In Vitro Characterization of a Novel Consensus Bacterial 6-Phytase and One of its Variants

Author(s): Trine Christensen*, Yueming Dersjant-Li, Vincent Sewalt, Rie Mejldal, Svend Haaning, Sina Pricelius, Igor Nikolaev, Robin A. Sorg, Arno de Kreij

Journal Name: Current Biochemical Engineering

Volume 6 , Issue 3 , 2020


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Abstract:

Background: Microbial phytases are added to animal feed to hydrolyze phytic acid (myoinositol hexakisphosphate, IP6) and phytate (salt of phytic acid) increasing phosphorus bioavailability. Novel phytases with enhanced bio-efficacy are being developed.

Objective: To characterize the biochemical and enzymatic properties of a novel consensus bacterial 6- phytase and its variant (PhyG), produced in Trichoderma reesei.

Methods: The in vitro specific activity, kinetic parameters, pH-activity profiles (relative to pH5.5), IP6 degradation, hydrolysis products and phosphate release of the phytases were determined using sodium phytate substrate. Melting point (Tm) was determined by differential scanning calorimetry and thermostability assessed by measuring residual activity at different temperatures. In vivo effects of PhyG supplementation at 0 to 1,000 FTU/kg on ileal IP6 digestibility and IP ester concentrations were determined in piglets.

Results: Both phytases exhibited pH optima of 3.5-4.5, high relative activity over a wide pH range (pH2.0-5.0), and substantial relative activity at pH1.5. At pH3.0, the specific activity of the PhyG variant was 1487 U/mg protein and at pH3.5 the kinetic constants were 240 μM (Km) and 1873 s-1 (Kcat). The hydrolysis of IP6 by both phytases was rapid. The major initial hydrolysis product was DLI( 1,2,3,4,5)P5, designating the phytases as bacterial 6-phytases (EC 3.1.3.26). Hydrolysis occurred at the D-3 (L-1) position in ~30% of instances, indicating a dual-specificity.

Conclusion: Both phytases showed high thermostability compared to wild type and existing commercial bacterial 6-phytases; PhyG exhibited 95% residual activity after 20 min incubation at 85.4ºC (pH5.5), Tm50 of ~93.2ºC and Tm of 98.8ºC. In vivo, PhyG at 1,000 FTU/kg achieved an ileal digestibility of IP6 of 89.3%.

Keywords: Buttiauxella sp., consensus bacterial 6-phytase, pH optimum, phosphate, thermostability, ileal phytate degradation, phytate degradation pathway.

