Cloning, expression, and characterization of a novel nitrilase, PaCNit, from Pannonibacter carbonis Q4.6

Thermoresponsive Gigaporous Microspheres Facilitate the Efficient Refolding of Recombinant Nitrilase Inclusion Bodies

“… 13 − 15 Recently, our laboratory has identified a novel nitrilase from Pannonibacter carbonis Q4.6, whose conservative triplet catalytic sit “Glu-Lys-Cys” has changed into “Glu-Ser-Cys.” To explore the properties of the enzyme, the gene encoding the nitrilase was cloned and expressed in Escherichia coli BL21 (DE3). 16 However, most of the expressed nitrilases formed inclusion bodies (IBs), which need to undergo refolding process to restore their bioactive structures.…”

Purification and Characterization of Nitphym, a Robust Thermostable Nitrilase From Paraburkholderia phymatum

“…( Zhang et al, 2018 ) or BGC4 from Paraburkholderia graminis ( Fan et al, 2017a ) that show half-lives under 4 h at 50°C. Nit phym is similar to PaCNit reported to maintain more than 50% of its activity after 24 h at 60°C when heterogeneously expressed in E. coli ( Liu et al, 2019 ), while it was isolated from Pannonibacter carbonis , a proteobacteria with optimum growth at 30–35°C ( Xi et al, 2018 ). Thermoactive nitrilases are commonly obtained from thermophilic and hyperthermophilic microorganisms ( Mueller et al, 2006 ; Chen et al, 2015 ; Dennett and Blamey, 2016 ; Cabrera and Blamey, 2017 ) with activity range usually up to 60–90°C and high stability even at high temperature.…”

“…In terms of pH and buffer profile, Nit phym has a behavior similar to those of many enzymes with maximum activity around pH 7 (Shen et al, 2021). The activity at this pH is of the same order as those between 6.0 and 8.0, unlike which was observed for the nitrilases PaCNit (Liu et al, 2019) or the one from Rhodobacter sphaeroides (Wang et al, 2014) which exhibited narrow pH profiles. For this feature, Nit phym is similar to PaNIT (Zhang et al, 2018) and to the nitrilase from Pseudomonas fluorescens strain EBC191 (Kiziak et al, 2005; Table 2).…”

“…The stability of Nit phym is impressive, with a half-live of 18 h at 60 • C, compared to these nitrilases and the one from Pantoea sp. (Zhang et al, 2018) or BGC4 from Paraburkholderia graminis (Fan et al, 2017a) that show half-lives under 4 h at 50 • C. Nit phym is similar to PaCNit reported to maintain more than 50% of its activity after 24 h at 60 • C when heterogeneously expressed in E. coli (Liu et al, 2019), while it was isolated from Pannonibacter carbonis, a proteobacteria with optimum growth at 30-35 • C (Xi et al, 2018). Thermoactive nitrilases are commonly obtained from FIGURE 8 | Effect of 3-phenylpropionitrile concentration on conversion.…”

“…These results are roughly similar to the ones we previously described despite some differences mainly due to solubility issues and inconsistencies occurring in 96-microwell plates screening ( Vergne-Vaxelaire et al, 2013 ). This characterization expands the list of enzymes with broad substrate specificity, which mainly includes PaCNit ( Liu et al, 2019 ; Table 2 ), NIT278 from Syntrophobacter fumaroxidans ( Vergne-Vaxelaire et al, 2013 ), NIT28 from Sphingomonas witichii ( Vergne-Vaxelaire et al, 2013 ), NIT191 from Burkholderia xenovorans ( Vergne-Vaxelaire et al, 2013 ), BGC4 from Paraburkholderia graminis ( Fan et al, 2017a ), bll6402 and blr3397 from Bradyrhizobium japonicum ( Zhu et al, 2007 , 2008 ), NitA from Pseudomonas fluorescens EBC191 ( Brunner et al, 2018 ; Table 2 ), and the recently nitrilases mined from Zobellia galactanivorans , Achromobacter insolitus , and Cupriavidus necator ( Sunder et al, 2020 ). The growing number of described nitrilases active on varying classes of nitriles supports the irrelevance of the previously widely used classification of nitrilases based on their main substrates (arylacetonitrilases, aromatic, and aliphatic nitrilases).…”