Effect of Physical and Chemical Parameters on the Activity of Purified Phosphatase Enzyme Produced by Bacillus cereus


  • Enas N. Danial Biochemistry Department, Faculty of Science- Al Faisaliah King Abdulaziz University, Jeddah
  • Maha I. Alkhalf Chemistry of Natural and Microbial Products, National Research Center, Cairo




Acid phosphatase, kinetic studies, enzyme purification, Bacillus cereus


Phosphorus is one of the most important nutrients for plant growth and development. This study aimed to survey the optimum condition for phosphatase enzyme production, purified and studied its properties. Phosphatase enzyme activity was determined by end point method. Phosphatase enzyme was obtained from Bacillus cereus EME 66 isolate. The enzyme was purified and characterized, using a three-step purification procedure with 12.3-fold. The phosphatase enzyme was partially purified using ammonium sulfate fractionation followed by ultrafiltration. The acid phosphatase is a monomer protein purified gel filtration to 5.6 fold. Results showed that the optimum temperature for the purified enzyme activity was 60 °C and it was stable at temperatures below 60 °C. This enzyme was stable between pH 4.0-6.0, and the optimal pH activity was found to 5.0. The activity of the enzyme enhanced by heavy metals (Fe3+, Cu2+, Ca2+, Mg2+ and K+). The enzyme activity was strongly inhibited by heavy metals Zn2+. The present article reveals on enzymatic characterization of acid phosphatase enzyme. 


