Liu Ming-Yu,Wang Yu-Yue,Li Han-Zhi,Luo Xue-Mei,Feng Jia-Xun,Zhao Shuai
Nitriles exhibit acute cytotoxicity to human and animal cells. Nitrilase is a green biocatalyst that can directly convert nitrile into nontoxic carboxylic acids and ammonia. However, the nitrilases capable of degrading 3-butenenitrile and 4-pentenenitrile derived from glucosinolate present in rapeseed meals are still limited. This study expressed the nit rilase 2 gene from bacterium Paraburkholderia g raminis ( PgNIT2 ) in Escherichia coli . The purified recombinant nitrilase rPgNIT2 showed specific activities of 5.94 U/mg and 6.66 U/mg when using 3-butenenitrile and 4-pentenenitrile as substrates at the optimal pH 7.0 and temperature 45 °C. The substrate binding pocket of PgNIT2 comprising 12 amino acids was required for rPgNIT2 activity. Remarkably, the specific activity of mutant enzyme A190I increased by 68.3% and 57.3%, respectively, against 3-butenenitrile and 4-pentenenitrile compared with the wild-type enzyme. Furthermore, the A190I showed improved thermostability and hydrolysis ability towards 3-butenenitrile and 4-pentenenitrile compared with rPgNIT2 but shared the same optimal pH and temperature. These results provided theoretical guidance for engineering of bacterial nitrilases used for rapeseed meal detoxification.
A combined experimental and theoretical study was conducted to investigate the isomerization of 2-methyl-3-butenenitrile (2M3BN) to 3-pentenenitrile (3PN) and to 2-methyl-2-butenenitrile (2M2BN) catalyzed by nickel diphosphine complexes. Ni(1,4-bis(diphenylphosphino)butane) (dppb) was identified as the most reactive catalyst among the complexes that we examined experimentally. Quantum mechanics (density functional theory) was then used to study the two isomerization mechanisms catalyzed by this complex. We find that for the 2M3BN → 3PN isomerization, the reaction is initiated with C CN bond cleavage, followed by an allyl direct rotation and C CN bond reformation. For the 2M3BN → 2M2BN isomerization, the most energetically favorable pathway begins with C H bond activation, followed by a π-σ-σ-π allyl rearrangement and C H bond reformation. Our proposed mechanism for the 2M3BN → 2M2BN isomerization is slightly different (yet energetically more favorable) than that described in previous studies, where it has been suggested that 2M2BN is obtained through a π-σ-σ allyl rearrangement rather than a π-σ-σ-π type rearrangement. Additionally, we investigated the effect of Lewis acids in the 2M3BN → 3PN isomerization, which has been shown in most experiments to attenuate the reaction. Notably, our calculations indicated that ZnCl 2 , which is used as a model Lewis acid, actually reduces the barriers for all elementary steps. However, the effective kinetic barrier for the isomerization increases from 23.7 (without ZnCl 2 ) to 24.0 kcal/mol because of the formation of a very stable Ni(π-allyl) (CN ZnCl 2 ) intermediate, causing a decrease in the reaction rate. This theoretical result was further confirmed by our own experiments.
The catalytic isomerization of 2-methyl-3-butenenitrile (2M3BN), a model reaction in the DuPont process, has been performed using NiL 4 (L=tri- O - p -tolyl phosphite) as a catalyst. The lowered catalytic activity in the isomerization with coexistence of 2-pentenenitrile (2PN) and 2-methyl-2-butenenitrile (2M2BN) indicates that both 2PN and 2M2BN are the catalyst inhibitors , and the quantitative relationship between the conversion of 2M3BN and the content of 2M2BN and 2PN is provided. DFT calculation results suggest that the inhibition effect is attributed to the generation of dead-end intermediates (2PN)NiL 2 and (2M2BN)NiL 2 , both of which take nickel atom out of the catalytic cycle in the isomerization process. To suppress the inhibition effect, new catalytic intermediates are rationally designed based on their computational %V bur . An efficient method that adding extra ligand 1, 5-bis(diphenylphosphino)pentane (dppp5) to the NiL 4 catalyst is selected experimentally. Compared to the results obtained with NiL 4 as catalyst, the (dppp5)NiL 2 increases the conversion of 2M3BN from 74.5 % to 93.4 % at 3 h of reaction and provides a high selectivity to 3PN (> 98 %) at optimal conditions.
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