[1]
U. Schlemmer, K.D. Jany, A. Berk, E. Schulz, and G. Rechkemmer, "Degradation of phytate in the gut of pigs--pathway of gastro-intestinal inositol phosphate hydrolysis and enzymes involved", Arch. Tierernahr., vol. 55, no. 4, pp. 255-280, 2001.
[http://dx.doi.org/10.1080/17450390109386197] [PMID: 12357589]
[2]
R. Greiner, and U. Konietzny, Phytase: biochemistry, enzymology and characteristics relevant to animal feed use.Enzymes in Farm Animal Nutrition., 2nd ed CAB International: London, UK, 2011, pp. 96-128.
[3]
P.H. Selle, and V. Ravindran, "Microbial phytase in poultry nutrition", Anim. Feed Sci. Technol., vol. 135, pp. 1-41, 2007.
[http://dx.doi.org/10.1016/j.anifeedsci.2006.06.010]
[4]
X.G. Lei, J.D. Weaver, E. Mullaney, A.H. Ullah, and M.J. Azain, "Phytase, a new life for an “old” enzyme", Annu. Rev. Anim. Biosci., vol. 1, pp. 283-309, 2013.
[http://dx.doi.org/10.1146/annurev-animal-031412-103717] [PMID: 25387021]
[5]
P. Hanlon, and V. Sewalt, "GEMs: genetically engineered microorganisms and the regulatory oversight of their uses in modern food production", Crit. Rev. Food Sci. Nutr., vol. 71, pp. 1-12, 2020.
[http://dx.doi.org/10.1080/10408398.2020.1749026] [PMID: 32274948]
[6]
Y. Dersjant-Li, A. Awati, H. Schulze, and G. Partridge, "Phytase in non-ruminant animal nutrition: A critical review on phytase activities in the gastrointestinal tract and influencing factors", J. Sci. Food Agric., vol. 95, no. 5, pp. 878-896, 2015.
[http://dx.doi.org/10.1002/jsfa.6998] [PMID: 25382707]
[7]
D. Menezes-Blackburn, S. Gabler, and R. Greiner, "Performance of seven commercial phytases in an in vitro simulation of poultry digestive tract", J. Agric. Food Chem., vol. 63, no. 27, pp. 6142-6149, 2015.
[http://dx.doi.org/10.1021/acs.jafc.5b01996] [PMID: 26111064]
[8]
P.H. Selle, A.J. Cowieson, and V. Ravindran, "Consequences of calcium interactions with phytate and phytase for poultry and pigs", Livest. Sci., vol. 124, pp. 126-141, 2009.
[http://dx.doi.org/10.1016/j.livsci.2009.01.006]
[9]
T.M. Shafey, M.W. McDonald, and J.G. Dingle, "Effects of dietary calcium and available phosphorus concentration on digesta pH and on the availability of calcium, iron, magnesium and zinc from the intestinal contents of meat chickens", Br. Poult. Sci., vol. 32, no. 1, pp. 185-194, 1991.
[http://dx.doi.org/10.1080/00071669108417339] [PMID: 2049622]
[10]
Y. Dersjant-Li, M.W.A. Verstegen, H. Schulze, T. Zandstra, H. Boer, J.W. Schrama, and J.A.J. Verreth, "Performance, digesta characteristics, nutrient flux, plasma composition, and organ weight in pigs as affected by dietary cation anion difference and nonstarch polysaccharide", J. Anim. Sci., vol. 79, no. 7, pp. 1840-1848, 2001.
[http://dx.doi.org/10.2527/2001.7971840x] [PMID: 11465371]
[11]
Z. Li, G. Yi, J. Yin, P. Sun, D.F. Li, and C. Knight, "Effects of organic acids on growth performance, gastrointestinal pH, intestinal microbial populations and immune responses of weaned pigs", Asian-Australas. J. Anim. Sci., vol. 21, pp. 252-261, 2008.
[http://dx.doi.org/10.5713/ajas.2008.70089]
[12]
S. Yu, M.F. Kvidtgaard, M.F. Isaksen, and S. Dalsgaard, "Characterization of a mutant Buttiauxella phytase using phytic acid and phytic acid-protein complex as substrates", Anim. Sci. Lett., vol. 1, pp. 18-32, 2014.
[13]
Y. Dersjant-Li, G. Archer, A.M. Stiewert, A.A. Brown, E.G. Sobotik, A. Jasek, L. Marchal, A. Bello, R.A. Sort, T. Christensen, H-S. Kim, R. Mejldal, I. Nkolaev, S. Pricelius, S. Haaning, J.F. Søresnesn, A. de Kreij, and V. Sewalt, "Functionality of a next generation biosynthetic bacterial 6-phytase in enhancing phosphorus availability to broilers fed a corn-soybean meal-based diet", Anim. Feed Sci. Technol, vol. 264, 2020.114481
[http://dx.doi.org/10.1016/j.anifeedsci.2020.114481]
[14]
