J. Technology and Education, Vol.17, No.1, pp.35-43, 2010
研究論文
Bacillus cereus新規株のスクリーニング
およびコルテックス溶解酵素のクローニングと解析
川村 敏之*，安田 太樹
福井工業高等専門学校物質工学科（〒916-8507福井県鯖江市下司町）
*kawamura@fukui-nct.ac.jp
Cloning and Analyzing of Cortex Lytic Enzyme from the novel strain of Bacillus cereus
Toshiyuki KAWAMURA* and Taiki YASUDA
Department of Chemistry & Biology Engineering, Fukui National College of Technology
(Geshi-cho, Sabae-shi, Fukui 916-8507, Japan)
(Received February 3, 2010; Accepted June 1, 2010)
Abstract
Bacillus and Clostridium form endospore which enable to survive themselves from environmental stress such as high temperature and chemical damage. The endospore is covered with the cortex layer consist of thick peptidoglycan. In cooking and food manufacturing, despite the heat- treated food rarely remain the endospore and cause food poisoning.
In this study, we initially screened Bacillus cereus from the water of medaka breeding aquarium and cloned by sowing the LB agar plate after treatment of saturated urea solution. We isolated of genomic DNA from screened bacteria and amplified 1.4kbp DNA sequence coding 16s-rRNA by PCR. The amplified fragment was determined sequense and queried in the database . As a result of the above search, the screened bacteria showed high homology to Bacillus cereus.
Secondary we approach to understand the mechanism of spore germination using B.cereus which originally screened. The cortex lytic enzyme (CLE) is known that it drills in the cortex layer consisted thick peptidoglycan during germination. We cloned CLE gene from screened B.cereus by PCR amplification and determined the sequense. As a result of querying, the cloned CEL has high homology and several mutation with presently known.
We also cloned CLE gene into the arabinose-inducible vector to product recombinant CLE protein in E.coli. The recombinant E.coli produced the 70kDa protein speculative CLE. But the most of production found degradation fragment. Endospore and proteins stored in it can endure the stress, such as heat and drugs. However our study indicates that protein itself was unstable and susceptible to degradation.
Keywords:
Bacillus cereus, cortex lytic enzyme (CLE), endospore
