Lüthi E, Jasmat N B, Bergquist P L
Department of Cellular and Molecular Biology, University of Auckland, New Zealand.
Appl Environ Microbiol. 1990 Sep;56(9):2677-83. doi: 10.1128/aem.56.9.2677-2683.1990.
A xylanase encoded by the xynA gene of the extreme thermophile "Caldocellum saccharolyticum" was overexpressed in Escherichia coli by cloning the gene downstream from the temperature-inducible lambda pR and pL promoters of the expression vector pJLA602. Induction of up to 55 times was obtained by growing the cells at 42 degrees C, and the xylanase made up to 20% of the whole-cell protein content. The enzyme was located in the cytoplasmic fraction in E. coli. The temperature and pH optima were determined to be 70 degrees C and pH 5.5 to 6, respectively. The xylanase was stable for at least 72 h if incubated at 60 degrees C, with half-lives of 8 to 9 h at 70 degrees C and 2 to 3 min at 80 degrees C. The enzyme had high activity on xylan and ortho-nitrophenyl beta-D-xylopyranoside and some activity on carboxymethyl cellulose and para-nitrophenyl beta-D-cellobioside. The gene was probably expressed from its own promoter in E. coli. Translation of the xylanase overproduced in E. coli seemed to initiate at a GTG codon and not at an ATG codon as previously determined.
通过将嗜热栖热放线菌（“Caldocellum saccharolyticum”）xynA基因编码的木聚糖酶基因克隆到表达载体pJLA602的温度诱导型λ pR和pL启动子下游，使其在大肠杆菌中过表达。在42℃培养细胞可实现高达55倍的诱导，且木聚糖酶占全细胞蛋白质含量的20%。该酶定位于大肠杆菌的细胞质部分。确定其最适温度和pH分别为70℃和pH 5.5至6。如果在60℃孵育，木聚糖酶至少稳定72小时，在70℃半衰期为8至9小时，在80℃半衰期为2至3分钟。该酶对木聚糖和邻硝基苯基β - D - 吡喃木糖苷具有高活性，对羧甲基纤维素和对硝基苯基β - D - 纤维二糖苷具有一些活性。该基因可能在大肠杆菌中从其自身启动子表达。在大肠杆菌中过量产生的木聚糖酶的翻译似乎起始于GTG密码子，而不是如先前确定的起始于ATG密码子。
