scholar 

  25. Hubenova Y, Georgiev D, Mitov M (2014) Stable current outputs and phytate degradation by yeast-based biofuel cell. Yeast 31(9):343–348. https://doi.org/10.1002/yea.3027

    CAS  Article  PubMed  Google Scholar 

  26. Islam S, Mate DM, Martinez R, Jakob F, Schwaneberg U (2018) A robust protocol for directed aryl sulfotransferase evolution towards the carbohydrate building block GlcNAc. Biotechnol Bioeng 115(5):1106–1115. https://doi.org/10.1002/bit.26535

    CAS  Article  PubMed  Google Scholar 

  27. Jakob F, Martinez R, Mandawe J, Hellmuth H, Siegert P, Maurer K-H, Schwaneberg U (2013) Surface charge engineering of a Bacillus gibsonii subtilisin protease. Appl Microbiol Biotechnol 97(15):6793–6802. https://doi.org/10.1007/s00253-012-4560-8

    CAS  Article  PubMed  Google Scholar 

  28. Jones DT (1999) Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 292(2):195–202. https://doi.org/10.1006/jmbi.1999.3091

    CAS  Article  Google Scholar 

  29. Kim M-S, Lei XG (2008) Enhancing thermostability of Escherichia coli phytase AppA2 by error-prone PCR. Appl Microbiol Biotechnol 79(1):69–75. https://doi.org/10.1007/s00253-008-1412-7

    CAS  Article  PubMed  Google Scholar 

  30. Kim T, Mullaney EJ, Porres JM, Roneker KR, Crowe S, Rice S, Ko T, Ullah AHJ, Daly CB, Welch R, Lei XG (2006) Shifting the pH profile of Aspergillus niger PhyA phytase to match the stomach pH enhances its effectiveness as an animal feed additive. Appl Environ Microbiol 72(6):4397–4403. https://doi.org/10.1128/aem.02612-05

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. Krieger E, Koraimann G, Vriend G (2002) Increasing the precision of comparative models with YASARA NOVA—a self-parameterizing force field. Proteins 47(3):393–402. https://doi.org/10.1002/prot.10104

    CAS  Article  Google Scholar 

  32. Kumar V, Sinha AK, Makkar HPS, Becker K (2010) Dietary roles of phytate and phytase in human nutrition: a review. Food Chem 120(4):945–959. https://doi.org/10.1016/j.foodchem.2009.11.052

    CAS  Article  Google Scholar 

  33. Kumar V, Yadav AN, Verma P, Sangwan P, Saxena A, Kumar K, Singh B (2017) β-Propeller phytases: diversity, catalytic attributes, current developments and potential biotechnological applications. Int J Biol Macromol 98:595–609. https://doi.org/10.1016/j.ijbiomac.2017.01.134

    CAS  Article  PubMed  Google Scholar 

  34. Labrou NE, Rigden DJ, Clonis YD (2004) Engineering the pH-dependence of kinetic parameters of maize glutathione S-transferase I by site-directed mutagenesis. Biomol Eng 21(2):61–66. https://doi.org/10.1016/j.bioeng.2003.10.002

    CAS  Article  PubMed  Google Scholar 

  35. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) PROCHECK: a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26:283–291. https://doi.org/10.1107/S0021889892009944

    CAS  Article  Google Scholar 

  36. Lehmann C, Sibilla F, Maugeri Z, Streit WR, Dominguez de Maria P, Martinez R, Schwaneberg U (2012) Reengineering CelA2 cellulase for hydrolysis in aqueous solutions of deep eutectic solvents and concentrated seawater. Green Chem 14(10):2719–2726. https://doi.org/10.1039/C2GC35790A

    CAS  Article  Google Scholar 

  37. Lei XG, Porres JM (2003) Phytase enzymology, applications, and biotechnology. Biotechnol Lett 25(21):1787–1794. https://doi.org/10.1023/a:1026224101580

    CAS  Article  PubMed  Google Scholar 

  38. Lei XG, Weaver JD, Mullaney E, Ullah AH, Azain MJ (2013) Phytase, a new life for an “old” enzyme. Annu Rev Anim Biosci 1(1):283–309. https://doi.org/10.1146/annurev-animal-031412-103717

    CAS  Article  PubMed  Google Scholar 

  39. Liao Y, Zeng M, Wu Z-f, Chen H, Wang H-n WQ, Shan Z, X-y H (2012) Improving phytase enzyme activity in a recombinant phyA mutant phytase from Aspergillus niger N25 by error-prone PCR. Appl Biochem Biotechnol 166(3):549–562. https://doi.org/10.1007/s12010-011-9447-0

