Preparation method and application of new enzyme strains and enzyme catalysts
A bacterial strain and restriction endonuclease technology, applied in the fields of biological enzyme engineering and microbial application, to achieve the effects of simplified preparation, reduced environmental pollution, and uncomplicated steps
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Embodiment 1
[0053] Example 1: Construction of prokaryotic expression system
[0054] Ketoreductase Lk Kred gene fragment was synthesized by Nanjing GenScript Biotechnology Co., Ltd., and recombined into pET21a vector. The positive recombinant plasmid Lk Kred-pET21a (+) was transformed into the expression host strain BL21 (DE3) (purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.), and the prokaryotic expression strain Lk Kred-pET21a (+) / BL21 (DE3) was obtained. Primary strains used as subsequent catalytic reactions.
[0055] The glucose dehydrogenase (BsGDH) and alcohol dehydrogenase (TbADH) genes used for coenzyme regeneration were synthesized by Nanjing GenScript Biotechnology Co., Ltd. The construction of the subsequent recombinant expression plasmid was the same as that of the Lk Kred-pET21a(+) plasmid , BsGDH-pET21a(+) / BL21(DE3), TbADH-pET21a(+) / BL21(DE3) expression strains were respectively obtained after being transferred into BL21(DE3).
Embodiment 2
[0056] Embodiment two: the fermentation preparation of enzyme
[0057] The expression strains Lk Kred-pET21a(+) / BL21(DE3), BsGDH-pET21a(+) / BL21(DE3) and TbADH-pET21a(+) / BL21(DE3) constructed above were added with a final concentration of 100ug / mL Ampicillin in 5mL LB liquid medium [10g / L tryptone (OXIOD), 5g / L yeast powder (OXIOD), 10g / L sodium chloride (Sinopharm Reagent)] was shaken at 37°C and 200rpm overnight, then press The 1% (V / V) ratio was inoculated in 500 mL LB liquid medium containing ampicillin with a final concentration of 100 ug / mL, and cultured with shaking at 37 ° C and 200 rpm. When the OD600 was between 0.8-1.0, the inducer IPTG (isopropyl-β-D-thiogalactopyranoside, IPTG) was added at a final concentration of 0.1 mM, and induced overnight at 25°C. The bacteria were collected by centrifugation at 8000rpm, then suspended in 50mM pH7.0 sodium phosphate buffer, ultrasonically disrupted (200W, 3s / 5s, 10min), centrifuged at 12000rpm at 4°C for 20min, and the super...
Embodiment 3
[0058] Embodiment three: detection of the catalytic activity of enzyme
[0059] The enzyme solution obtained above is used for the substrate-catalyzed reaction. Dissolve 1g of substrate in 8mL of 50mM pH7.0 sodium phosphate buffer containing 10% isopropanol, then add 1.5g of glucose, stir and dissolve, add 2mg of NADP+, 2ml of enzyme solution, and replenish the volume to 10mL with buffer. The reaction solution was placed in a constant temperature water bath at 37°C, and stirred by magnetic force for reaction. During the reaction, the pH of the system was maintained at 7.0 with 1M sodium hydroxide solution. After 24 hours of reaction, samples were taken and detected by HPLC. The conversion rate of the substrate reached 96%, and the chiral purity value of the product was >98%.
[0060] Coenzyme regeneration using alcohol dehydrogenase: dissolve 1g of substrate in 8mL of 50mM pH7.0 sodium phosphate buffer containing 10% isopropanol, add 2mg of NADP+ and 2ml of enzyme solution a...
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