Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Purification and Characterization of a Novel Alkaline Protease from Bacillus horikoshii

  • Received : 2011.09.05
  • Accepted : 2011.09.26
  • Published : 2012.01.28
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Abstract

An investigation was conducted on the enhancement of production and purification of an oxidant and SDS-stable alkaline protease (BHAP) secreted by an alkalophilic Bacillus horikoshii, which was screened from the body fluid of a unique Korean polychaeta (Periserrula leucophryna) living in the tidal mud flats of Kwangwha Island in the Korean West Sea. A prominent effect on BHAP production was obtained by adding 2% maltose, 1% sodium citrate, 0.8% NaCl, and 0.6% sodium carbonate to the culturing medium. The optimal medium for BHAP production contained (g/l) SBM, 15; casein, 10; $K_2HPO_4$, 2; $KH_2PO_4$, 2; maltose, 20; sodium citrate, 10; $MgSO_4$, 0.06; NaCl, 8; and $Na_2CO_3$, 6. A protease yield of approximately 56,000 U/ml was achieved using the optimized medium, which is an increase of approximately 5.5-fold compared with the previous optimization (10,050 U/ml). The BHAP was homogenously purified 34-fold with an overall recovery of 34% and a specific activity of 223,090 U/mg protein using adsorption with Diaion HPA75, hydrophobic interaction chromatography (HIC) on Phenyl-Sepharose, and ion-exchange chromatography on a DEAE- and CM-Sepharose column. The purified BHAP was determined a homogeneous by SDS-PAGE, with an apparent molecular mass of 28 kDa, and it showed extreme stability towards organic solvents, SDS, and oxidizing agents. The $K_m$ and $k_{cat}$ values were 78.7 ${\mu}M$ and $217.4s^{-1}$ for N-succinyl-Ala-Ala-Pro-Phe-pNA at $37^{\circ}C$ and pH 9, respectively. The inhibition profile exhibited by PMSF suggested that the protease from B. horikoshii belongs to the family of serine proteases. The BHAP, which showed high stability against SDS and $H_2O_2$, has significance for industrial application, such as additives in detergent and feed industries.

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References

  1. Amoozegar, M. A., A. Z. Fatemi, H. R. Karbalaei-Heidari, and M. R. Razavi. 2007. Production of an extracellular alkaline metalloprotease from a newly isolated, moderately halophile, Salinivibrio sp. strain AF-2004. Microbiol. Res. 162: 369-377. https://doi.org/10.1016/j.micres.2006.02.007
  2. Banerjee, U. C., R. K. Sani, W. Azmi, and R. Soni. 1999. Thermostable alkaline protease from Bacillus brevis and its characterization as a laundry detergent additive. Process Biochem. 35: 213-219. https://doi.org/10.1016/S0032-9592(99)00053-9
  3. Beg, Q. K. and R. Gupta. 2003. Purification and characterization of an oxidation-stable, thiol-dependent serine alkaline protease from Bacillus mojavensis. Enzyme Microb. Technol. 32: 294-304. https://doi.org/10.1016/S0141-0229(02)00293-4
  4. Bradford, M. M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254. https://doi.org/10.1016/0003-2697(76)90527-3
  5. Bryan, P. N. 2000. Protein engineering of subtilisin. Biochim. Biophys. Acta 1543: 203-222. https://doi.org/10.1016/S0167-4838(00)00235-1
  6. Chu, W.H. 2007. Optimization of extracellular alkaline protease production from species of Bacillus. J. Ind. Microbiol. Biotechnol. 34: 241-245. https://doi.org/10.1007/s10295-006-0192-2
  7. Estell, D. D., T. P. Graycar, and J. A. Wells. 1985. Engineering an enzyme by site-directed mutagenesis to be resistant to chemical oxidation. J. Biol. Chem. 260: 6518-6521.
