Construction Of A Genomic DNA Library Of Pyrococcus Horikoshii OT₃ And Screening Of The Novel Enzymes

Hyperthermophiles, represented by bacterial and archaeal species, grow optimally at temperatures between 80℃ and 110℃. These organisms have been isolated from all types of terrestrial and marine hot environments, including natural and man-made environments. Hyperthermophilic enzymes developed unique structure-function properties of high thermostability and optimal activity at temperatures above 80℃. Some of these enzymes are active at temperatures as high as 110℃ and above. Hyperthermophilic enzymes have become model systems to study enzyme evolution, enzyme stability and activity mechanisms, protein structure-function relationships, and biocatalysis under extreme conditions. It is quietly valuable to exploit the enzymes, which have the degradation for pesticide from the hyperthermophilic enzymes for the prevention and cure of pollution.Pyrococcus horikoshii OT3 is a kind of hyperthermophilic archaea. It grows with an optimal temperature of 98℃. 90.72% of the total genome of pyrococcus horikoshii OT3 is the coding region. About 50% of coding gene is no similarity to database. The total genome length of pyrococcus horikoshii OT3 is 1738505bp.To study the coding genes that are no similarity to database, the genome library from the pyrococcus horikoshii OT3 was established firstly. The total DNA of pyrococcus horikoshii OT3 was extracted and incomplete digested with Sau3A I .2-6kb fragment was recovered by agarose gel DNA purification kit and cloned into pET15b vector. The recombinant vectors were transformed into E. coli BL21 (DE3) Codon Plus competent cells.7144 transductants were selected. The probability of finding a specific gene from the genome library was over 99.9%. The first target screened was esterase, because we found five genes with lipase motif from Aeropyrum pernix K1 that also is a kind of hyperthermophilic archaea. Esterases are widely distributed in animals, plants and microorganisms. For their activities in both aqueous and nonaqueous solvent systems, esterases have developed into the most widely used class of enzymes in various industrial processes, finding use in stereospecific hydrolysis, transesterification, ester synthesis, modification of physicochemical properties of triglycerides for the fats and other organic biosynthesis reactions.As the positive control, APE1547 which is a esterase come from Aeropyrum pernix K1 was used for establishing a system with substrate colored to screen the genome library. The genome library was conserved into 96-well microplates, which could be screened by Multiskan Ascent. Moreover, passing by reproducing the library, inducting to express the protein, collecting the cells, crumbling the cells by freezing and thawing reduplicative and heat inactivation, the suspension of every single clone was obtained. The suspension was mixed with p-nitrophenol octoate as substrate into the 96-well microplates. The temperature of esterase reaction was setted at 75oC. Fast scanning OD405 by Multiskan Ascent after reaction was proceeded to screen the positive clones. By the screening to the positive control, the screen system was confirmed to be accurate, significant and feasible.The genome library also was blended and parted to conserve at -80oC. The blended library could be screened by nitrocellulose film. With Naphthol AS Nonanoate as substrate and Fast Blue BB Salt as the color-developing reagent, the screen system has preliminarily procured achievement.