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

本研究以耐冷细菌希瓦氏菌（Shewanella sp.）西比（SIBI）为出发菌株，在E.大肠杆菌，纯化，并表征。该酶在40℃时活性最高。该最适温度比E. coli APase。西比APase在80℃孵育后几乎完全失活。5 min，而E. coliAPase在80℃保温1h后仍保持60%的活性。西<cat>比APase的k值高于E. coli APase在40℃和20℃下分别扩增6.2倍和9.6倍，并从希瓦氏菌SIB 1中克隆了DNA连接酶基因的一部分。根据希瓦氏菌（Shewanella oneidensis）MR-1的DNA连接酶基因序列设计了一对PCR引物。以MR-1基因组DNA为模板，利用设计的引物进行PCR扩增，得到600 bp的DNA片段。该DNA片段编码的氨基酸序列与MR-1 DNA连接酶的同源性为77%。为了筛选Bgl II和RNase A的热不稳定突变体，我们尝试将这两种酶的基因在其N-末端带有6 × His-标签的情况下插入到外壳蛋白pIII基因的5 '-末端，从而在M13噬菌体上展示这两种酶。噬菌粒由E. coli菌株，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不成功。

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.