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1.
Indian J Biochem Biophys ; 46(4): 281-8, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19788059

RESUMO

A serine residue Ser463, required for proper function of E. coli y-glutamyltranspeptidase (EcGGT) was identified by site-directed mutagenesis on the basis of sequence alignment of human, pig, rat, and three bacterial enzymes. Thr-, Asp-, and Lys-substituted variants were overexpressed in E. coli M15 cells and the recombinant proteins were purified to near homogeneity by nickel-chelate chromatography. With the exception of S463T, the other two variants completely lost GGT activity, implying the importance of this residue in EcGGT. Moreover, substitution of Ser463 with either Lys or Asp impaired the capability of autocatalytic processing of the precursor into alpha- and beta-subunit. Computer modeling showed that the critical bonding distance of Gln390 C-Thr391 OG1 was significantly increased in S463D and S463K, indicating that these distance changes might be responsible for the lack of enzyme maturation. Measurements of intrinsic tryptophan fluorescence revealed alteration of the microenvironment of aromatic amino acid residues in S463D and S463K, while circular dichroism (CD) spectra were nearly identical for wild-type and all mutant enzymes. The temperature-dependent signal in the far-UV region for S463T was consistent with that of wild-type enzyme, but S463D and S463K showed a different sensitivity towards temperature-induced denaturation. These results implied that a significant conformational change occurred as a result of Asp- and Lys-substitution.


Assuntos
Escherichia coli/enzimologia , Serina/química , gama-Glutamiltransferase/química , Sequência de Aminoácidos , Ácido Aspártico/química , Catálise , Dicroísmo Circular , Glutamina/química , Lisina/química , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência/métodos , Treonina/química , Triptofano/química , gama-Glutamiltransferase/genética
2.
J Colloid Interface Sci ; 334(1): 42-9, 2009 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-19364609

RESUMO

A class of acrylic copolymers with narrow molecular weight distribution from butyl methacrylate and glycidyl methacrylate comonomers via atom transfer radical polymerization was synthesized. Various types of polarities including hydroxyl-amines, glycols, and carboxylic acids were then grafted onto the oxirane side groups. The resultant comb-like copolymers with different polar pendants were tested for homogenizing a representative Yellow pigment in 1,6-hexanediol diacrylate medium. Specifically, the polyacrylates with 1,3-diamine pendants (7-10 multiplicity on each polymer strain) enabled to homogeneously disperse the pigment than the analogous copolymers with hydroxyl or carboxylic acid groups. Ultimately, the pigment dispersion with an average size of ca. 20 nm in diameter, high transmittance and low viscosity was achieved. Furthermore, the pigment dispersion was allowed to UV-cure into a film, and for the first time, the primary structures of the pigment particles (ca. 50 nm in diameter) were observed by transmission electronic microscope.

3.
Appl Biochem Biotechnol ; 150(2): 157-70, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18483700

RESUMO

The recombinant Escherichia coli gamma-glutamyltranspeptidase (EcGGT) was immobilized in Ca-alginate-kappa-carrageenan beads. Effects of alginate concentration, amount of loading enzyme, and bead size on the entrapped activity were investigated. Optimum alginate concentration for EcGGT immobilization was found to be 2% (w/v). Using a loading enzyme concentration of 1.5 mg/g alginate, maximum enzyme activity was observed. With increase in bead size from 1.9 to 3.1 mm, the immobilization efficiency was decreased significantly because of mass transfer resistance. Thermal stability of the free EcGGT was increased as a result of the immobilization. Ca-alginate-kappa-carrageenan-EcGGT beads were suitable for up to six repeated uses, losing only 45% of their initial activity. Upon 30 days of storage the preserved activity of free and immobilized enzyme were found as 4% and 68%, respectively. The synthesis of L: -theanine was performed in 50 mM Tris-HCl buffer (pH 10) containing 25 mM L: -glutamine, 40 mM ethylamine, and 1.5 mg EcGGT/g alginate at 40 degrees C for 12 h, and a conversion rate of 27% was achieved.


Assuntos
Alginatos/química , Cálcio/química , Carragenina/química , Escherichia coli/enzimologia , gama-Glutamiltransferase/química , Enzimas Imobilizadas , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Microesferas
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