    CAS  Article  PubMed  Google Scholar 

  40. Lim D, Golovan S, Forsberg CW, Jia Z (2000) Crystal structures of Escherichia coli phytase and its complex with phytate. Nat Struct Biol 7(2):108–113. https://doi.org/10.1038/72371

    CAS  Article  PubMed  Google Scholar 

  41. Lülsdorf N, Vojcic L, Hellmuth H, Weber TT, Mußmann N, Martinez R, Schwaneberg U (2015) A first continuous 4-aminoantipyrine (4-AAP)-based screening system for directed esterase evolution. Appl Microbiol Biotechnol 99(12):5237–5246. https://doi.org/10.1007/s00253-015-6612-3

    CAS  Article  PubMed  Google Scholar 

  42. Martínez R, Schwaneberg U (2013) A roadmap to directed enzyme evolution and screening systems for biotechnological applications. Biol Res 46:395–405. https://doi.org/10.4067/S0716-97602013000400011

    Article  PubMed  Google Scholar 

  43. Mate Diana M, Gonzalez-Perez D, Falk M, Kittl R, Pita M, De Lacey AL, Ludwig R, Shleev S, Alcalde M (2013) Blood tolerant laccase by directed evolution. Chem Biol 20(2):223–231. https://doi.org/10.1016/j.chembiol.2013.01.001

    CAS  Article  PubMed  Google Scholar 

  44. Mate DM, Gonzalez-Perez D, Mateljak I, Gomez de Santos P, Vicente AI, Alcalde M (2016) The pocket manual of directed evolution: tips and tricks. In: Brahmachari G, Demain A, Adrio JL (eds) Biotechnology of microbial enzymes: production, biocatalysis and industrial applications. Academic Press Elsevier, Amsterdam, pp 185–214. https://doi.org/10.1016/B978-0-12-803725-6.00008-X

    Chapter  Google Scholar 

  45. Meyer AS (2010) Enzyme technology for precision functional food ingredient processes. Ann N Y Acad Sci 1190(1):126–132. https://doi.org/10.1111/j.1749-6632.2009.05255.x

    CAS  Article  PubMed  Google Scholar 

  46. Pagano AR, Roneker KR, Lei XG (2007) Distribution of supplemental Escherichia coli AppA2 phytase activity in digesta of various gastrointestinal segments of young pigs. J Anim Sci 85(6):1444–1452. https://doi.org/10.2527/jas.2006-111

    CAS  Article  PubMed  Google Scholar 

  47. Park S-C, Choi Y-W, Oh T-K (1999) Comparative enzymatic hydrolysis of phytate in various animal feedstuff with two different phytases. J Vet Med Sci 61(11):1257–1259. https://doi.org/10.1292/jvms.61.1257

    CAS  Article  PubMed  Google Scholar 

  48. Qiu J, Elber R (2006) SSALN: an alignment algorithm using structure-dependent substitution matrices and gap penalties learned from structurally aligned protein pairs. Proteins 62(4):881–891. https://doi.org/10.1002/prot.20854

    CAS  Article  PubMed  Google Scholar 

  49. Rao DECS, Rao KV, Reddy TP, Reddy VD (2009) Molecular characterization, physicochemical properties, known and potential applications of phytases: an overview. Crit Rev Biotechnol 29(2):182–198. https://doi.org/10.1080/07388550902919571

    CAS  Article  PubMed  Google Scholar 

  50. Reddy CS, Achary VMM, Manna M, Singh J, Kaul T, Reddy MK (2015) Isolation and molecular characterization of thermostable phytase from Bacillus subtilis (BSPhyARRMK33). Appl Biochem Biotechnol 175(6):3058–3067. https://doi.org/10.1007/s12010-015-1487-4

    CAS  Article  PubMed  Google Scholar 

  51. Romero PA, Arnold FH (2009) Exploring protein fitness landscapes by directed evolution. Nat Rev Mol Cell Biol 10:866–876. https://doi.org/10.1038/nrm2805

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. Sáez-Jiménez V, Acebes S, Garcia-Ruiz E, Romero A, Guallar V, Alcalde M, Medrano FJ, Martínez AT, Ruiz-Dueñas FJ (2016) Unveiling the basis of alkaline stability of an evolved versatile peroxidase. Biochem J 473:1917–1928. https://doi.org/10.1042/bcj20160248

    Article  PubMed  Google Scholar 

  53. Sandberg A-S, Brune M, Carlsson N-G, Hallberg L, Skoglund E, Rossander-Hulthén L (1999) Inositol phosphates with different numbers of phosphate groups influence iron absorption in humans. Am J Clin Nutr 70(2):240–246. https://doi.org/10.1093/ajcn.70.2.240

