Immobilization of Carboxypeptidase A into Modified Chitosan Matrixes by Covalent Attachment

Carboxypeptidase A (CPA) is a metalloexopeptidase that catalyzes the hydrolysis of the peptide bonds that are adjacent to the C-terminal end of a polypeptide chain. The enzyme preferentially cleaves over C-terminal L-amino acids with aromatic or branched side chains. This is of main importance for food industry because it can be employed for manufacturing functional foods from different protein sources with reduced hydrophobic amino acid content for patients with deficiencies in the absorption or digestion of the corresponding amino acids. In that way, strategies for effective multipoint covalent immobilization of CPA metalloenzyme on chitosan beads have been developed. The study of the ability to produce several chemical modifications on chitosan molecules before, during and after its coagulation to form carrier beads lead in a protective effect of the polymer matrix. The chemical modification of chitosan through the use of an N-alkylation strategy produced the best derivatives. N-alkyl chitosan derivative beads with D-fructose presented values of 0.86 for immobilization yield, 314.6 IU g^?1 bead for initial activity of biocatalyst and were 5675.64-fold more stable than the free enzyme at 55 °C. Results have shown that these derivatives would present a potential technological application in hydrolytic processes due to both their physical properties, such as low swelling capacity, reduced metal chelation ability and bulk mesoporosity, and increased operational stability when compared with soluble enzyme.     

Stückelino dark matter in anomalous U(1)′ models

We study a possible dark matter candidate in the framework of a minimal anomalous U(1)′ extension of the MSSM. It turns out that in a suitable decoupling limit the Stückelino, the fermionic degree of freedom of the Stückelberg multiplet, is the lightest supersymmetric particle (LSP). We compute the relic density of this particle including coannihilations with the next to lightest supersymmetric particle (NLSP) and with the next to next to lightest supersymmetric particle (NNLSP), which are assumed to be almost degenerate in mass. This assumption is needed in order to satisfy the stringent limits that the Wilkinson Microwave Anisotropy Probe (WMAP) puts on the relic density. We find that the WMAP constraints can be satisfied by different NLSP and NNLSP configurations as a function of the mass gap with the LSP. These results hold in the parameter space region where the model remains perturbative.     

One-Step Immobilization and Stabilization of a Recombinant Enterococcus faecium DBFIQ E36 l-Arabinose Isomerase for d-Tagatose Synthesis

A recombinant l-arabinose isomerase from Enterococcus faecium DBFIQ E36 was immobilized onto multifunctional epoxide supports by chemical adsorption and onto a chelate-activated support via polyhistidine-tag, located on the N-terminal (N-His-L-AI) or on the C-terminal (C-His-L-AI) sequence, followed by covalent bonding between the enzyme and the support. The results were compared to reversible L-AI immobilization by adsorption onto charged agarose supports with improved stability. All the derivatives presented immobilization yields of above 75%. The ionic interaction established between agarose gels containing monoaminoethyl-N-aminoethyl structures (MANAE) and the enzyme was the most suitable strategy for L-AI immobilization in comparison to the chelate-activated agarose. In addition, the immobilized biocatalysts by ionic interaction in MANAE showed to be the most stable, retaining up to 100% of enzyme activity for 60 min at 60 °C and with K_m values of 28 and 218 mM for MANAE-N-His-L-AI and MANAE-C-His-L-AI, respectively.

     

Operational and Thermal Stability Analysis of <Emphasis Type="Italic">Thermomyces lanuginosus</Emphasis> Lipase Covalently Immobilized onto Modified Chitosan Supports

The aim of this paper was to evaluate different strategies of chitosan activation using cross-linking reagent like glycidol, epichlorohydrin, and glutaraldehyde for Thermomyces lanuginosus lipase (TLL) immobilization. Operational activity and stability by esterification of oleic acid with ethanol and thermal inactivation using these derivatives were investigated. Derivative obtained by sequentially activation with glycidol, ethylenediamine, and glutaraldehyde and subsequent TLL immobilization showed the best performance, with high hydrolytic activity value. Its stability was 15-fold higher than solubilized TLL in the evaluated inactivation conditions (60 °C, 25 mM sodium phosphate buffer pH 7). After 5 cycles of oleic acid esterification, only a few percentage of its conversion has reduced. On the other hand, glycidol-activated chitosan derivative showed very low hydrolytic activity value. Epichlorohydrin-activated chitosan derivative showed regular hydrolytic activity value. Both derivatives showed low immobilization yields. Operational stability of this last derivative was very low, where after the first cycle of oleic acid esterification, only 56% of its initial conversion was obtained.

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The reaction of 3,3-dichloroallyltrimethylsilane with n-butyllithium

n-Butyllithium reacts with 3,3-dichloroallyltrimethylsilane to metalate the vinyl proton. Under the reaction conditions the Me₃SiCH₂C(Li)CCl₂ formed undergoes β-elimination of LiCl to give ClCCCH₂SiMe₃ whose subsequent reaction with n-butyllithium produces LiCCCH₂SiMe₃. Addition of trimethylchlorosilane gives Me₃SiCCCH₂SiMe₃. When two molar equivalents of n-butyllithium are used, further metalation of LiCCCH₂SiMe₃ gives LiCCCH(Li)SiMe₃. The action of N-bromosuccinimide on Me₃SiCH₂CHCCl₂ resulted in formation of Me₃SiCHCHCCl₂Br.