Enantioselective resolution of (R,S)-DMPM to prepare (R)-DMPM by an adsorbed-covalent crosslinked esterase PAE07 from Pseudochrobactrum asaccharolyticum WZZ003.

(R)-N-(2,6-dimethylphenyl) aminopropionic acid methyl ester ((R)-DMPM) is an important chiral intermediate of the fungicide N-(2,6-Dimethylphenyl)-N-(methoxyacetyl)-alanine methyl ester ((R)-Metalaxyl). In this study, (1) D3520 (macroporous acrylic anion resin), selected from the ten resins, was used to immobilize the esterase from Pseudochrobactrum asaccharolyticum WZZ003 (PAE07) for resoluting the (R,S)-DMPM to obtain (R)-DMPM. (2) Up to 20 g/L PAE07 could be immobilized onto D3520 with a high enzymatic activity of 32.4 U/g. Moreover, the Km and Vmax values of 19.1 mM and 2.8 mM/min for D3520-immobilized PAE07 indicated its high activity and stereoselectivity. (3) The optimal temperature and pH for the immobilized PAE07 were 40 ℃ and 8.0, and substrate concentration was up to 0.35 M. After 15 h reaction, the conversion rate from (R,S)-DMPM to (R)-DMPM was 48.0% and the e.e.p and E values were 99.5% and 1393.0, respectively. In scale-up resolution, 200 g/L substrate and 12.5 g immobilized esterase PAE07 condition, a conversion rate from substrate to product of 48.1% and a product e.e.p of 98% were obtained within 12 h, with the activity of immobilized PAE07 retained 80.2% after 5 cycles of reactions. These results indicated that the D3520-immobilized esterase PAE07 had great potential for enzymatic resolution of (R,S)-DMPM to prepare (R)-Metalaxyl.

Purification, identification and characterization of an esterase with high enantioselectivity to (S)-ethyl indoline-2-carboxylate.

OBJECTIVE: To purify an esterase which can selectively hydrolyze (R,S)-ethyl indoline-2-carboxylate to produce (S)-indoline-2-carboxylic acid and characterize its enzymatic properties. RESULTS: An intracellular esterase from Bacillus aryabhattai B8W22 was isolated and the purified protein was identified as a carboxylesterase by MALDI-TOF mass spectrometry. The enzyme (named BaCE) was 59.03-fold purification determined to be of approximately 35 kDa. Its specific activity was 0.574 U/mL with 20% yield. The enzyme showed maximum activity at pH 8.5 and 30 °C and was stable at 20-30 °C using pNPB as the substrate. The Km, Vmax, kcat and kcat/Km of the esterase were 0.52 mM, 6.39 µM/min, 26.87 min-1 and 51.67 mM/min, respectively. The esterase demonstrated high enantioselectivity toward (S)-ethyl indoline-2-carboxylate with 96.55% e.e.p at 44.39% conversion, corresponding to an E value of 133.45. CONCLUSIONS: In this study, a new esterase BaCE with an apparent molecular mass of 35 kDa was purified to homogeneity for the first time. The esterase from Bacillus aryabhattai B8W22 was isolated with a purification more than 59-fold and a yield of 20% by anion exchange chromatography and hydrophobic interaction chromatography. And its biochemical characterization were described in detail with pNPB as substrate. It displayed high enantioselectivity toward (S)-ethyl indoline-2-carboxylate. We next plan to highly express esterase BaCE in Escherichia coli, and apply it to industrial production of (S)-indoline-2-carboxylic acid.

High-level expression and characterization of a stereoselective lipase from Aspergillus oryzae in Pichia pastoris.

Pichia pastoris expression is a mature and efficient eukaryotic expression system. In this work, Aspergillus oryzae lipase (AOL, with the molecular mass of 28 kDa), which can perform highly stereoselective hydrolysis of (R, S)-methyl 2-(4-hydroxyphenoxy) propanoate, was expressed in P. pastoris X-33. The specific activity of AOL was 432 U/mg, which was obtained by fed-batch cultivation in a 5 L bioreactor using a methanol feeding strategy. After fermentation, the supernatant was concentrated by ultrafiltration with a 10 kDa cut-off membrane and purified with DEAE-Sepharose™ FF ion-exchange chromatography and phenyl Seflnose™ 6 FF hydrophobic interaction chromatography. The purified lipase activity reached 5509 U/mg. AOL showed high activity toward short-chain triacylglyceride (C4), and the optimum temperature and pH were 40 °C and 8.0, respectively. The purified enzyme activity was inhibited by Zn2+ and Cu2+. Moreover, the Km and Vmax values were 1 mM and 32.89 mmol/min, respectively.

