Purification of Alkaline Pectinase in Engineering Bacillus subtilis
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
- PDF / 406,245 Bytes
- 12 Pages / 439.37 x 666.142 pts Page_size
- 24 Downloads / 225 Views
Purification of Alkaline Pectinase in Engineering Bacillus subtilis Ming Li, Xin Sun, Liying Zhou, Hongxin Wang, Dongxia Li, Shuya Wang and Fuping Lu
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. Keywords Alkaline pectinase Chromatography
Isolation and purification
Ultrafiltration
151.1 Introduction Alkaline pectinase refers to the pectinase with higher activity under basic condition. Pectate lyase mainly acts in alkaline environment, occupying a large proportion in alkaline pectinase, thus alkaline pectinase generally refers to pectate lyase [1]. M. Li X. Sun L. Zhou H. Wang D. Li S. Wang F. Lu (&) Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, National Engineering Laboratory for Industrial Enzymes, The College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, People’s Republic of China e-mail: [email protected]
T.-C. Zhang et al. (eds.), Proceedings of the 2012 International Conference on Applied Biotechnology (ICAB 2012), Lecture Notes in Electrical Engineering 251, DOI: 10.1007/978-3-642-37925-3_151, Ó Springer-Verlag Berlin Heidelberg 2014
1419
1420
M. Li et al.
Alkaline pectinase is widely used in leather and silk industry, and shows wide application prospect in food, feed, and particularly in the extraction and washing for plant drugs in the field of medicine. A strain of Bacillus subtilis TCCC11485 with high yield of alkaline pectinase was constructed in our laboratory. The enzyme produced by the engineering bacteria has high expression activity, short fermentation period, fast degumming for pectinase, and small influence on fiber quality, which has potential application in the textile industry. The purification of alkaline pectinase is the prerequisite and foundation for the effective utilization of alkaline pectinase. The purification methods for alkaline pectinase produced by bacteria, mold, and yeast reported at home
Data Loading...
