Development of heat-labile and psychrophilic enzymes for clean and efficient recombinant DNA experiment

在重组DNA实验中，我们试图开发出不耐热的碱性磷酸酶、BglII和RNaseA，以使这些酶在高温下失活，并分离出嗜冷性DNA连接酶基因，以便在低温下高效连接DNA。在大肠杆菌中大量生产了sibi，对其进行了纯化和鉴定。该酶在40℃时活性最高。与大肠杆菌APase相比，该最适温度下移了20℃。在80℃孵育时，Sibi APase几乎完全失活。而大肠杆菌APase在80℃下孵育1h仍能保持60%的活性。在40℃和20℃时，Sibi APase的klt；cat&gt；值分别是大肠杆菌APase的6.2倍和9.6倍。SIB1.根据与新城疫志贺氏菌紧密相关的DNA连接酶基因序列，设计了一对聚合酶链式反应引物。SIB1.以MR-1基因组DNA为模板，设计引物，通过聚合酶链式反应扩增出一条600bp的DNA片段。该DNA片段编码一个氨基酸序列，与MR-1 DNA连接酶有77%的同源性。为了筛选Bgl II和RNaseA的不耐热突变体，我们试图通过在外壳蛋白PIII基因的5‘端插入这些酶的N端带有6×His标签的基因来展示这些酶在M13噬菌体上的表达。从携带这些杂交基因的大肠杆菌菌株中生产噬菌体颗粒，并分析其与Ni-NTA平板的结合。然而，没有观察到特异性结合，可能是因为这些酶的噬菌体展示没有成功。

We tried to develop heat-labile alkaline phosphatase, BglII, and RNase A to inactivate these enzymes by heat in recombinant DNA experiment, and to isolate psychrophilic DNA ligase gene for efficient DNA ligation at low temperatures.An alkaline phosphatase (APase) from psychrotrophic bacterium Shewanella sp. SIBI was overproduced in E. coli, purified, and characterized. The enzyme exhibited the highest activity at 40℃. This optimum temperature is shifted downward by 20℃, as compared to that of E. coli APase. SIBI APase was almost fully inactivated upon incubation at 80℃. for 5min, whereas E. coli APase retained 60% of the activity upon incubation at 80℃ for 1h. The k_<cat> value of SIBI APase was higher than that of E. coli APase by 6.2 times at 40℃ and 9.6 times at 20℃.A part of DNA ligase gene was isolated from Shewanella sp. SIB1. A pair of PCR primers was designed based on the DNA sequence of the DNA ligase gene of Shewanella oneidensis MR-1, which is closely related to Shewanella sp. SIB1. A 600-bp DNA fragment was amplified by PCR using MR-1 genomic DNA as a template and the designed primers. The DNA fragment encoded an amino acid sequence with 77% identity to MR-1 DNA ligase. Isolation of the entire gene is now under progress.To select heat-labile mutant of Bgl II and RNase A, we tried to display these enzymes on M13 phage by inserting the genes of these enzymes with a 6 × His-tag at their N-termini to the 5'-terminus of the coat protein pIII gene. Phagemid particles were produced from E. coli strains harboring these hybrid genes, and were analyzed for the binding to a Ni-NTA plate. However, no specific binding was observed, possibly because phage display of these enzymes was unsuccessful.