Y. Dersjant-Li, B. Villca, V. Sewalt, A. de Kreij, L. Marchal, D.E. Velayudhan, R.A. Sorg, T. Christensen, R. Mejldal, I. Nikolaev, S. Pricelius, H-S. Kim, S. Haaning, J.F. Sørensen, and R. Lizardo, "Functionality of a next generation biosynthetic bacterial 6-phytase in enhancing phosphorus availability to weaned piglets fed a corn-soybean meal-based diet without added inorganic phosphate", Anim Nutr, vol. 6, no. 1, pp. 24-30, 2020.
[http://dx.doi.org/10.1016/j.aninu.2019.11.003] [PMID: 32211525]
[15]
L.M. Babe, T. Christensen, S. Haaning, H-S. Kim, R. Mejldal, I. Nikolaev, J.C. Prasad, S. Pricelius, J.F. Sorensen, and R.A. Sorg, Engineered Robust High Tm-Phytase Clade Polypeptides and Fragments Thereof Patent Application Number WO/2020/106796, 2020.http://patentscope.wipo.int/search/en/detail.jsf?docId=WO2020106796
[16]
G.S. Ladics, K-H. Han, M.S. Jang, H. Park, V. Marshall, Y. Dersjant-Li, and V.J. Sewalt, "Safety evaluation of a novel variant of consensus bacterial phytase", Toxicol. Rep., vol. 7, pp. 844-851, 2020.
[http://dx.doi.org/10.1016/j.toxrep.2020.07.004] [PMID: 32714839]
[17]
S. Kumar, G. Stecher, M. Li, C. Knyaz, and K. Tamura, "MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms", Mol. Biol. Evol., vol. 35, no. 6, pp. 1547-1549, 2018.
[http://dx.doi.org/10.1093/molbev/msy096] [PMID: 29722887]
[18]
P. Shi, H. Huang, Y. Wang, H. Luo, B. Wu, K. Meng, P. Yang, and B. Yao, "A novel phytase gene appA from Buttiauxella sp. GC21 isolated from grass carp intestine", Aquaculture, vol. 275, pp. 70-75, 2008.
[http://dx.doi.org/10.1016/j.aquaculture.2008.01.021]
[19]
H. Nielsen, K.D. Tsirigos, S. Brunak, and G. von Heijne, "A brief history of protein sorting prediction", Protein J., vol. 38, no. 3, pp. 200-216, 2019.
[http://dx.doi.org/10.1007/s10930-019-09838-3] [PMID: 31119599]
[20]
S. Whelan, and N. Goldman, "A general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach", Mol. Biol. Evol., vol. 18, no. 5, pp. 691-699, 2001.
[http://dx.doi.org/10.1093/oxfordjournals.molbev.a003851] [PMID: 11319253]
[21]
A.J. Engelen, F.C. van der Heeft, P.H.G. Randsdorp, W.A.C. Somers, J. Schaefer, and B.J. van der Vat, "Determination of phytase activity in feed by a colorimetric enzymatic method: Collaborative interlaboratory study", J. AOAC Int., vol. 84, no. 3, pp. 629-633, 2001.
[http://dx.doi.org/10.1093/jaoac/84.3.629] [PMID: 11417623]
[22]
E. Skoglund, N.G. Carlsson, and A.S. Sandberg, "High performance chromatographic separation of inositol phosphate isomers on strong anion exchange columns", J. Agric. Food Chem., vol. 46, pp. 1877-1882, 1998.
[http://dx.doi.org/10.1021/jf9709257]
[23]
Thermo Fisher Scientific, “Determination of inositol phosphates in dried grains with solubles”, Application Note 1070, Written by: K. Oates, B., De Borba, and J. Rohrer, 2014.
[24]
B.Q. Phillippy, and J.M. Bland, "Gradient ion chromatography of inositol phosphates", Anal. Biochem., vol. 175, no. 1, pp. 162-166, 1988.
[http://dx.doi.org/10.1016/0003-2697(88)90374-0] [PMID: 3245565]
[25]
E. Skoglund, N-G. Carlsson, and A-S. Sandberg, "Determination of isomers of inositol mono- to hexaphosphates in selected foods and intestinal contents using high-performance ion chromatography", J. Agric. Food Chem., vol. 45, pp. 431-436, 1997.
[http://dx.doi.org/10.1021/jf9603238]
[26]
F.J. Short, P. Gorton, J. Wiseman, and K.N. Boorman, "Determination of titanium dioxide added as an inert marker in chicken digestibility studies", Anim. Feed Sci. Technol., vol. 59, pp. 215-221, 1996.
[http://dx.doi.org/10.1016/0377-8401(95)00916-7]
[27]
V. Kumar, and A.K. Sinha, General aspects of phytases., Enzymes in Human and Animal Nutrition Principles and Perspectives. V. and Ku, 2018.
[http://dx.doi.org/10.1016/B978-0-12-805419-2.00003-4]
[28]
Y. Dersjant-Li, M. Hruby, C. Evans, and R. Greiner, "A critical review of methods used to determine phosphorus and digestible amino acid matrices when using phytase in poultry and pig diets", J. Appl. Anim. Nutr., vol. 7, no. e2, 2019.
[http://dx.doi.org/10.1017/JAN.2019.1]
