Cross‐linked protein complex exhibiting asymmetric oxidation activities in the absence of added cofactor

A protein complex (PC) suspension exhibits asymmetric biooxidation activities in the absence of any added cofactor such as NAD(P) + or FAD. It can be extracted from pea protein (PP)‐gel (PP encapsulated with Ca 2+ alginate gel and aerated in air for several hours) using hot water by rotary shaking and powdered by the following three steps: (1) forming precipitates from the suspension using 30% (w/v) aqueous (NH 4 ) 2 SO 4 , (2) crosslinking the precipitates with 0.25% (v/v) GA, and (3) preparing the cross‐linked powder by freeze‐drying. The cross‐linked PC (CLPC) performed asymmetric oxidation of the toward ( R )‐isomers of rac ‐ 1 and rac ‐2 in 50 mM glycine–NaOH (pH 9.0) buffer/DMSO cosolvent [2.07% (v/v)] with high enantioselectivity; thus, the ( S )‐isomers can be obtained in greater than 99% ee from the corresponding rac ‐ p ‐substituted naphthyl methyl carbinol ( rac ‐ 1 and rac ‐2 ). The CLPC activity was not only competitively inhibited by addition of either 1.0 mM ZnCl 2 or a chelating agent such as 1.0 mM EDTA but also denatured by pretreatments: autoclaving at 121°C (20 min) or using 6.0 M guanidine–HCl containing 50 mM DTT. These results indicated that the PC catalytic process may utilize an electron transfer system incorporating a redox cation (e.g., Fe 2+ ⇄ Fe 3+ or Zn). Therefore, the newly introduced CLPC can asymmetrically oxidize the substrates without the addition of any cofactor resulting in a low‐cost organic method. Overall, our results show that the CLPC is an easily prepared, low‐cost reagent that can function under mild conditions and afford stereoselectivity, regioselectivity, and substrate specificity. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 953–961, 2012