    CAS  Article  PubMed  Google Scholar 

  54. Schröder B, Hattenhauer O, Breves G (1998) Phosphate transport in pig proximal small intestines during postnatal development: lack of modulation by calcitriol. Endocrinology 139(4):1500–1507. https://doi.org/10.1210/endo.139.4.5922

    Article  PubMed  Google Scholar 

  55. Sebastian S, Touchburn SP, Chavez ER (1998) Implications of phytic acid and supplemental microbial phytase in poultry nutrition: a review. Worlds Poult Sci J 54(1):27–47. https://doi.org/10.1079/wps19980003

    Article  Google Scholar 

  56. Selle PH, Ravindran V (2007) Microbial phytase in poultry nutrition. Anim Feed Sci Technol 135(1):1–41. https://doi.org/10.1016/j.anifeedsci.2006.06.010

    CAS  Article  Google Scholar 

  57. Shivange AV, Schwaneberg U (2017) Recent advances in directed phytase evolution and rational phytase engineering. In: Alcalde M (ed) Directed enzyme evolution: advances and applications. Springer, Cham, pp 145–172. https://doi.org/10.1007/978-3-319-50413-1_6

    Chapter  Google Scholar 

  58. Shivange AV, Marienhagen J, Mundhada H, Schenk A, Schwaneberg U (2009) Advances in generating functional diversity for directed protein evolution. Curr Opin Chem Biol 13(1):19–25. https://doi.org/10.1016/j.cbpa.2009.01.019

    CAS  Article  PubMed  Google Scholar 

  59. Shivange AV, Schwaneberg U, Roccatano D (2010) Conformational dynamics of active site loop in Escherichia coli phytase. Biopolymers 93(11):994–1002. https://doi.org/10.1002/bip.21513

    CAS  Article  PubMed  Google Scholar 

  60. Shivange AV, Serwe A, Dennig A, Roccatano D, Haefner S, Schwaneberg U (2012) Directed evolution of a highly active Yersinia mollaretii phytase. Appl Microbiol Biotechnol 95(2):405–418. https://doi.org/10.1007/s00253-011-3756-7

    CAS  Article  PubMed  Google Scholar 

  61. Shivange AV, Dennig A, Schwaneberg U (2014) Multi-site saturation by OmniChange yields a pH- and thermally improved phytase. J Biotechnol 170:68–72. https://doi.org/10.1016/j.jbiotec.2013.11.014

    CAS  Article  PubMed  Google Scholar 

  62. Shivange AV, Hoeffken HW, Haefner S, Schwaneberg U (2016a) Protein consensus-based surface engineering (ProCoS): a computer-assisted method for directed protein evolution. Biotechniques 61(6):305–314. https://doi.org/10.2144/000114483

    CAS  Article  PubMed  Google Scholar 

  63. Shivange AV, Roccatano D, Schwaneberg U (2016b) Iterative key-residues interrogation of a phytase with thermostability increasing substitutions identified in directed evolution. Appl Microbiol Biotechnol 100(1):227–242. https://doi.org/10.1007/s00253-015-6959-5

    CAS  Article  PubMed  Google Scholar 

  64. Sippl MJ (1993) Recognition of errors in three-dimensional structures of proteins. Proteins 17(4):355–362. https://doi.org/10.1002/prot.340170404

    CAS  Article  PubMed  Google Scholar 

  65. Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41(1):207–234. https://doi.org/10.1016/j.pep.2005.01.016

    CAS  Article  Google Scholar 

  66. Suzuki U, Yoshimura K, Takaishi M (1907) Über ein enzym “phytase” das anhydro-oxy-methylen-diphosphosaure spaltet. Tokyo Imp Univ Coll Agric Bull 7:503–512

    Google Scholar 

  67. Svihus B (2014) Function of the digestive system. J Appl Poult Res 23(2):306–314. https://doi.org/10.3382/japr.2014-00937

    CAS  Article  Google Scholar 

  68. Tee L, Schwaneberg U (2006) A screening system for the directed evolution of epoxygenases: importance of position 184 in P450 BM3 for stereoselective styrene epoxidation. Angew Chem Int Ed Engl 45(32):5380–5383. https://doi.org/10.1002/anie.200600255

    CAS  Article  PubMed  Google Scholar 

  69. Torres-Salas P, Mate DM, Ghaz II, Plou FJ, Ballesteros AO, Alcalde M (2013) Widening the pH activity profile of a fungal laccase by directed evolution. ChemBioChem 14(8):934–937. https://doi.org/10.1002/cbic.201300102