[29]
E. Humer, C. Schwarz, and K. Schedle, "Phytate in pig and poultry nutrition", J. Anim. Physiol. Anim. Nutr. (Berl.), vol. 99, no. 4, pp. 605-625, 2015.
[http://dx.doi.org/10.1111/jpn.12258] [PMID: 25405653]
[30]
P.H. Selle, A.J. Cowieson, N.P. Cowieson, and V. Ravindran, "Protein-phytate interactions in pig and poultry nutrition: A reappraisal", Nutr. Res. Rev., vol. 25, no. 1, pp. 1-17, 2012.
[http://dx.doi.org/10.1017/S0954422411000151] [PMID: 22309781]
[31]
M. Amerah, C. Gilbert, P.H. Simmins, and V. Ravindran, "Influence of feed processing on the efficacy of exogenous enzymes in broiler diets", Worlds Poult. Sci. J., vol. 67, pp. 29-46, 2011.
[http://dx.doi.org/10.1017/S0043933911000031]
[32]
E.G. Kiarie, and A. Mills, "Role of feed processing on gut health and function in pigs and poultry: conundrum of optimal particle size and hydrothermal regimens", Front. Vet. Sci., vol. 6, no. 19, p. 19, 2019.
[http://dx.doi.org/10.3389/fvets.2019.00019] [PMID: 30838217]
[33]
I.B. Durowoju, K.S. Bhandal, J. Hu, B. Carpick, and M. Kirkitadze, "Differential scanning calorimetry – a method for assessment the thermal stability and conformation of protein antigen", J. Vis. Exp., vol. 121, no. 121, 2017.
[http://dx.doi.org/10.3791/55262] [PMID: 28287565]
[34]
S. Ortwin, and F. Igbasan, "In vitro properties of phytases from various microbial origins", Int. J. Food Sci. Technol., vol. 37, pp. 813-822, 2002.
[http://dx.doi.org/10.1046/j.1365-2621.2002.00621.x]
[35]
Y. Dersjant-Li, and G. Dusel, "Increasing the dosing of a Buttiauxella phytase improves phytate degradation, mineral, energy, and amino acid digestibility in weaned pigs fed a complex diet based on wheat, corn, soybean meal, barley, and rapeseed meal1", J. Anim. Sci., vol. 97, no. 6, pp. 2524-2533, 2019.
[http://dx.doi.org/10.1093/jas/skz151] [PMID: 31056701]
[36]
S. Yu, A. Cowieson, C. Gilbert, P. Plumstead, and S. Dalsgaard, "Interactions of phytate and myo-inositol phosphate esters (IP1-5) including IP5 isomers with dietary protein and iron and inhibition of pepsin", J. Anim. Sci., vol. 90, no. 6, pp. 1824-1832, 2012.
[http://dx.doi.org/10.2527/jas.2011-3866] [PMID: 22228039]
[37]
L.A. Beeson, C.L. Walk, M.R. Bedford, and O.A. Olukosi, "Hydrolysis of phytate to its lower esters can influence the growth performance and nutrient utilization of broilers with regular or super doses of phytase", Poult. Sci., vol. 96, no. 7, pp. 2243-2253, 2017.
[http://dx.doi.org/10.3382/ps/pex012] [PMID: 28204754]
[38]
B.Q. Phillippy, "Identification by two-dimensional NMR of myo-inositol tris- and tetrakis (phosphates) formed from phytic acid by wheat phytase", J. Agric. Food Chem., vol. 37, pp. 1261-1265, 1989.
[http://dx.doi.org/10.1021/jf00089a013]
[39]
R. Greiner, N. Carlsson, and M.L. Alminger, "Stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme of Escherichia coli", J. Biotechnol., vol. 84, no. 1, pp. 53-62, 2001.
[http://dx.doi.org/10.1016/S0168-1656(00)00331-X] [PMID: 11035187]
[40]
A. Ariza, O.V. Moroz, E.V. Blagova, J.P. Turkenburg, J. Waterman, S.M. Roberts, J. Vind, C. Sjøholm, S.F. Lassen, L. De Maria, V. Glitsoe, L.K. Skov, and K.S. Wilson, "Degradation of phytate by the 6-phytase from Hafnia alvei: A combined structural and solution study", PLoS One, vol. 8, no. 5, p. e65062, 2013.
[http://dx.doi.org/10.1371/journal.pone.0065062] [PMID: 23741456]
[41]
K. Pontoppidan, V. Glitsoe, P. Guggenbuhl, A.P. Quintana, C.S. Nunes, D. Pettersson, and A-S. Sandberg, "In vitro and in vivo degradation of myo-inositol hexakisphosphate by a phytase from Citrobacter braakii", Arch. Anim. Nutr., vol. 66, no. 6, pp. 431-444, 2012.
[http://dx.doi.org/10.1080/1745039X.2012.735082] [PMID: 23098167]


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VOLUME: 6
ISSUE: 3
Year: 2020
Page: [156 - 171]
Pages: 16
DOI: 10.2174/2212711906999201020201710

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