Screening Of Microorganisms And Its Application On Asymmetric Synthesis Of Chiral Piperidinol Via Biocatalysis

Many bioactive compounds?natural or non-natural?have nitrogen-containing heterocyclic structure.Chiral hydroxy group in the piperidine ring of hexa-nitrogenous heterocycle compounds plays an important role in their biological activity.Chemical methods for the preparation of chiral piperidinol usually require harsh operating conditions and have lower stereoselectivity in comparison with enzymatic methods.Here,an excellent biocatalyst—Rhodococcus erythropolis WZ010 was isolated and demonstrated the potentials on the asymmetric reduction of N-Boc-3-piperidonetoopticallypure?S?-N-Boc-3-piperidinol.Furthermore,the targeted gene encoding for N-Boc-3-piperidone reductase?ReADH?from R.erythropolis WZ010 was cloned and over-expressed in Escherichia coli BL21?DE3?.In addition,enzymatic properties of ReADH were characterized,and enzymatic and whole-cell catalyzed biocatalysis for asymmetric reduction of N-Boc-3-piperidone was investigated in details.The results are as follows:1.The isolated biocatalyst R.erythropolis WZ010 asymmetrically reduced N-Boc-3-piperidone to?S?-N-Boc-3-piperidinol with high e.e.value of 99%,following the Prelog's rule.The optimum temperature for N-Boc-3-piperidone reduction was 30?.The stain showed high catalytic activity within the pH range from 7.5 to 10.0.The reaction required no exogenous coenzyme and the product yield was significantly improved when isopropanol was used as co-substrate in the reaction.R.erythropolis WZ010 also showed good stereoselectivity in the reduction of N-Boc-3-pyrrolidinone to?S?-N-Boc-3-Pyrrolidinol,offering the product e.e.value of 97.8%.2.The gene readh encoding N-Boc-3-piperidone reductase from R.erythropolis WZ010 was cloned and over-expressed in E.coli BL21?DE3?.The structure analysis of ReADH showed that the enzyme belonged to medium-chain alcohol dehydrogenase family and was homotetrameric.The subunit size of ReADH was around 38kDa,and each subunit contained two binding sites of catalytic and structural zincs.The enzyme showed the highest specific activity of 207.5U/mg at 60?and pH 6.0 and demonstrated an e.e.value of>99%in the N-Boc-3-piperidone reduction.The values of V_max and k_cat/K_m reached 270.3U/mg and 529.1s^-1mM^-1,respectively.The enzyme specific activity for ketone reduction was much higher than that of alcohol oxidation.Particularly,the activity for?S?-N-Boc-3-piperidinol oxidation was barely detectable.Therefore,ReADH could also be defined as piperidone reductase.3.When isopropanol was used as co-substrate for coenzyme regeneration,both the purified enzyme ReADH and whole cells of pEASY-E₂-readh/E.coli BL21?DE3?showed great efficiency in asymmetric synthesis of?S?-N-Boc-3-piperidinol.The purified ReADH-catalyzed system consisted of 10U ReADH,100mM N-Boc-3-piperidone,4%?v/v?isopropanol and 0.1mM NAD⁺.After 3h of reaction under the conditions of37?,pH 8.5 and 200rpm,the space-time yield of the product reached up to147g L^-1d^-11 and the TTN value was as high as 920.The whole-cell catalyzed system consisted of 1.0g wet cells,10%?w/v?N-Boc-3-piperidone?500mM?and 2.5M isopropanol.After 6h of reaction under the conditions of 37?,pH 8.5 and 200rpm,the space-time yield of the product and TTN value were 299.4g L^-1d^-1 and 936.3,respectively.Taken together,TTN values from both enzymatic and whole-cell catalyzed approaches were satisfactory,indicating that coenzyme recycling regeneration was effective.