    CAS  Article  PubMed  Google Scholar 

  70. Veum TL, Bollinger DW, Buff CE, Bedford MR (2006) A genetically engineered Escherichia coli phytase improves nutrient utilization, growth performance, and bone strength of young swine fed diets deficient in available phosphorus. J Anim Sci 84(5):1147–1158. https://doi.org/10.2527/2006.8451147x

    CAS  Article  PubMed  Google Scholar 

  71. Viader-Salvadó JM, Gallegos-López JA, Carreón-Treviño JG, Castillo-Galván M, Rojo-Domínguez A, Guerrero-Olazarán M (2010) Design of thermostable beta-propeller phytases with activity over a broad range of pHs and their overproduction by Pichia pastoris. Appl Environ Microbiol 76(19):6423–6430. https://doi.org/10.1128/aem.00253-10

    Article  PubMed  PubMed Central  Google Scholar 

  72. Walton J, Gray TK (1979) Absorption of inorganic phosphate in the human small intestine. Clin Sci (Lond) 56(5):407–412. https://doi.org/10.1042/cs0560407

    CAS  Article  Google Scholar 

  73. Wang W, Malcolm BA (2002) Two-stage polymerase chain reaction protocol allowing introduction of multiple mutations, deletions, and insertions, using QuikChange site-directed mutagenesis. Methods Mol Biol 182:37–43. https://doi.org/10.1385/1-59259-194-9:037

    CAS  Article  PubMed  Google Scholar 

  74. Weaver JD, Mullaney EJ, Lei XG (2007) Altering the substrate specificity site of Aspergillus niger PhyB shifts the pH optimum to pH 3.2. Appl Microbiol Biotechnol 76(1):117–122. https://doi.org/10.1007/s00253-007-0975-z

    CAS  Article  PubMed  Google Scholar 

  75. Yi Z, Kornegay ET (1996) Sites of phytase activity in the gastrointestinal tract of young pigs. Anim Feed Sci Technol 61(1):361–368. https://doi.org/10.1016/0377-8401(96)00959-5

    CAS  Article  Google Scholar 

  76. Yi Z, Kornegay ET, Ravindran V, Lindemann MD, Wilson JH (1996) Effectiveness of Natuphos phytase in improving the bioavailabilities of phosphorus and other nutrients in soybean meal-based semipurified diets for young pigs. J Anim Sci 74(7):1601–1611. https://doi.org/10.2527/1996.7471601x

    CAS  Article  PubMed  Google Scholar 

  77. Zhang R, Yang P, Huang H, Yuan T, Shi P, Meng K, Yao B (2011) Molecular and biochemical characterization of a new alkaline β-propeller phytase from the insect symbiotic bacterium Janthinobacterium sp. TN115. Appl Microbiol Biotechnol 92(2):317–325. https://doi.org/10.1007/s00253-011-3309-0

    CAS  Article  PubMed  Google Scholar 

  78. Zhao Q, Liu H, Zhang Y, Zhang Y (2010) Engineering of protease-resistant phytase from Penicillium sp.: high thermal stability, low optimal temperature and pH. J Biosci Bioeng 110(6):638–645. https://doi.org/10.1016/j.jbiosc.2010.08.003

    CAS  Article  PubMed  Google Scholar 

  79. Zhu L, Tee KL, Roccatano D, Sonmez B, Ni Y, Sun ZH, Schwaneberg U (2010a) Directed evolution of an antitumor drug (arginine deiminase PpADI) for increased activity at physiological pH. ChemBioChem 11(5):691–697. https://doi.org/10.1002/cbic.200900717

    CAS  Article  PubMed  Google Scholar 

  80. Zhu W, Qiao D, Huang M, Yang G, Xu H, Cao Y (2010b) Modifying thermostability of appA from Escherichia coli. Curr Microbiol 61(4):267–273. https://doi.org/10.1007/s00284-010-9606-5

    CAS  Article  PubMed  Google Scholar 

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Funding

This study was financed by the German Federal Ministry of Education and Research (BMBF) under the program Basistechnologien für die nächste Generation biotechnologischer Verfahren (FKZ 031A165).

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Correspondence to Ulrich Schwaneberg.

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Georgette Körfer and Catalina Novoa shared first authorship.

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Körfer, G., Novoa, C., Kern, J. et al. Directed evolution of an acid Yersinia mollaretii phytase for broadened activity at neutral pH. Appl Microbiol Biotechnol 102, 9607–9620 (2018). https://doi.org/10.1007/s00253-018-9308-7

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Keywords

  • Directed evolution
  • Phytase
  • High throughput screening
  • Site-saturation mutagenesis
  • pH profile
  • Semi-rational design