Cloning and recombinant expression of chitosanase gene from Bacillus sp. S-1
会议名称：《2010 First International Conference on Cellular,Molecular Biology, Biophysics and Bioengineering(CMBB 2010)》
会议日期：2010年
学科分类：0710[理学-生物学] 07[理学] 08[工学] 09[农学] 071007[理学-遗传学] 0901[农学-作物学] 0836[工学-生物工程] 090102[农学-作物遗传育种]
基 金：supported by Open-end Funds of Jiangsu Key Laboratory of Marine Biotechnology Huaihai Institute of Technology(Grant No.2009HS01)
关 键 词：chitosanase Bacillus recombinant expression chitooligosaccharides
摘 要：In this work,we report the characterization of a chitosanase-producing bacterium isolated from *** strain was grouped under the genus Bacillus by virtue of its morphological,physiological properties and 16S rDNA gene sequence and named it Bacillus *** to the information of chitosanase full-length sequences deposited in NCBI,a pair of degenerated primes was designed and a partial sequence of chitosanase gene was obtained by polymerase chain reaction(PCR) using Bacillus sp.S-1 genome DNA as the template.A genome walking library was constructed followed as the protocol provided by CLONTECH *** flanking sequences of the 5’and 3’terminal was obtained by genome walking method and two-step PCR *** overlapped and confirmed,the full-length sequence of chitosanase from Bacillus sp.S-1 was achieved and it contained 1362 bp coding 453 amino acids(accession number is EU924147) .The predicted amino acid sequence was 96% similar to that of Bacillus cereus ATCC 14579(accession number is NC04722) .The fusion protein containing BSCHITO was produced in Escherichia coli and purified using Ni-NTA affinity *** purified rBSCHITO degraded the chitosan(the degree of deacetylation of 99%) to produce mixture of *** BSCHITO is thus an endo-chitosanase.