BIOCHEMICAL CHARACTERIZATION OF RECOMBINANT PHYTASE ENZYME (phyK) FROM Klebsiella sp. ASR1 ENCAPSULATED WITH ALGINATE

Muhammad Eka Hidayatullah, . Sajidan, Ari Susilowati, Baraka Stewart Mkumbe, Ralf Greiner

Abstract


Karakterisasi Biokimia Enzim Fitase Rekombinan (phyK) dari Klebsiella sp. ASR1 Yang Dienkapsulasi Dengan Alginat

Enzim fitase melepas molekul fosfor pada atom C dari benzena Inositol fitat. Tetapi fitase memiliki kelemahan tidak mampu bertahan terhadap kondisi ekstrim dalam lambung nonruminansia. Solusi dalam penelitian ini yaitu fitase dienkapsulasi menggunakan alginat. Penelitian ini bertujuan mengkarakterisasi fitase setelah dienkapsulasi menggunakan alginate. Hasil penelitian ini yaitu fitase yang dienkapsulasi memiliki aktivitas tertinggi pada pH 6,0, sedangkan fitase tanpa enkapsulasi pada pH 5,0. Suhu optimum untuk aktivitas tertinggi fitase yang dienkapsulasi yaitu 70ºC, sedangkan fitase tanpa enkapsulasi 37ºC. Untuk perlakuan penambahan ion logam, aktivitas tertinggi fitase yang dienkapsulasi terjadi dengan penambahan 0,1 mM Fe2+ dan 1,0 mM Ca2+, sedangkan fitase tanpa enkapsulasi dengan penambahan 0,1 mM Fe2+. Berdasarkan hasil penelitian ini, fitase yang dienkapsulasi memiliki keunggulan lebih banyak dibandingkan dengan fitase tanpa enkapsulasi, karena mampu bertahan pada pH dan suhu tinggi, dan beberapa efek ion logam.

Kata Kunci: alginat, asam fitat, enkapsulasi, fitase, fitase rekombinan

ABSTRACT

Phytase enzymes release phosphorus molecules on the C atom from benzene inositol phytate. But phytase has the disadvantage of being unable to withstand extreme conditions in the non-ruminant stomach. The solution in this research was phytase encapsulated using alginate. This study aims to characterize phytase after being encapsulated using alginate. The results of this study were the encapsulated phytase had the highest activity at pH 6.0, while the unencapsulated phytase at pH 5.0. The optimum temperature for the highest activity of the encapsulated phytase was 70ºC, while the unencapsulated phytase 37ºC. For treatment of metal ion addition, the highest activity of the encapsulated phytase occurred with the addition of 0.1 mM Fe2+ and 1.0 mM Ca2+, while the unencapsulated phytase with the addition of 0.1 mM Fe2+. Based on the results of this study, the encapsulated phytase had more advantages compared to the unencapsulated phytase, as the former withstand high pH and temperature, and some metal ion effects.


Keywords


alginate; encapsulation; characterization; phytate; recombinant phytase

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References


Ali S, Zafar W, Shafiq S, Manzoor M (2017) Enzymes immobilization: An overview of techniques, support materials and its applications. Int J Sci Technol Res 6:64–72

Chen C, Cheng K, Ko T, Guo R (2015) Current progress in phytase research: Three-dimensional structure and protein engineering. ChemBioEng Rev 2:76–86. doi: 10.1002/cben.201400026

Cowieson AJ, Ruckebusch JP, Sorbara JOB, Wilson JW, Guggenbuhl P, Tanadini L, Roos FF (2017) A systematic view on the effect of microbial phytase on ileal amino acid digestibility in pigs. Anim Feed Sci Technol 231:138–149. doi: 10.1016/j.anifeedsci.2017.07.007

Fei B, Xu H, Zhang F, Li X, Ma S, Cao Y, Xie J, Qiao D, Cao Y (2013) Relationship between Escherichia coli AppA phytase’s thermostability and salt bridges. J Biosci Bioeng 115:623–627. doi: 10.1016/j.jbiosc.2012.12.010

Fernandez-Lafuente R (2017) Special issue: Enzyme immobilization 2016. Molecules 22:E601. doi: 10.3390/molecules22040601

Gerke J (2015) Phytate (inositol hexakisphosphate) in soil and phosphate acquisition from inositol phosphates by higher plants. A Review. Plants 4:253–266. doi: 10.3390/plants4020253

Godoi RR, Andrade SFV, Machado JM, de Souza TPP, Gonçalves AFA, Lopes TB, Parachin NS, Naves LP, Lima WJN, Pereira VV, Galdino AS (2017) Fungal phytase production in different hosts: A brief review. J Microbiol Biotechnol 2:000120. doi: 10.23880/oajmb-16000120