  8. Fang, Y. Y., W. B. Yang, S. L. Ong, J. Y. Hu, and W. J. Ng. 2001. Fermentation of starch for enhanced alkaline protease production by constructing an alkalophilic Bacillus pumilus strain. Appl. Microbiol. Biotechnol. 57: 153-160. https://doi.org/10.1007/s002530100765
  9. Gattinger, L. D., Z. Duvnjak, and A. W. Khan. 1990. The use of canola meal as a substrate for xylanase production by Trichoderma reesei. Appl. Microbiol. Biotechnol. 33: 21-25.
  10. Gencka, H. and C. Tari. 2006. Alkaline protease production from alkalophilic Bacillus sp. isolated from natural habitats. Enzyme Microb. Technol. 39: 703-710. https://doi.org/10.1016/j.enzmictec.2005.12.004
  11. Gessesse, A. 1997. The use of nug meal as low-cost substrate for the production of alkaline protease by the alkaliphilic Bacillus sp. AR-009 and some properties of the enzyme. Bioresour. Technol. 62: 59-61. https://doi.org/10.1016/S0960-8524(97)00059-X
  12. Gupta, R., K. Gupta, R. K. Saxena, and S. Khan. 1999. Bleachstable alkaline protease from Bacillus sp. Biotechnol. Lett. 21: 135-138. https://doi.org/10.1023/A:1005478117918
  13. Gupta, R., Q. K. Beg, S. Khan, and B. Chauhan. 2002. An overview on fermentation, downstream processing and properties of microbial alkaline proteases. Appl. Microbiol. Biotechnol. 60: 381-395. https://doi.org/10.1007/s00253-002-1142-1
  14. Hmidet, N., N. E. Ali, A. Haddar, S. Kanoun, S. K. Alya, and M. Nasri. 2009. Alkaline proteases and thermostable $\alpha$-amylase co-produced by Bacillus licheniformis NH1: Characterization and potential application as detergent additive. Biochem. Eng. J. 47: 71-79. https://doi.org/10.1016/j.bej.2009.07.005
  15. Horikoshi, K. 1971. Production of alkaline enzymes by alkalophilic microorganisms. I. Alkaline protease produced by Bacillus no. 221. Agric. Biol. Chem. 35: 1407-1414.
  16. Horikoshii, K. 1999. Alkalophiles: Some applications of their products for biotechnology. Microbiol. Mol. Biol. Rev. 63: 735-750.
  17. Ito, S., T. Kobayashi, K. Ara, K. Ozaki, S. Kawai, and Y. Hatada. 1998. Alkaline detergent enzymes from alkaliphiles: Enzymatic properties, genetics, and structures. Extremophiles 2: 185-190. https://doi.org/10.1007/s007920050059
  18. Jacobs, M. F. 1995. Expression of the subtilisin Carlsbergencoding gene in Bacillus licheniformis and Bacillus subtilis. Gene 152: 67-74.
