Wang Yapin, Liu Yihan, Wang Zhengxiang, Lu Fuping
Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education, Tianjin, 300457, People's Republic of China,
Biotechnol Lett. 2014 Sep;36(9):1783-9. doi: 10.1007/s10529-014-1538-x. Epub 2014 May 4.
To achieve efficient expression and secretion of a biologically-active pullulanase, the effect of promoter and signal peptide on the production of pullulanase was studied. Three types of promoters (PP43, P apr and P amy ) and four types of signal peptides (SP sacB , SP amy , SP aprl and SP aprs ) were combined to construct twelve expression cassettes for pullulanase in Bacillus subtilis. The pullulanase activity assay was employed to quantify the level of differential expression, and a real-time PCR assay was applied to comparatively track the transcriptional level. Under the same experimental conditions, the potency ratios among the three promoters were P apr > P amy > PP43. The secretion efficiency ratios mediated by the signal peptides were SP sacB > SP amy > SP aprs > SP aprl . The highest yield of pullulanase could be achieved under the promotion mediated by P apr and secretion by SP sacB .
为实现具有生物活性的普鲁兰酶的高效表达与分泌，研究了启动子和信号肽对普鲁兰酶产生的影响。将三种类型的启动子（PP43、P apr和P amy）和四种类型的信号肽（SP sacB、SP amy、SP aprl和SP aprs）进行组合，构建了12个用于在枯草芽孢杆菌中表达普鲁兰酶的表达盒。采用普鲁兰酶活性测定法对差异表达水平进行定量，并应用实时PCR测定法对转录水平进行比较跟踪。在相同实验条件下，三种启动子的效力比为P apr > P amy > PP43。信号肽介导的分泌效率比为SP sacB > SP amy > SP aprs > SP aprl。在P apr的促进作用和SP sacB的分泌作用下，可实现普鲁兰酶的最高产量。