Gull I, Hameed A, Aslam MS, Athar MA (2013) Optimization of phytase production in solid state fermentation by different fungi. Afr J Microbiol Res 7:5207–5212. doi: 10.5897/AJMR2013.6136

Homaei AA, Sariri R, Vianello F, Stevanato R (2013) Enzyme immobilization: An update. J Chem Biol 6:185–205. doi: 10.1007/s12154-013-0102-9

Irshad M, Asgher M, Bhatti KH, Zafar M, Anwar Z (2017) Anticancer and nutraceutical potentialities of phytase/phytate. Int J Pharmacol 13:808–817. doi: 10.3923/ijp.2017.808.817

Kaur R, Saxena A, Sangwan P, Yadav AN, Kumar V, Dhaliwal HC (2017) Production and characterization of a neutral phytase of Penicillium oxalicum EUFR-3 isolated from Himalayan region. Nusantara Biosci 9:68–76. doi: 10.13057/nusbiosci/n090112

Keerti, Gupta A, Kumar V, Dubey A, Verma AK (2014) Kinetic characterization and effect of immobilized thermostable β-glucosidase in alginate gel beads on sugarcane juice. ISRN Biochem 2014:178498. doi: 10.1155/2014/178498

Kyzioł A, Mazgała A, Michna J, Regiel-Futyra A, Sebastian V (2017) Preparation and characterization of alginate/chitosan formulations for ciprofloxacin-controlled delivery. J Biomater Appl 32:162–174. doi: 10.1177/0885328217714352

Ma X-F, Tudor S, Butler T, Ge Y, Xi Y, Bouton J, Harrison M, Wang Z-Y (2012) Transgenic expression of phytase and acid phosphatase genes in alfalfa (Medicago sativa) leads to improved phosphate uptake in natural soils. Mol Breeding 30:377–391. doi: 10.1007/s11032-011-9628-0

McKie VA, McCleary BV (2016) A novel and rapid colorimetric method for measuring total phosphorus and phytic acid in foods and animal feeds. J AOAC Int 99:738–743. doi: 10.5740/jaoacint.16-0029

Megazyme (2017) Phytic acid (Phytate )/Total phosphorus – Measured as phosphorus released by phytase and alkaline phosphatase. Assay procedure. Megazyme. Wicklow, Ireland

Mogal C, Singh D, Mehta A, Ahmad T, Suthar K (2017) Isolation and biochemical characterization of phytase from different sources. J Appl Biotechnol Bioeng 2:152–155. doi: 10.15406/jabb.2017.02.00039

Naves L de P, Corrêa AD, Bertechini AG, Gomide EM, Santos CD dos (2012) Effect of pH and temperature on the activity of phytase products used in broiler nutrition. Rev Bras Ciênc Avíc 14:181–185. doi: 10.1590/S1516-635X2012000300004

Neira-Vielma AA, Aguilar CN, Ilyina A, Contreras-Esquivel JC, Carneiro-da-Cunha MG, Michelena-Alvarez G, Martínez-Hernández JL (2018) Optimized production of phytase by solid-state fermentation using triticale as substrate and source of inducer. Afr J Biotechnol 17:81–90. doi: 10.5897/AJB2017.16267

Cardoso FSN, Koblitz MGB, Ortiz GMD, de Carvalho JLV, de Carvalho LMJ (2019) Study of the parameters used in the encapsulation of commercial pectinase in calcium alginate and its effect on its catalytic activity. Food Sci Technol Campinas 39:247–252. doi: 10.1590/fst.31518

Niu C, Yang P, Luo H, Huang H, Wang Y, Yao B (2017) Engineering the residual side chains of HAP phytases to improve their pepsin resistance and catalytic efficiency. Sci Rep 7:42133. doi: 10.1038/srep42133

Nuhriawangsa AMP, Bachruddin Z, Sajidan, Wibowo A (2008) Karakterisasi fitase kasar (ekstraseluler) dari bakteri rekombinan E. coli BL21 (DE3)+pEAS1. Media Kedokt Hewan 24:124–131

Oktavia R, Suharti, Susanti E (2013) Karakterisasi enzim bromelin yang diamobilisasi dalam agar komersial. J Online Kimia Universitas Negeri Malang 2:1–10

Mupondi LT, Mnkeni PNS, Muchaonyerwa P, Mupambwa A (2018) Vermicomposting manure-paper mixture with igneous rock phosphate enhances biodegradation, phosphorus bioavailability and reduces heavy metal concentrations. Heliyon 4:e00749. doi: 10.1016/j.heliyon.2018.e00749

