Croux C, García J L
Département de Génie Biochimique et Alimentaire, Institut National des Sciences Appliquées, Toulouse, France.
FEMS Microbiol Lett. 1992 Aug 1;74(1):13-20. doi: 10.1016/0378-1097(92)90730-c.
The complete lyc gene encoding the autolytic lysozyme of Clostridium acetobutylicum ATCC 824 was reconstructed from two overlapping DNA fragments and cloned into a suitable plasmid enabling Escherichia coli to produce this lytic enzyme under the control of the lac promoter. A polypeptide with an apparent M(r) of 35,000, corresponding to that predicted from the nucleotide sequence, was observed by maxicell analysis of whole-cell extracts of E. coli harboring the clostridial gene. The enzyme yield was shown to depend on the pH of the culture medium, since the protein was unstable at alkaline pH. The expression of the lyc gene was not increased by using the E. coli strong promoter, lpp-lac, probably due to the limit imposed by the extreme differences in codon usage. Although the LYC lysozyme does not contain a cleavable signal peptide, most of the protein was found in the periplasmic fraction of E. coli suggesting that this enzyme was secreted through a specific mechanism, as already observed for other autolysins.
编码丙酮丁醇梭菌ATCC 824自溶溶菌酶的完整lyc基因由两个重叠的DNA片段重建，并克隆到合适的质粒中，使大肠杆菌能够在lac启动子的控制下产生这种裂解酶。通过对携带梭菌基因的大肠杆菌全细胞提取物进行大细胞分析，观察到一种表观分子量为35,000的多肽，与核苷酸序列预测的一致。结果表明，酶产量取决于培养基的pH值，因为该蛋白质在碱性pH下不稳定。使用大肠杆菌强启动子lpp-lac并没有增加lyc基因的表达，这可能是由于密码子使用的极端差异所造成的限制。尽管LYC溶菌酶不包含可裂解的信号肽，但大部分蛋白质存在于大肠杆菌的周质部分，这表明该酶是通过一种特定机制分泌的，这与其他自溶素的情况已观察到的一样。
