Abstract
The purpose of this investigation was to study the effect ofBacillus subtilis PE-11 cells immobilized in various matrices, such as calcium alginate, k-Carrageenan, ployacrylamide, agar-agar, and gelatin, for the production of alkaline protease. Calcium alginate was found to be an effective and suitable matrix for higher alkaline protease productivity compared to the other matrices studied. All the matrices were selected for repeated batch fermentation. The average specific volumetric productivity with calcium alginate was 15.11 U/mL/hour, which was 79.03% higher production over the conventional free-cell fermentation. Similarly, the specific volumetric productivity by repeated batch fermentation was 13.68 U/mL/hour with k-Carrageenan, 12.44 U/mL/hour with agar-agar, 11.71 U/mL/hour with polyacrylamide, and 10.32 U/mL/hour with gelatin. In the repeated batch fermentations of the shake flasks, an optimum level of enzyme was maintained for 9 days using calcium alginate immobilized cells. From the results, it is concluded that the immobilized cells ofB subtilis PE-11 in calcium alginate are more efficient for the production of alkaline protease with repeated batch fermentation. The alginate immobilized cells ofB subtilis PE-11 can be proposed as an effective biocatalyst for repeated usage for maximum production of alkaline protease.
Keywords: Alkaline protease production, B subtilis PE-11, immobilized cells, repeated batch fermentation
Full Text
The Full Text of this article is available as a PDF (224.1 KB).
References
- 1.Prakasham RS, Subba Rao Ch, Sreenivas Rao R, Rajesham S, Sarma PN. Optimization of alkaline protease production byBacillus sp using Taguchi methodology. Appl Biochem Biotechnol. 2005;120:133–144. doi: 10.1385/ABAB:120:2:133. [DOI] [PubMed] [Google Scholar]
- 2.Adinarayana K, Ellaiah P. Response surface optimisation of the critical medium components for the production of alkaline protease from a newly isolatedBacillus subtilis PE-11. J Pharm Pharma Sci. 2002;5:281–287. [PubMed] [Google Scholar]
- 3.Ellaiah P, Srinivasulu B, Adinarayana K. A review on microbial alkaline proteases. J Sci Ind Res (India). 2002;61:690–704. [Google Scholar]
- 4.Ellaiah P, Adinarayana K, Pardhasaradhi SV, Srinivasulu B. Isolation of alkaline protease producing bacteria from Visakhapatnam soil. Ind J Microbiol. 2002;42:173–175. [Google Scholar]
- 5.Beshay U. Production of alkaline protease byTeredinobacter turnirae cells immobilized in calcium alginate beads. Afrian J Biotechnol. 2003;2:60–65. [Google Scholar]
- 6.Adinarayana K, Ellaiah P. Production of alkaline protease by immobilized cells of alkalophilicBacillus sp. J Sci Ind Res (India) 2003;62:589–592. [Google Scholar]
- 7.Adinarayana K, Ellaiah P, Siva Prasad D. Production and partial peharacterization of thermostable serine alkaline protease from a newly isolatedBacillus subtilis PE-11. AAPS PharmSciTech. 2003;4:E56–E56. doi: 10.1208/pt040456. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Adinarayana K, Ellaiah P. Investigations on alkaline protease production withB subtilis PE-11 immobilised in calcium alginate gel beads. Process Biochem. 2004;39:1331–1339. doi: 10.1016/S0032-9592(03)00263-2. [DOI] [Google Scholar]
- 9.Sen S, Satyanarayana T. Optimization of alkaline protease production by thermophilicBacillus licheniformis S-40. Indian J Microbiol. 1993;33:43–47. [Google Scholar]
- 10.Fortin C, Vuillemards JC. Culture flourescence monitoring of immobilized cells. In: Bont JAM, Visser J, Mattiasson B, Tramper J, editors. Physiology of immobilized cells. Amsterdam: Elsevier; 1990. pp. 45–55. [Google Scholar]
- 11.Kukubu T, Karube I, Suzuki S. Protease production by immobilized mycelia ofStreptomyces fradiae. Biotechnol Bioeng. 1981;23:29–37. doi: 10.1002/bit.260230104. [DOI] [Google Scholar]
- 12.Linko S, Haapala R. Progress in Biotechnology. In: Wijffels RH, Buitellar RM, Bucke C, Tramper J, editors. Immobilized Cells: Basics and Applications. Amsterdam, The Netherlands: Elsevier; 1996. pp. 40–53. [Google Scholar]
- 13.Ramakrishna SV, Jamuna R, Emery AN. Production of ethanol by immobilized yeast cells. Appl Biochem Biotechnol. 1992;37:275–282. doi: 10.1007/BF02788879. [DOI] [Google Scholar]
