To express feruloyl esterase A from Aspergillus oryzae in Pichia pastoris expression system and study its hydrolysis function, explore the conditions and effects of purification for ferulic acid extracts by macroporos resin. Using the total RNA from A. oryzae CICC 40186 as the template, we amplified coding sequence AorfaeA encoding a mature feruloyl esterase A (AorFaeA) by RT-PCR technique. Then, the coding sequence AorfaeA was successfully expressed in Pichia pastoris GS115 mediated by an expression plasmid pPIC9K. The purified recombinant AorFaeA (reAorFaeA) showed one single band on SDS-PAGE with an apparent molecular weight of 39.0 kDa. The maximum activity of reAorFaeA to methyl ferulate, measured by high-performance liquid chromatography (HPLC), was 58.35 U/mg. Then, reAorFaeA was used to release ferulic acid from de-starched wheat bran in the presence of xylanase. The purification tests for ferulic acid from the enzymatic hydrolysate were carried out with preselected macroporous resins. The results showed that macroporous resin HPD-300 had much higher adsorption and desorption capacities. Ferulic acid could be quantitatively recovered by 50% of the eluent concentration at a flow speed of 1 mL/min. Under the purification condition, the recovery ratio of ferulic acid was 92%, and the content of ferulic acid was increased from 0.13% in the raw material to 10.55%. This work exploits the breakdown of ferulic acid by recombinant enzymeand provids a good strategy to its "green production".
Aspergillus oryzae ; enzymology ; Carboxylic Ester Hydrolases ; biosynthesis ; genetics ; Cloning, Molecular ; Coumaric Acids ; chemistry ; Electrophoresis, Polyacrylamide Gel ; Hydrolysis ; Molecular Weight ; Pichia ; genetics ; metabolism

A mesophilic xylanase from Aspergillus oryzae, abbreviated to AoXyn11A, belongs to glycoside hydrolase family 11. Using AoXyn11A as the parent, the thermotolerant hybrid xylanase, we constructed AEx11A by substituting its N-terminus with the corresponding region of a hyperthermostable family 11 xylanase, EvXyn11(TS). AoXyn11A- and AEx11A-encoding genes were expressed in Pichia pastoris GS115 separately, and effects of temperatures on expressed products were determined and compared. The optimum temperature (T(opt)) of AEx11A was 75 degrees C and its half-life at 70 degrees C (t1/2(70)) was 197 min, improved as compared with those (T(opt) = 50 degrees C, t1/2(70) = 1.0 min) of AoXyn11A. Homology modeling of the AEx11A's structure and comparison between structures of AEx11A and AoXyn11A revealed that one disulfide bridge (Cys5-Cys32) was introduced into AEx11A resulted from N-terminus substitution. To explore the effect of the disulfide bridge on the thermostability of AEx11A, it was removed from AEx11A by site-directed mutagenesis (C5T). Analytical results show that the T(opt) of the mutant AEx11A (AEx11A(C5T)) dropped to 60 degrees C from 75 degrees C of AEx11A, and its t1/2(70) and t1/2(80) also decreased to 3.0 and 1.0 min from 197 and 25 min.
Amino Acid Sequence ; Amino Acid Substitution ; Aspergillus oryzae ; enzymology ; Base Sequence ; Disulfides ; chemistry ; metabolism ; Endo-1,4-beta Xylanases ; biosynthesis ; chemistry ; genetics ; Enzyme Stability ; genetics ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; methods ; Pichia ; genetics ; metabolism ; Protein Engineering ; methods ; Recombinant Proteins ; biosynthesis ; chemistry ; genetics

Racemases have been applied for the synthesis of enantiomerically pure compounds through the deracemization methods. Mandelate racemase from Pseudomonas putida was the only enzyme that catalyzes the interconversion of mandelate enantiomers. Using genome mining approaches, we identified 9 mandelate racemases (MRs). A novel racemase named ArMR with higher activity and better soluble protein expression, was isolated from Agrobacterium radiobacter. ArMR displayed the optimum catalytic activity at 50 ℃, pH 7.8 in Tris-HCl buffer. The half-life of ArMR at 50, 40 and 30 ℃ was 0.17, 27.2 and 70.7 h, respectively. KM parameter of ArMR towards (R)- and (S)-mandelic acid was 1.44 and 0.81 mmol/L, respectively; the corresponding kcat value was 410 s⁻¹ and 218 s⁻¹. In addition, KM of ArMR towards (R)- and (S)-2-chloro mandelic acid was 6.48 and 6.37 mmol/L, and the corresponding kcat value 0.22 s⁻¹ and 0.23 s⁻¹, respectively. Meanwhile, Mg²⁺ and Mn²⁺ could activate the enzyme whereas Zn²⁺ inactivated the enzyme completely. Discovery of more novel MRs provides supports further research and development of these racemases.