Potential Application ofN-Carbamoyl-β-Alanine Amidohydrolase fromAgrobacterium tumefaciensC58 for β-Amino Acid Production

Abstract

An N-carbamoyl-␤-alanine amidohydrolase of industrial interest from Agrobacterium tumefaciens C58 (␤car At ) has been characterized. ␤car At is most active at 30°C and pH 8.0 with N-carbamoyl-␤-alanine as a substrate. The purified enzyme is completely inactivated by the metal-chelating agent 8-hydroxyquinoline-5-sulfonic acid (HQSA), and activity is restored by the addition of divalent metal ions, such as Mn 2؉ , Ni 2؉ , and Co 2؉ . The native enzyme is a homodimer with a molecular mass of 90 kDa from pH 5.5 to 9.0. The enzyme has a broad substrate spectrum and hydrolyzes nonsubstituted N-carbamoyl-␣-, -␤-, -␥-, and -␦-amino acids, with the greatest catalytic efficiency for N-carbamoyl-␤-alanine. ␤car At also recognizes substrate analogues substituted with sulfonic and phosphonic acid groups to produce the ␤-amino acids taurine and ciliatine, respectively. ␤car At is able to produce monosubstituted ␤ 2 -and ␤ 3 -amino acids, showing better catalytic efficiency (k cat /K m ) for the production of the former. For both types of monosubstituted substrates, the enzyme hydrolyzes N-carbamoyl-␤-amino acids with a short aliphatic side chain better than those with aromatic rings. These properties make ␤car At an outstanding candidate for application in the biotechnology industry.

Enhanced stability of newly isolated trimeric l‐methionine‐N‐carbamoylase from Brevibacillus reuszeri HSN1 by covalent immobilization

“…Thermostabilization of proteins after immobilization was also reported earlier [42]. l-Hydantoinase and carbamoylase activities are reported to remain constant at the maximum level in the range of [45][46][47][48][49][50][51][52][53][54][55][56][57][58][59][60] • C [32]. The soluble d-hydantoinase lost 60% activity in 10 Min when the reaction was conducted at 70…”

“…The properties of onestep partially purified l-N-carbamoylase from this strain were investigated. The trimeric enzyme, high specific activity (purified enzyme, 145 μmol/Min/mg), broad substrate specificity, better temperature-pH optima [44][45][46], and productivity have suggested the further stabilization of this enzyme as a potential biocatalyst. Here, we used one-step partially purified (anion-exchange chromatography step) l-N-carbamoylase with the specific activity of 12 U/mg, for covalent immobilization on Eupergit R C. This technique has been used in previous studies [31,32], but it could not become industry friendly for l-N-carbamoylase because of the unstable nature of the enzyme.…”

Mutational and Structural Analysis ofl-N-Carbamoylase Reveals New Insights into a Peptidase M20/M25/M40 Family Member

“…Their relationship with other amidohydrolases and with the peptidase M20/M25/M40 family has been established based on sequence similarity (21,29,42). A closer relationship among L-carbamoylases and ␤-ureidopropionases is supported by the findings of two ␤-ureidopropionases that are able to hydrolyze N-carbamoyl-L-␣-amino acids (40,41,47).In a previous work, we were able to show the involvement in catalysis of several highly conserved residues in L-carbamoylases, as well as the relationship of these enzymes with several peptidases (42). Dimerization was also proved to be necessary for enzymatic activity, as the residues involved in catalysis come from both monomers.…”

“…Their relationship with other amidohydrolases and with the peptidase M20/M25/M40 family has been established based on sequence similarity (21,29,42). A closer relationship among L-carbamoylases and ␤-ureidopropionases is supported by the findings of two ␤-ureidopropionases that are able to hydrolyze N-carbamoyl-L-␣-amino acids (40,41,47).…”

“…This is further supported by the cacodylate molecule found in the environment of this triad. Asp 284 is also highly conserved in L-carbamoylases and other members of the peptidase M20/M25/M40 family (such as hAcy1 [41]), and in BsLcar it interacts directly with Arg 286 through a hydrogen bonding network (Fig. 2).…”