[1]Chang CH, Yang SS. Thermo-tolerant phosphate-solubilizing microbes for multi-functional biofertilizer preparation. Bioresour Technol 2009; 100: 1648-58.https://doi.org/10.1016/j.biortech.2008.09.00`9
[2]Spiers G A, McGill WB. Effects of phosphorus addition and energy supply on acid phosphatase production and activity in soils. Soil Biol and Biochem 1979; 11(1): 3-8.https://doi.org/10.1016/0038-0717(79)90110-X
[3]Fitriatin BN, Arief DH, Simarmata T, Santosa DA, Joy B. Phosphatase-producing bacteria isolated from Sanggabuanaforest and their capability to hydrolyze organic phosphate. J Soil Sci Environm Mang 2011; 2(10): 299-303.https://doi.org/10.5897/JSSEM
[4]Gawas SP, Singh SM, Naik S, Ravindra R. High-temperature optima phosphatases from the cold-tolerant Arctic fungus Penicillium citrinum. Polar Res 2012; 31:1-7. http://dx.doi.org/10.3402/polar.v31i0.11105
[5]Hu XJ, Li ZJ, Cao YC, Zhang J, Gong YX, Yang YF. Isolation and identification of a phosphate-solubilizing bacterium Pantoea stewartii subsp. stewartii g6, and effects of temperature, salinity, and pH on its growth under indoor culture conditions. Aquaculture Int 2010; 18(6): 1079-91. https://doi.org/10.1007/s10499-010-9325-8
[6]Mahesh M, Somashekhar R, Preenon B, Puttaiah ET. Optimization for the Production of Extracellular Alkaline Phosphatase from Proteus mirabilis J. Bioproc Biotech 2015; 5(3): 1-5.https://doi.org/10.4172/2155-9821.1000213
[7]Kerovuo J, Tynkkynen S. Expression of Bacillus subtilis phytase in Lactobacillus plantarum 755. Lett Appl Microbiol 2000; 30(5): 325-9.https://doi.org/10.1046/j.1472-765x.2000.00660.x
[8]Jain R, Garg V, Dangwal K, Madhuri KL. Purification and characterization of acid phosphatase from monocrotophos (MCP) hydrolyzing Aspergillus niger ITCC 7782.10 isolated from local agricultural field. Turk J Biochem 2013; 38(4): 396-402.https://doi.org/10.5505/tjb.2013.19870
[9]Ruangsanka S. Identification of phosphate-solubilizing fungi from the asparagus rhizosphere as antagonists of the root and crown rot pathogen Fusarium oxysporum. Science Asia 2014; 40: 16-20.https://doi.org/10.2306/scienceasia1513-1874.2014.40.016
[10]Kannaiyram S, Vedhachalam R, Thanigaimalai M. Production and Characterization of Alkaline Phosphatase Produced by Bacillus Species. J Appl Biol Biotechnol 2015; 3(5): 31-4.https://doi.org/10.7324/JABB.2015.3506
[11]Surchandra TH, Roy SS, Rakesh SN, Sahoo MR, Prakash N. Partial purification and biochemical characterization of acid phosphatase from germinated mung bean (Vigna radiata) seeds. Afr J Biotechnol 2012; 11(103): 16777-82.https://doi.org/10.5897/AJB12.2552
[12]Pradhan N, Sukla LB. Solubilization of inorganic phosphates by fungi isolated from agriculture soil. Afr J Biotechnol 2006; 5(10): 850-4.https://doi.org/10.5897/AJB2006.000-5050
[13]Rombola TH, Pedrinho EA, Macedo LEG, Gonçalves AM, Santos LF, Pizauro JM. Identification and enzymatic characterization of acid phosphatase from Burkholderia gladioli. BMC Res Notes 2014; 7: 221.https://doi.org/10.1186/1756-0500-7-221
[14]Kim DH, Oh BC, Choi WC, Lee JK, Oh TK. Enzymatic evaluation of Bacillus amyloliquefaciens phytase as a feed additive. Biotechnol Lett 1999; 2: 925-7.https://doi.org/10.1023/A:1005602717835
[15]Greiner R, Konietzny U, Jany KD. Purification and properties of a phytase from rye. J Food Biochem 1998; 22:143-61.https://doi.org/10.1111/j.1745-4514.1998.tb00236.x
[16]Sarmidi MR, El Enshasy HA. Biotechnology for wellness industry: Concepts and biofactories. Int J Biotechn Well Ind 2012; 1: 3-28. http://dx.doi.org/10.6000/1927-¬?3037.2012.01.01.01
[17]Beshay U, El Enshasy H. Production of ?-amylase by Bacillus amyloliquefaciens during batch cultivation in shake flask and stirred tank bioreactor. Deutsche Lebensmittel Rundshau 2002; 98: 5-9.
[18]El Enshasy H, Aboul-Enein A, Helmy S, El Azaly Y. Optimization of the industrial production of alkaline protease by Bacillus licheniformis in different production scales. Aust J Basic Appl Sci 2008; 2: 583-93.
[19]Abdel Fattah YR, El Enshasy HA, Soliman NA, El-Gendi H. Bioprocess development for the production of alkaline protease by Bacillus pseudoformus Mn6 through statistical experimental designs. J Microbiol Biotechnol 2009; 19: 378-86. http://dx.doi.org/10.4014/jmb.0806.380
[20]Esawy M, Mansour S, Ahmed E, Naziha M, El Enshasy H. Characterization of extracellular dextranase from novel halophilic Bacillus subtilis NRC-B233b a mutagenic honey isolate under solid state fermentation. Elect J Chem 2012; 9:1494-510. http://dx.doi.org/10.1155/2012/860619
[21]Elsayed EA, Omar HG, Abdel Galil S, El Enshasy HA. Optimization of fed-batch cultivation for extracellular ?-amylase production by Bacillus amyloliquefaciens in submerged culture. J Sci Ind Res 2016; 75: 480-6.
[22]Tham S, Ching D C, Huang H J, Lee Y F, H T S, Ching CC. Bioscience, biochemical characterization of an acid phosphatase from Thermos thermophiles. Biotechnol Biochem 2010; 74(4): 727-35.https://doi.org//10.1271/bbb.90773
[23]Parhamfar M, Dalfard AB, Parhamfar M, Rad SF. Purification and characterization of an extracellular phosphatase enzyme From Bacillus spp. J Cell Mol Res 2016; 8(2): 90-7.https://doi.org/10.22067/jcmr.v8i2.58676
[24]Ullah HJA, Gibson MD. Purification and characterization of acid phosphatase from cotyledons of germinating soybean seeds. Arch Biochem Biophys 1988; 260(2): 514-20https://doi.org/10.1016/0003-9861(88)90476-6
[25]Mohammed A. Purification and biochemical characterization of acid phosphatase from Vigna aconitifolia. Am J Plant Physiol 2010; 5(6): 361-70.https://doi.org/10.3923/ajpp.2010.361.370
[26]Sharma SB, Sayyed RZ, Trivedi MH, Gobi TA. Phosphate solubilizing microbes: sustainable approach for managing phosphorus deficiency in agricultural soils. Springer Plus 2013; 2: 587. https://doi.org/10.1186/2193-1801-2-587
[27]Shekhar NC. An efficient microbiological growth medium for screening phosphate solubilizing microorganisms. FEMS Microbiol Lett 1999; 170(1): 265-70.https://doi.org/10.1111/j.1574-6968.1999.tb13383.x
[28]Shen J, Yuan L, Zhang J, Li H, Bai Z, Chen X, Zhang W, Zhang F. Phosphorus dynamics: From soil to plant. Plant Physiol 2011; 156: 997-1005.https://doi.org/10.1104/pp.111.175232