Engineered purine nucleoside phosphorylase variant enzymes
一种化嘌呤核苷磷酸化酶变体、化嘌呤核苷磷酸化酶的技术，应用在工程化嘌呤核苷磷酸化酶变体酶领域，能够解决核苷类似物化学复杂性等问题
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Embodiment 1
[0204] Preparation of wet cell pellets containing HTP-PNP
[0205] The parental gene for the PNP (SEQ ID NO:2) enzyme used to generate the variants of the present invention was obtained from the E. coli genome and cloned into the pCK110900 vector. W3110 E. coli cells were transformed with the corresponding plasmid containing the PNP-encoding gene, plated on LB agar plates containing 1% glucose and 30 μg / ml chloramphenicol (CAM), and grown overnight at 37°C. Pick a monoclonal colony and inoculate into 180 μl of LB containing 1% glucose and 30 μg / mL chloramphenicol, and place in a well of a 96-well shallow-well microtiter plate. Use the plate with O 2 A permeable seal was sealed and the culture was grown overnight at 30°C, 200 rpm and 85% humidity. Then, 10 μl of each cell culture was transferred to wells of a 96-well deep-well plate containing 390 μl TB and 30 μg / ml CAM. with O 2 Seal the deep well plate with a permeable seal and incubate at 30 °C, 250 rpm and 85% humidity ...
Embodiment 2
[0207] Preparation of cell lysates containing HTP PNP
[0208] Use 200 μl buffer containing 100 mM triethanolamine pH 7.5, 1 mg / mL lysozyme, 0.5 mg / mL PMBS and 5 mM MnCl 2 Lysis buffer prepared as described in Example 1 was used to lyse the cryoprecipitate. The cleavage mixture was shaken for 2 hours at room temperature. Plates were then centrifuged at 4000 rpm and 4°C for 15 min. The supernatants were then used as clarified lysates in biocatalytic reactions to determine activity levels.
Embodiment 3
[0210] Preparation of lyophilized lysates from shake flask (SF) cultures
[0211] Single colonies containing the desired gene picked from LB agar plates with 1% glucose and 30 μg / ml CAM and incubated overnight at 37°C were transferred to 6 ml LB with 1% glucose and 30 μg / ml CAM. Grow the culture at 30 °C, 250 rpm for 18 h, and subculture into 250 ml of TB containing 30 μg / ml CAM at about 1:50 to reach a final OD of about 0.05 600 . Grow the subculture at 30 °C, 250 rpm for approximately 195 min to reach an OD between 0.6-0.8 600 , and induced with 1 mMIPTG. Subcultures were then grown for 20 h at 30°C, 250 rpm. The subculture was centrifuged at 4000 rpm for 20 min. Discard the supernatant, and resuspend the pellet in 35 ml with 5 mM MnCl 2 25 mM triethanolamine buffer pH 7.5. use Cells were lysed by a processor system (Microfluidics) at 18,000 psi. The lysate was precipitated (10,000 rpm x 60 min), then the supernatant was frozen and lyophilized to produce a shake fla...
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