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

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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.