Abstract
The alkaline pectinase in fermentation liquid of engineering Bacillus subtilis was isolated and purified by using centrifugation, ultrafiltration, ammonium sulfate precipitation, and ion exchange chromatography, and the optimum conditions of ion exchange chromatography were determined. The results showed that, the recovery rate of crude enzymes ultrafiltrated by an ultrafiltration membrane with molecular weight cut-off 10000 Dalton reached 73.3 %, and the specific activity was 259.7 U/mg; after fractional salting out, the recovery rate was up to 62.9 %, and the specific activity was 1084 U/mg. With the DEAE-Sephorose CL-4B anion exchange chromatography under gradient elution with 0–1.0 mol/L NaCl (buffer solution of Gly-NaOH at pH8.6), the specific activity increased to 1230 U/mg. Subsequently, Sephadex-G75 column chromatography was applied and the specific activity increased to 2352 U/mg. The purity was 12.5 times of the crude enzyme, with the recovery rate of 21.6 %. The molecular weight of finally obtained alkaline pectinase was 43 kDa and the SDS polyacrylamide gel electrophoresis displayed that it was electrophoretically pure.
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References
Zhang JH, Li Y, Liu H et al (2005) Isolation, phylogenetic analysis of a bacterium with high yield of alkaline pectate lyase and optimization of its culture conditions. Chinese J App And 11(3):354–358
Schejterand A, Marcus L (1988) Isozymes of pectinesterase and polygalacturonase from Botrytis cinerea Pers. Methods Enzymol 161:366–373
Haruyoshi K (1998) Endopectate lyase from Erwinia aroideae. Methods Enzymol 161:381–385
Konno H (1988) Galacturan, 1,4-α-Galactuionidase from Carrot Daucus carota and Liverwort Marchantia polymorpha. Methods Enzymol 161:373–380
Pitt D (1988) Pectin lyase from Phoma medicaginisrar pinodella. Methods Enzymol 161:350–354
Forster H (1988) Pectinesterases from Phytophthora infestans. Methods Enzymol 161:355–361
Rombouts FM, Wissenburg AK, Pilnik W (1979) Chromtographic separation of orange pectinesterase isoenzymes on pectates with different degrees of crosslinking. J Chromatogr, 168:151-161
Lubomira RB, Vladimir TB (1972) Selective purification of Aspergillus niger endopolygalacturonase by affinity chromatography on cross-linked pectic acid. Acta Bioch Bioph Sin 268:187–193
Tagawa K, Kaji A (1988) Polygalacturonase from Corticium rolfsii. Methods Enzymol 161:361–365
Wu HM, Zhou J (1997) The purification and characterization of pectinases from Cladosporium herbarum and its mutants. Guizhou Science 15(3):163–170
Li S, Dong RA, He YM (2007) Study on the detection conditions of pectate lyase produced by Bacillus. Life Sci Instrum 5(9):18–21
Wang JZ, Fan M (2001) Protein technology handbook. Science Press, Beijing
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dyebinding. Anal Biochem 72:248–254
Acknowledgments
This work was supported by Key Technology Research and Development Program of Tianjin, China (No. 11ZCKFSY00900), Tianjin Research Program of Application Foundation and Advanced Technology (No. 11JCYBJC 09600) and National Natural Science Foundation of China (No. 21176190).
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Li, M. et al. (2014). Purification of Alkaline Pectinase in Engineering Bacillus subtilis . In: Zhang, TC., Ouyang, P., Kaplan, S., Skarnes, B. (eds) Proceedings of the 2012 International Conference on Applied Biotechnology (ICAB 2012). Lecture Notes in Electrical Engineering, vol 251. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-37925-3_151
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DOI: https://doi.org/10.1007/978-3-642-37925-3_151
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