1. Alteration of substrate specificity of aspartase by directed evolution
Yasuhisa Asano, Ikuo Kira, Kenzo Yokozeki Biomol Eng. 2005 Jun;22(1-3):95-101. doi: 10.1016/j.bioeng.2004.12.002.
Aspartase (l-aspartate ammonia-lyase, EC 4.3.1.1), which catalyzes the reversible deamination of l-aspartic acid to yield fumaric acid and ammonia, is highly selective towards l-aspartic acid. We screened for enzyme variants with altered substrate specificity by a directed evolution method. Random mutagenesis was performed on an Escherichia coli aspartase gene (aspA) by error-prone PCR to construct a mutant library. The mutant library was introduced to E. coli and the transformants were screened for production of fumaric acid-mono amide from l-aspartic acid-alpha-amide. Through the screening, one mutant, MA2100, catalyzing deamination of l-aspartic acid-alpha-amide was achieved. Gene analysis of the MA2100 mutant indicated that the mutated enzyme had a K327N mutation. The characteristics of the mutated enzyme were examined. The optimum pH values for the l-aspartic acid and l-aspartic acid-alpha-amide of the mutated enzyme were pH 8.5 and 6.0, respectively. The K(m) value and V(max) value for the l-aspartic acid of the mutated enzyme were 28.3 mM and 0.26 U/mg, respectively. The K(m) value and V(max) value for the l-aspartic acid-alpha-amide of the mutated enzyme were 1450 mM and 0.47 U/mg, respectively. This is the first report describing the alteration of the substrate specificity of aspartase, an industrially important enzyme.
2. Methods for syntheses of N-methyl-DL-aspartic acid derivatives
M Boros, J Kökösi, J Vámos, I Kövesdi, B Noszál Amino Acids. 2007 Nov;33(4):709-17. doi: 10.1007/s00726-006-0453-4. Epub 2007 Mar 2.
A novel practical method for the synthesis of N-methyl-DL-aspartic acid 1 (NMA) and new syntheses for N-methyl-aspartic acid derivatives are described. NMA 1, the natural amino acid was synthesized by Michael addition of methylamine to dimethyl fumarate 5. Fumaric or maleic acid mono-ester and -amide were regioselectively transformed into beta-substituted aspartic acid derivatives. In the cases of maleamic 11a or fumaramic esters 11b, the alpha-amide derivative 13 was formed, but hydrolysis of the product provided N-methyl-DL-asparagine 9 via base catalyzed ring closure to DL-alpha-methylamino-succinimide 4, followed by selective ring opening. Efficient methods were developed for the preparation of NMA-alpha-amide 13 from unprotected NMA via sulphinamide anhydride 15 and aspartic anhydride 3 intermediate products. NMA diamide 16 was prepared from NMA dimethyl ester 6 and methylamino-succinimide 4 by ammonolysis. Temperature-dependent side reactions of methylamino-succinimide 4 led to diazocinone 18, resulted from self-condensation of methylamino-succinimide via nucleophyl ring opening and the subsequent ring-transformation.
3. Synthesis of antimicrobial peptoids
Paul R Hansen, Jens K Munk Methods Mol Biol. 2013;1047:151-9. doi: 10.1007/978-1-62703-544-6_11.
Peptoids (N-substituted glycines) are mimics of α-peptides in which the side chains are attached to the backbone N (α) -amide nitrogen instead of the C (α) -atom. Peptoids hold promise as therapeutics since they often retain the biological activity of the parent peptide and are stable to proteases. In recent years, peptoids have attracted attention as new potential antibiotics against multiresistant bacteria. Here we describe the submonomer solid-phase synthesis of an antimicrobial peptoid, H-Nmbn-Nlys-Nlys-Nnap-Nbut-Nmbn-Nlys-NH2.
