Isolation and Identification of a Bacterial Strain with Pullulanase Activity and the Cloning of the Pullulanase Gene
A novel bacterial strain OPF-0031 with pullulanase activity was isolated from mud sample of the sea. With the analysis of the 16S rDNA sequence and the characterization of biochemical reactions, the strain was identified to be Bacillus cereus. A 2641 bp D
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Isolation and Identification of a Bacterial Strain with Pullulanase Activity and the Cloning of the Pullulanase Gene Guixiu Dong, Tao Xia, Yongqian Qiu, Xiaoliang Si, Yiyu Liu, Pingping Yang, Xiangzhao Mao and Lizhong Guo
Abstract A novel bacterial strain OPF-0031 with pullulanase activity was isolated from mud sample of the sea. With the analysis of the 16S rDNA sequence and the characterization of biochemical reactions, the strain was identified to be Bacillus cereus. A 2641 bp DNA fragment including a pullulanase gene from the genome of OPF-0031, was present. The nucleotide sequence of the gene encoding pullulanase was cloned and analyzed in Escherichia coli. Keywords Gene cloning Phylogenetic dendrogram
Isolation 16SrDNA Pullulan hydrolase activity
143.1 Introduction Pullulanases are widely distributed among animals, plants, fungi, and bacteria, which hydrolyze the a-1,6-glucosidic linkages in pullulan, amylopectin, starch and related oligosaccharides [1]. Since a variety of microbial pullulanases have been obtained, they have been classified into four groups based on substrate specificities and reaction products [2, G. Dong T. Xia Y. Qiu X. Si Y. Liu X. Mao (&) College of Food Science and Engineering, Ocean University of China, Qingdao 266003, People’s Republic of China e-mail: [email protected] G. Dong P. Yang School of Food and Bioengineering, Shandong Institute of Light Industry, Jinan 250353, People’s Republic of China L. Guo (&) Shandong Province Key Lab of Applied Mycology, Qingdao Agricultural University, Qingdao 266109, 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_143, Springer-Verlag Berlin Heidelberg 2014
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3]: (i) neopullulanase (EC 3.2.1.135): pullulan hydrolase type I attacks a-1,4 glycosidic linkages in pullulan, forming panose; (ii) isopullulanase (EC 3.2.1.57): pullulan hydrolase type II can hydrolyze a-1,4 glycosidic linkages in pullulan, forming isopanose; (iii) In branched oligosaccharides or pullulan, pullulanase type I (EC 3.2.1.41) specifically attacks a-1,6 glycosidic linkages, linear oligomers or maltotriose were formed, respectively; and (iv) amylopullulanase: pullulanase type II attacks both a-1,6 glycosidic linkages in pullulan and branched substrates and the a-1,4 glycosidic linkages in polysaccharides. Based on its characteristic, pullulanase has attracted significant attention as a useful tool in starch industry. So far, high value of pullulanase has been applied in the preparing resistant starch, cyclodextrin, maltotriose syrup, beer brewing, and maltotriose syrup. To date, a large number of different microorganisms with pullulanases activity have been characterized [4–7]. Some of the isolates have been identified to be moderately aerobic thermophilic bacteria Bacillus acidopullulyticus [8], Bacillus flavocaldarius KP 1228 [9], Therm
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