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

Abstract

Despite the success of some nitrilases in industrial applications, there is a constant demand to broaden the catalog of these hydrolases, especially robust ones with high operational stability. By using the criteria of thermoresistance to screen a collection of candidate enzymes heterologously expressed in Escherichia coli, the enzyme Nitphym from the mesophilic organism Paraburkholderia phymatum was selected and further characterized. Its quick and efficient purification by heat treatment is of major interest for large-scale applications. The purified nitrilase displayed a high thermostability with 90% of remaining activity after 2 days at 30°C and a half-life of 18 h at 60°C, together with a broad pH range of 5.5–8.5. Its high resistance to various miscible cosolvents and tolerance to high substrate loadings enabled the quantitative conversion of 65.5 g⋅L–1 of 3-phenylpropionitrile into 3-phenylpropionic acid at 50°C in 8 h at low enzyme loadings of 0.5 g⋅L–1, with an isolated yield of 90%. This study highlights that thermophilic organisms are not the only source of industrially relevant thermostable enzymes and extends the scope of efficient nitrilases for the hydrolysis of a wide range of nitriles, especially trans-cinnamonitrile, terephthalonitrile, cyanopyridines, and 3-phenylpropionitrile.

Prospects of formamide as nitrogen source in biotechnological production processes

“…Phylogenetic analysis revealed that formamidases belong to at least two different groups of enzymes, namely the acetamidase/formamidase super family (FmdA-AmdA, Pfam PF03069), which also includes amidohydrolases of acetamide (Draper 1967 ), and the nitrilase family, which is a subfamily of the carbon-hydrogen nitrilase superfamily and hydrolyses various nitriles, producing ammonia and the respective carboxylic acid (Bessonnet et al 2021 ; Teepakorn et al 2021 ). Although both have certain sequence similarities with members of the major amidase families like aliphatic amidases, acylamide aminohydrolases, and nitrilase/cyanide hydratases, these two groups are demarcated (Fig.…”