Reis CLB, de Sousa EYA, de Franca Serpa J, Oliveira RC, dos Santos JCS (2019) Design of immobilized enzyme biocatalysts: Drawbacks and opportunities. Quim Nova 42:768–783. doi: 10.21577/0100-4042.20170381

Rezakhani N, Molaei A, Parivar K, Khayati M, Etemadzade S (2014) Immobilization of protease in biopolymers (mixture of alginate-chitosan). J Paramed Sci 5:108–113. doi: 10.22037/jps.v5i4.7858

Sajidan (2002) Molekulare Charakterisierung einer Phytase (Myo-Inositol Hexakisphosphate Hydrolase) und von Phosphatasen aus Bakterienisolaten indonesischer Reisfelder (Klebsiella pneumoniae). Dissertation. Institut für Biologie, Humboldt-Universität zu Berlin

Sajidan A, Farouk A, Greiner R, Jungblut P, Müller E-C, Borriss R (2004) Molecular and physiological characterisation of a 3-phytase from soil bacterium Klebsiella sp. ASR1. Appl Microbiol Biotechnol 65:110–118. doi: 10.1007/s00253-003-1530-1

Sajidan, Wulandari R, Sari EN, Ratriyanto A, Weldekiros H, Greiner R (2015) Phytase-producing bacteria from extreme regions in Indonesia. Braz Arch Biol Technol 58:711–717. doi: 10.1590/S1516-89132015050173

Savita PD, Suvarna VC, Annu T, Balakrishna AN, Kanchanashri B, Yallappa M (2017) Characterization and identification of phytate solubilizing yeasts isolated from food grains. Int J Curr Microbiol Appl Sci 6:1184–1192. doi: 10.20546/ijcmas.2017.604.145

Saxena R, Kumar M, Tomar RS (2017) Nanobiotechnology: A new paradigm for crop production and sustainable agriculture. Res J Pharm Biol Chem Sci 8:823–832

Shah K, Desai P (2017) Isolation and characterization of phytase enzyme from Escherichia coli. Int J Pharm Bio Sci 8:474–478. doi: 10.22376/ijpbs.2017.8.2.b474-478

Suleimanova AD, Beinhauer A, Valeeva LR, Chastukhina IB, Balaban NP, Shakirov EV, Greiner R, Sharipova MR (2015) Novel glucose-1-phosphatase with high phytase activity and unusual metal ion activation from soil bacterium Pantoea sp. strain 3.5.1. Appl Environ Microbiol 81:6790–6799. doi: 10.1128/AEM.01384-15

Tsai CT, Meyer AS (2014) Enzymatic cellulose hydrolysis: Enzyme reusability and visualization of β-glucosidase immobilized in calcium alginate. Molecules 19:19390–19406. doi: 10.3390/molecules191219390

Wang T, Hao Y, Li W, Liu Y (2015) Enzymatic properties of phytase from Escherichia Coli DH5α. Proceedings of the International Conference on Chemical, Material and Food Engineering:88–92. doi: 10.2991/cmfe-15.2015.22

Widowati S, Andriani D, Riyanti EI, Raharto P, Sukarno L (2001) Karakterisasi fitase dari Bacillus coagulans. Dalam: Mariska I, Somantri IH, Sutrisno, Machmud M, Simanungkalit RDM,
Suyono, Orbani IN (eds) Prosiding Seminar Hasil Penelitian Rintisan dan Bioteknologi Tanaman, 30-31 Januari 2001. BPBTP, Bogor, hal 245–255

Wulandari R (2011) Analisis gen 16S rRNA pada bakteri penghasil enzim fitase. Tesis, Universitas Sebelas Maret Surakarta

Yao M, Lu W, Chen T, Wang W, Fu Y, Yang B, Liang A (2014) Effect of metals ions on thermostable alkaline phytase from Bacillus subtilis YCJS isolated from soybean rhizosphere soil. Ann Microbiol 64:1123–1131. doi: 10.1007/s13213-013-0751-5

Zdarta J, Jedrzak A, Klapiszewski Ł, Jesionowski T (2017) Immobilization of cellulase on a functional inorganic-organic hybrid support: Stability and kinetic study. Catalysts 7:374. doi: 10.3390/catal7120374

Zhang Z, Zhang R, Zou L, McClements DJ (2016) Protein encapsulation in alginate hydrogel beads: Effect of pH on microgel stability, protein retention and protein release. Food Hydrocolloids 58:308–315. doi: 10.1016/j.foodhyd.2016.03.01




DOI: https://doi.org/10.29122/jbbi.v7i1.2997

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