Biosynthesis Chiral Intermediate Of Duloxetine

(S)-Duloxetine known as a inhibitor of 5-HT and NE receptors has strikingly curative effect in the treatment of Major Depression,Diabetic Peripheral NeuroPathic Pain,Female Stress Urinary Incontinence,Fibromyalgia Syndrome,Premenstrual Dysphoric Disorder.Comparing to traditional chemical metods biosynthesis?S?-DMAA owns advantages like excellent product optical purity,a much smaller pollution,and mild reaction conditions.Humble catalytic efficiency due to low expression of ketoreductase in wild strains,high costs owing to lack of co-enzyme regeneration system,and low conversion because of substrate inhibition largely hinder it applies to practical production.In order to deal problems in biocatalysis,we constructed two recombinat strains and developed two noval methods of biosynthesis?S?-DMAA based on substrate-coupling and enzyme-copling.We optimized expression conditions of recombination engineering strains to make sure two kinds ketoreductase soluble high expression.In addition,recombination engineering strains can concurrently achieve substrate reduction and coenzyme regeneration with help of substrate-coupling and enzyme-coupling coenzyme system in synthesis of?S?-DMAA,which makes it easy to lower the production cost.Finally to improve catalytic efficiency and conversion,reaction conditions are optimized.Solutions specifically including:1.Construction of recombinant plasmids and optimization of expression conditions.Alcohol dehydrogenase from Lactobacillus kefir?Lk-ADH?,Rhodosporidium toruloides?Rt-ADH?were chosen as ketoredutase and glucose dehydrogenase from Bacillus megaterium were chosen as co-enzyme regenerated enzyme.In order to acquire recombinant plasmids of pET-28?-lk-adh,pET28?-rt-adh and pET28?-gdh,intact gene fragments were synthesized and recombinated with pET-28a.Optimal expression conditions of recombinant plasmids are Lk-ADH with induction time of 48 h under 15?,0.4 mM IPTG,obtained maximum enzyme activity 524 U/L and protein concentration of 1.41 mg/mL.Rt-ADH with induction time of 24 h under 30?,0.4mM IPTG,obtained maximum enzyme activity 813 U/L and protein concentration of1.85 mg/mL.SDS-PAGE results shown that two kinds ketoreductase own high level of soulable expression.2.Asymmetric biosynthesis of?S?-DMAA.Optimization of reaction temperatu re,substrate concentration,co-enzyme concentration and co-substrate concentration were carried out.Isopropanol and glucose dehydrogenase are respectively used in substrate-coupling and enzyme-coupling co-enzyme regeneration system.Lk-ADH with 4 hours catalysis,under optimal reaction conditions of 50?,0mM extra co enzyme,pH7,16.6%?V/V?isopropanol,and 1.6:1substrate/cell mass ratio possess ed 99.4%conversion according to HPLC analysis?substrate loading:72 mM?and85.3%final yield..Rt-ADH with 4 hours catalysis,under optimal reaction condit ions of 30?,0mM extra co-enzyme,pH 7,134 mM glucose,2.4:1substrate/cell mass ratio possessed 99.6%conversion according to HPLC analysis?substrate lo ading:100mM?and 85.8%final yield.FT-IR,LC-MS,¹H NMR,¹³C NMR and c hiral HPLC analysis all shown that extraction products share identical molecular s tructure and optical properties with target product.