- 14.Venkatasubramanian K. Immobilized Microbial Cells. In: Bull MJ, editor. Progress in Industrial Microbiology, Volume 15. New York, NY: Elsevier; 1979. pp. 61–95. [Google Scholar]
- 15.Colowick SP, Kaplan NO. Immobilized Enzymes. In: Mosbatch K, editor. Methods in Enzymology, Volume 44. New York, NY: Academic Press; 1976. pp. 169–190. [Google Scholar]
- 16.Kennedy JF, Melo EHM, Jumel K. Immobilized enzymes in cells. Chem Eng Prog. 1990;86:81–89. [Google Scholar]
- 17.Ramkrishna SV, Prakasham RS. Microbial fermentation with immobilized cells. Curr Sci. 1999;77:87–100. [Google Scholar]
- 18.Romo S, Perezmartinez C. The use of immobilization in alginate beads for long-term storage of Pseudoanabaena-Galeata (Cyanobacteria) in the laboratory. J Phycol. 1997;33:1073–1076. doi: 10.1111/j.0022-3646.1997.01073.x. [DOI] [Google Scholar]
- 19.Johnsen A, Flink JM. Influence of alginate properties and gel reinforcement on fermentation characteristics of immobilized yeast cells. Enz Microb Technol. 1986;8:737–748. doi: 10.1016/0141-0229(86)90162-6. [DOI] [Google Scholar]
- 20.Veelken M, Pape H. Production of tylosin and nikkomycin by immobilizedStreptomyces cells. Eur J Appl Microbiol Biotechnol. 1982;15:206–210. doi: 10.1007/BF00499956. [DOI] [Google Scholar]
- 21.Tsuchida O, Yamagota Y, Ishizuka J, et al. An alkaline proteinase of an alkalophilicBacillus sp. Curr Microbiol. 1986;14:7–12. doi: 10.1007/BF01568094. [DOI] [Google Scholar]
- 22.Abd-El-Haleem D, Beshay U, Abdelhamid A, Moawad H, Zaki S. Effects of nitrogen sources on biodegradation of phenol by immobilizedAcinetobacter sp strain W-17. Afr J Biotechnol. 2003;2:8–12. [Google Scholar]
- 23.Beshay U, Abd-El-Haleem D, Moawad H, Zaki S. Phenol biodegradation by free and immobilizedAcinetobacter. Biotechnol Lett. 2002;24:1295–1297. doi: 10.1023/A:1016222328138. [DOI] [Google Scholar]
- 24.Brodelius P, Vandamme EJ. Immobilized cell systems. In: Rehm HJ, Reed G, editors. Biotechnology. Weinheim: VCH Verlagsgesellschaft; 1987. pp. 405–464. [Google Scholar]
- 25.Kim DM, Kim GJ, Kim HS. Enhancement of operational stability of immobilized whole cell D-Hydantoinase. Biotechnol Lett. 1994;16:11–16. doi: 10.1007/BF01022616. [DOI] [Google Scholar]
- 26.Kierstan M, Bucke C. The immobilization of microbial cells, subcellular organelles, and enzymes in calcium alginate gels. Biotechnol Bioeng. 1977;19:387–397. doi: 10.1002/bit.260190309. [DOI] [PubMed] [Google Scholar]
- 27.Deo YM, Costerton JW, Gaucher RM. Semicontinuous and continuous production of antibiotics by immobilized fungal cells. Developments in Industrial microbiology. Appl Microbiol Biotechnol. 1985;21:220–225. doi: 10.1007/BF00295126. [DOI] [Google Scholar]
- 28.Anna V, Nigar B, Venko B, et al. Cyclodextrin glucanotransferase production by free and agar gel immobilized cells ofB circulans ATCC 21783. Proc Biochem. 2003;38:1585–1591. doi: 10.1016/S0032-9592(03)00060-8. [DOI] [Google Scholar]
- 29.Vuillemard JC, Terre S, Benoit S, Amiot J. Protease production by immobilized growing cells ofSerratia marcescens andMyxococcus xanthus in calcium alginate gel beads. Appl Microbiol Biotechnol. 1988;27:423–431. [Google Scholar]
- 30.Bandyopadhyay A, Das AK, Mandal SK. Erythromycin production byStreptomyces erythreus entrapped in calcium alginate beads. Biotechnol Lett. 1993;15:1003–1006. doi: 10.1007/BF00129926. [DOI] [Google Scholar]
- 31.Farid MAEL, Diwavey AI, Enshasy HA. Production of oxytetracycline by immobilizedStreptomyces rimosus cells in calcium alginate. Acta Biotechnol. 1994;14:303–309. doi: 10.1002/abio.370140316. [DOI] [Google Scholar]
- 32.Audet P, Paquin C, Lacroix C. Immobilized growing lactic acid bacteria with k-Carrageenan-locust bean gum gel. Appl Microbiol Biotechnol. 1988;29:11–18. doi: 10.1007/BF00258344. [DOI] [Google Scholar]
- 33.Audet P, Lacroix C, Paquin C. Continuous fermentation of a supplemented whey permeate medium with immobilizedStreptococcus salivarius subspThermophilus. Int Dairy J. 1992;2:1–15. doi: 10.1016/0958-6946(92)90040-S. [DOI] [Google Scholar]
- 34.Norton S, Lacroix C, Vuillemard JC. Kinetic study of continuous whey permeate fermentation by immobilizedLactobacillus helveticus for lactic acid production. Enzyme Microbiol Technol. 1994;16:457–466. doi: 10.1016/0141-0229(94)90015-9. [DOI] [Google Scholar]