  19. Jaouadi, B., S. Ellouz-Chaabouni, M. Rhimi, and S. Bejar. 2008. Biochemical and molecular characterization of a detergentstable serine alkaline protease from Bacillus pumilus CBS with high catalytic efficiency. Biochimie 90: 1291-1305. https://doi.org/10.1016/j.biochi.2008.03.004
  20. Jaouadi, B., N. Aghajari, R. Haser, and S. Bejar. 2010. Enhancement of the thermostability and the catalytic efficiency of Bacillus pumilus CBS protease by site-directed mutagenesis. Biochimie 92: 360-369. https://doi.org/10.1016/j.biochi.2010.01.008
  21. Joo, H. S., C. G. Kumar, G. C. Park, S. R. Paik, and C. S. Chang. 2002. Optimization of the production of an extracellular alkaline protease from Bacillus horikoshii. Process Biochem. 38: 155-159. https://doi.org/10.1016/S0032-9592(02)00061-4
  22. Joo, H. S., C. G. Kumar, G. C. Park, S. R. Paik, and C. S. Chang. 2003. Oxidant and SDS-stable alkaline protease from Bacillus clausii I-52: Production and some properties. J. Appl. Microbiol. 95: 267-272. https://doi.org/10.1046/j.1365-2672.2003.01982.x
  23. Joo, H. S., C. G. Kumar, G. C. Park, S. R. Paik, and C. S. Chang. 2004. Bleach-resistant alkaline protease produced by a Bacillus sp. isolated from the Korean polychaeta, Periserrula leucophryna. Process Biochem. 39: 1441-1447. https://doi.org/10.1016/S0032-9592(03)00260-7
  24. Joo, H. S. and C. S. Chang. 2005. Production of protease from a new alkalophilic Bacillus sp. I-312 grown on soybean meal: Optimization and some properties. Process Biochem. 40: 1263- 1270. https://doi.org/10.1016/j.procbio.2004.05.010
  25. Joshi, R. H., M. S. Dodia, and S. P. Singh. 2008. Production and optimization of a commercially viable alkaline protease from a haloalkaliphilic bacterium. Biotechnol. Bioprocess Eng. 13: 552-559. https://doi.org/10.1007/s12257-007-0211-9
  26. Kaur, S., R. M. Vohra, M. Kapoor, Q. K. Beg, and G. S. Hoondal. 2001. Enhanced production and characterization of a highly thermostable alkaline protease from Bacillus sp. P-2. World J. Microbiol. Biotechnol. 17: 125-129. https://doi.org/10.1023/A:1016637528648
  27. Klingeberg, M., B. Galunsky, C. Sjoholm, V. Kasche, and G. Antranikian. 1995. Purification and properties of a highly thermostable, sodium dodecyl sulfate-resistant and stereospecic proteinase from the extremely thermophilic archaeon Thermococcus stetteri. Appl. Environ. Microbiol. 61: 3098-3104.
  28. Kobayashi, T., Y. Hakamada, S. Adachi, J. Hitomi, T. Yoshimatsu, K. Koike, S. Kawai, and S. Ito. 1995. Purification and properties of an alkaline protease form alkalophilic Bacillus sp. KSM-16. Appl. Microbiol. Biotechnol. 43: 473-481. https://doi.org/10.1007/BF00218452
  29. Koo, K. B., H. S. Joo, and J. W. Choi. 2010. Decolorization method of crude alkaline protease preparation produced from an alkalophilic Bacillus clausii. Biotechnol. Bioprocess Eng. 16: 89-96.
  30. Kumar, C. G. and H. Takagi. 1999. Microbial alkaline proteases: From a bioindustrial viewpoint. Biotechnol. Adv. 17: 561-594. https://doi.org/10.1016/S0734-9750(99)00027-0
  31. Kumar, C. G., M. P. Tiwari, and K. D. Jany. 1999. Novel alkaline serine proteases from alkalophilic Bacillus sp.: Purification and characterization. Process Biochem. 34: 441-449. https://doi.org/10.1016/S0032-9592(98)00110-1
  32. Kumar, C. G. and P. Parrack. 2003. Activated charcoal: A versatile decolorization agent for the recovery and purification of alkaline protease. World J. Microbiol. Biotechnol. 19: 243-246. https://doi.org/10.1023/A:1023644615956
  33. Kunst, F., N. Ogasawara, I. Moszer, A. M. Albertini, G. Alloni, V. Azevedo, et al. 1997. The complete genome sequence of the Gram-positive bacterium Bacillus subtilis. Nature 390: 237-238.
  34. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 22: 680-685.
