Study On Resolution Of Chiral Aromatic Amine Compounds By Microbial Enzyme

Chiral aromatic amine compounds are not only used as the important intermediate for pharmaceuticals, but also as resolving agents and chiral auxiliaries. In this paper, we tried to isolate microorganisms producing enzyme with high stereoselectivity which can be used in resolution of chiral aromatic amine. The purpose was to provide a new, simpler and environmentally friendly method for preparation of chiral aromatic amine.A strain producing lipase was isolated from the soil samples. The lipase activity of this wild strain was172U/L after fermentation of36h. It was characterized and identified as Bacillus licheniformis J03. The fermentation experiments were carried out to optimize the lipase-producing conditions of J03. The cultivation medium consisted of sucrose1.2%, yeast extract0.5%, peptone0.5%, olein0.4%, K2HPO40.2%, NaCl0.5%, MgSO4-7H2O0.05%, pH7.0, and the culture condition were temperature30℃, shaking rate200rpm. Under the acquired conditions, the highest activity of lipase by J03was579U/L,3.37times higher than the original activity. Resolution process of the1-phenylethylamine with resting cells of J03were investigated. The biotransformation conditions were optimized:methyl tert-butyl ether (MTBE) as solvent,1-phenylethylamine concentration0.5%, methyl methoxyacetate concentration0.5%, resting cells of J030.02g/mL, temperature30℃, shaking rate200rpm, reaction time24h. Under the conditions mentioned above, the enantiomeric excess against1-phenylethylamine was33.5%and its conversion was as high as36.6%.The hydrolysis of2-chloro-N-(1-(4-chlorophenyl)ethyl) acetamie catalyzed by Aspergillus niger M041was carried out. The fermentation experiments were carried out by Plackett-Burman design and response surface methodology to optimize the enzyme production condition. Cultivated in the optimal condition:glucose1.8%, peptone1.79%, K2HPO40.2%, MgSO40.1%, NaCl0.5%, Al3+1.51mmol/L, no pH adjustment, the enzymatic activity of Aspergillus niger M041was48.9U/L, increased by50.9%.The enzymatic hydrolysis conditions were investigated:MTBE containing3%water as solvent, resting cells of M0410.02g/mL, substrate concentration0.2%, temperature35℃, shaking rate200rpm, reaction time24h. Under the optimal conditions mentioned above, the enantiomeric excess against substrate was36.4%and its conversation was as high as41.3%.