Rapid synthesis of flavone-based monoamine oxidase (MAO) inhibitors targeting two active sites using click chemistry.

A new library of flavone derivatives targeting two active sites of monoamine oxidases ("aromatic cage" and substrate cavity) were designed and synthesized using click chemistry (CuAAC reaction) between 6-N3 -2-phenyl chromones (Az1-Az2) and a series of alkynes (k1-k20). Their inhibitory activities against MAO isoforms (MAO-A and MAO-B) are evaluated. Compounds with fluorine, amide bonds, or amino bonds have shown better inhibition. The most potent flavone MAO inhibitor studied is Az2k19 (1.6 µm for MAO-A, 2.1 µm for MAO-B), while Az1k15 and Az2k15 displayed better selectivity toward MAO-B (SI > 10). Docking studies are in accordance with our hypothesis that these inhibitors are most likely located at both the substrate cavity and the "aromatic cage". Our results show that it is considerable to develop new MAO inhibitors from C6 substitution of flavone derivatives and that these compounds are also potential for the treatment of diseases associated with MAOs.

Influence of ammonium salts on the lipase/esterase activity assay using p-nitrophenyl esters as substrates.

p-Nitrophenyl esters with a short-chain carboxylic group, such as p-nitrophenyl acetate (p-NPA) and p-nitrophenyl butyrate (p-NPB), could be effectively hydrolyzed by ammonium salts. p-Nitrophenyl esters were usually used as substrates to assay the lipase/esterase activity. Ammonium sulfate precipitation was often used to purify proteins, and some ammonium salts were usually used as nitrogen sources or inorganic salts for the lipase/esterase production. To study the effect of ammonium salts on the assay of the lipase/esterase activity, the contributing factors of hydrolysis of p-NPA/p-NPB catalyzed by ammonium salts were investigated. The lipase activities were compared in the presence and absence of ammonium sulfate. The hydrolysis reaction could be catalyzed under neutral and alkaline circumstances. The hydrolysis rate increased with the increase in the reaction temperature or the concentration of ammonium ion. When p-NPA was employed as the substrate for the analysis of the lipase/esterase activity, the effect of ammonium sulfate on the analysis could be neutralized by setting a control when the concentration of ammonium sulfate was less than 40% saturation. However, when the concentration of ammonium sulfate increased from 40% to 100% saturation, the enzyme activities decreased about 13-40%, which could not be ignored for accurate analysis of the enzyme activity.

N-(2-Iodo-phen-yl)benzene-carbox-imid-amide.

The title compound, C(13)H(11)IN(2), crystallizes with two independent molecules (A and B) in the asymmetric unit. The two aromatic rings are inclined to one another by 73.3 (2)° in molecule A, and by 74.4 (1)° in molecule B. In molecule A, the iodophenyl and the phenyl rings are inlclined to the N=C-N plane by 88.0 (4) and 19.0 (4)°, respectively. In molecule B the corresponding angles are 85.0 (4) and 20.7 (4)°, respectively. In the crystal, the two molecules are not parallel but have a dihedral angle between the iodophenyl rings of 8.6 (1)°, and 44.5 (2)° between the phenyl rings. The A and B molecules are linked vvia N-H⋯N hydrogen bonds to form -A-B-A-B- chains propagating along direction [100].

4,5,6,7-Tetra-chloro-N-(2,3,4-trifluoro-phen-yl)phthalimide.

The asymmetric unit of the title compound, C(14)H(2)Cl(4)F(3)NO(2), contains two independent mol-ecules. In each mol-ecule, the phthalimide ring system is nearly planar [maximum atomic deviation = 0.031 (2) or 0.038 (2) Å] and oriented with respect to the benzene ring at 65.04 (7) or 71.76 (10)°. Weak inter-molecular C-H⋯O and C-H⋯F hydrogen bonding is present in the crystal structure.