  35. Manachini, P. L. and M. G. Fortina. 1998. Production in seawater of thermostable alkaline proteases by a halotolerant strain of Bacillus licheniformis. Biotechnol. Lett. 20: 565-568. https://doi.org/10.1023/A:1005349728182
  36. Maurer, K. H. 2004. Detergent proteases. Curr. Opin. Biotechnol. 15: 330-334. https://doi.org/10.1016/j.copbio.2004.06.005
  37. Patel, R., M. Dodia, and S. P. Singh. 2005. Extracellular alkaline protease from a newly isolated haloalkaliphilic Bacillus sp.: Production and optimization. Process Biochem. 40: 3569-3575. https://doi.org/10.1016/j.procbio.2005.03.049
  38. Phadatare, S. U., V. V. Deshpande, and M. C. Srinvasan. 1993. High activity alkaline protease from Conidiobolus coronatus(NCL 86.8.20): Enzyme production and compatibility with commercial detergents. Enzyme Microb. Technol. 15: 72-76. https://doi.org/10.1016/0141-0229(93)90119-M
  39. Rao, M. B., A. M. Tanksale, M. S. Ghatge, and V. V. Deshpande. 1998. Molecular and biotechnological aspects of microbial proteases. Microbiol. Mol. Biol. Rev. 62: 597-635.
  40. Rao, C. S., T. Sathish, P. Ravichandra, and R. S. Prakasham. 2009. Characterization of thermo- and detergent stable serine protease from isolated Bacillus circulans and evaluation of ecofriendly applications. Process Biochem. 44: 262-268. https://doi.org/10.1016/j.procbio.2008.10.022
  41. Sadana, A. and A. M. Beelaram. 1994. Efficiency and economics of bioseparation: Some case studies. Bioseparation 4: 221-235.
  42. Saeki, K., J. Hitomi, M. Okuda, Y. Hatada, Y. Kageyama, M. Takaiwa, et al. 2002. A novel species of alkalophilic Bacillus that produces an oxidatively stable alkaline serine protease. Extremophiles 6: 65-72. https://doi.org/10.1007/s007920100224
  43. Saeki, K., K. Ozaki, T. Kobayashi, and S. Ito. 2007. Detergent alkaline proteases: Enzymatic properties, genes, and crystal structures. J. Biosci. Bioeng. 103: 501-508. https://doi.org/10.1263/jbb.103.501
  44. Samal, B. B., B. Karan, and Y. Stabinsky. 1990. Stability of two novel serine proteinases in commercial laundry detergent formulations. Biotechnol. Bioeng. 28: 609-612.
  45. Shivanand, P. and G. Jayaraman. 2009. Production of extracellular protease from halotolerant bacterium, Bacillus aquimaris strain VITP4 isolated from Kumta coast. Process Biochem. 44: 1088- 1094. https://doi.org/10.1016/j.procbio.2009.05.010
  46. Sigma, D. S. and G. Mooser. 1975. Chemical studies of enzyme active sites. Annu. Rev. Biochem. 44: 889-931. https://doi.org/10.1146/annurev.bi.44.070175.004325
  47. Stepanov, V. M., G. N. Rudenskaya, L. P. Revina, Y. B. Gryanova, E. N. Lysogorskaya, and I. Y. Filippova. 1992. A serine proteinase of an archaebacterium, Halobacterium mediterranei. Biochem. J. 283: 281-286. https://doi.org/10.1042/bj2830281
  48. Studdert, C. A., M. K. H. Seitz, M. I. P. Gil, J. J. Sanchez, and R. E. de Castro. 2001. Purification and biochemical characterization of the haloalkaliphilic archaeon Natronococcus occultus extracellular serine protease. J. Basic Microbiol. 41: 375-383. https://doi.org/10.1002/1521-4028(200112)41:6<375::AID-JOBM375>3.0.CO;2-0
  49. Takami, H., K. Nakasone, Y. Takaki, G. Maeno, R. Sasaki, N. Masui, et al. 2000. Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis. Nucl. Acids Res. 28: 4317-4331. https://doi.org/10.1093/nar/28.21.4317
  50. Tunlid, A., S. Rosen, B. Ek, and L. Rask. 1994. Purification and characterization of an extracellular serine protease from the nematode-trapping fungus Arthrobotrys oligospora. Microbiology 140: 1687-1695. https://doi.org/10.1099/13500872-140-7-1687
  51. VijayAnand, S., J. Hemapriya, J. Selvin, and S. Kiran. 2010. Production and optimization of haloalkaliphilic protease by an extremophile-Halobacterium sp. Js1, isolated from thalassohaline environment. Global J. Biotechnol. Biochem. 5: 44-49.
