Tuler T R, Callanan M J, Klaenhammer T R
Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh 27695, USA.
J Dairy Sci. 2002 Oct;85(10):2438-50. doi: 10.3168/jds.S0022-0302(02)74326-9.
Walker and Klaenhammer (2001) developed a novel expression system in Lactococcus lactis that facilitated the release of beta-galactosidase (117 kDa monomer) without the need for secretion or export signals. The system is based on the controlled expression of integrated prophage holin and lysin cassettes via a lactococcal bacteriophage phi31 transcriptional activator (Tac31A) that resides on a high-copy plasmid. Approximately 85% of beta-galactosidase activity was detected in the supernatant of leaky lactococci without evidence of hindered growth, cell lysis, or membrane damage. The objective of this study was to determine if intracellular peptidases were externalized from leaky lactococci. Five L. lactis peptidases (PepA, PepC, PepN, PepO and PepXP) and two Lactobacillus helveticus peptidases (PepN and PepO) were cloned and overexpressed on two high-copy vectors. The lactococcal peptidases were also cloned into the high-copy vector that contained the Tac31A transcriptional activator to determine if they were externalized from the leaky prophage-containing L. lactis subsp. lactis strain NCK203. Two of the lactococcal peptidases (PepA and PepO) required an additional strong promoter (Lactobacillus paracasei P144) and optimized assay conditions to detect enzyme activity. Results showed different levels of enzymatic overexpression associated with the cellular fraction (2 to 250-fold increases in activity) and negligible amounts of activity present within the supernatant fraction (0 to 6% of total peptidase activity). The lactococcal phage-based protein release mechanism did not facilitate the externalization of the lactococcal peptidases investigated in this study.
沃克和克莱恩哈默（2001年）在乳酸乳球菌中开发了一种新型表达系统，该系统有助于β-半乳糖苷酶（117 kDa单体）的释放，而无需分泌或输出信号。该系统基于通过位于高拷贝质粒上的乳酸乳球菌噬菌体phi31转录激活因子（Tac31A）对整合的原噬菌体溶菌蛋白和溶素盒进行可控表达。在渗漏性乳酸乳球菌的上清液中检测到约85%的β-半乳糖苷酶活性，且没有生长受阻、细胞裂解或膜损伤的迹象。本研究的目的是确定细胞内肽酶是否从渗漏性乳酸乳球菌中释放到细胞外。克隆了五种乳酸乳球菌肽酶（PepA、PepC、PepN、PepO和PepXP）和两种瑞士乳杆菌肽酶（PepN和PepO），并在两种高拷贝载体上进行过表达。还将乳酸乳球菌肽酶克隆到含有Tac31A转录激活因子的高拷贝载体中，以确定它们是否从含有渗漏性原噬菌体的乳酸乳球菌亚种乳酸乳球菌菌株NCK203中释放到细胞外。两种乳酸乳球菌肽酶（PepA和PepO）需要额外的强启动子（副干酪乳杆菌P144）和优化的检测条件才能检测到酶活性。结果表明，与细胞组分相关的酶过表达水平不同（活性增加2至250倍），而上清液组分中的活性量可忽略不计（占总肽酶活性的0至6%）。本研究中所研究的基于乳酸乳球菌噬菌体的蛋白质释放机制并未促进乳酸乳球菌肽酶释放到细胞外。