  52. Wells, J. A., E. Ferrari, D. J. Henner, D. A. Estell, and E. Y. Chen. 1983. Cloning, sequencing, and secretion of Bacillus amyloliquefaciens subtilisin in Bacillus subtilis. Nucl. Acids Res. 11: 7911-7925. https://doi.org/10.1093/nar/11.22.7911
  53. Yang, J. K., I. L. Shih, Y. M. Tzeng, and S. L.Wang. 2000. Production and purification of protease from a Bacillus subtilis that can deproteinize crustacean wastes. Enzyme Microb. Technol. 26: 406-413. https://doi.org/10.1016/S0141-0229(99)00164-7
  54. Zambare, V. P., S. S. Nilegaonkar, and P. P. Kanekar. 2007. Production of an alkaline protease by Bacillus cereus MCM B- 326 and its application as a dehairing agent. World J. Microbiol. Biotechnol. 23: 1569-1574. https://doi.org/10.1007/s11274-007-9402-y
  55. Jaouadi, B., N. Aghajari, R. Haser, and S. Bejar. 2010. Enhancement of the thermostability and the catalytic efficiency of Bacillus pumilus CBS protease by site-directed mutagenesis. Biochimie 92: 360-369. https://doi.org/10.1016/j.biochi.2010.01.008
  56. Hadj-Ali, N. E., R. Agrebi, B. Ghorbel-Frikha, A. Sellami- Kamoun, S. Kanoun, and M. Nasri. 2007. Biochemical and molecular characterization of a detergent stable alkaline serineprotease from a newly isolated Bacillus licheniformis NH1. Enzyme Microb. Technol. 40: 515-523. https://doi.org/10.1016/j.enzmictec.2006.05.007
  57. Huang, Q., Y. Peng, X. Li, H. Wang, and Y. Zhang. 2003. Purification and characterization of an extracellular alkaline serine protease with dehairing function from Bacillus pumilus. Curr. Microbiol. 46: 169-173. https://doi.org/10.1007/s00284-002-3850-2
  58. Agrebi, R., A. Haddar, N. Hmidet, K. Jellouli, L. Manni, and M. Nasri. 2009. BSF1 fibrinolytic enzyme from a marine bacterium Bacillus subtilis A26: Purification, biochemical and molecular characterization. Process Biochem. 44: 1252-1259. https://doi.org/10.1016/j.procbio.2009.06.024
  59. Nakamura, T., Y. Yamagata, and E. Ichishima. 1992. Nucleotide sequence of the subtilisin NAT gene, aprN, of Bacillus subtilis (natto). Biosci. Biotechnol. Biochem. 56: 1869-1871. https://doi.org/10.1271/bbb.56.1869
  60. Ko, J. H., J. P. Yan, L. Zhu, and Y. P. Qi. 2004. Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02. Comp. Biochem. Physiol. C 137: 65-74.
  61. Liang, X., S. Jia, Y. Sun, M. Chen, X. Chen, and J. Zhong. 2007. Secretory expression of nattokinase from Bacillus subtilis YF38 in Escherichia coli. Mol. Biotechnol. 37: 187-194. https://doi.org/10.1007/s12033-007-0060-y
  62. Lee, S. K., D. H. Bae, T. J. Kwon, S. B. Lee, H. H. Lee, and J. H. Park. 2001. Purification and characterization of a fibrinolytic enzyme from Bacillus sp. KDO-13 isolated from soybean paste. J. Microbiol. Biotechnol. 11: 845-852.

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