Alternative Heterologous Expression of L-Arabinose Isomerase from Enterococcus faecium DBFIQ E36 By Residual Whey Lactose Induction.

This study reports an alternative strategy for the expression of a recombinant L-AI from Enterococcus faecium DBFIQ E36 by auto-induction using glucose and glycerol as carbon sources and residual whey lactose as inducer agent. Commercial lactose and isopropyl ß-D-1-thiogalactopyranoside (IPTG) were also evaluated as inducers for comparison of enzyme expression levels. The enzymatic extracts were purified by affinity chromatography, characterized, and applied in the bioconversion of D-galactose into D-tagatose. L-AI presented a catalytic activity of 1.67 ± 0.14, 1.52 ± 0.01, and 0.7 ± 0.04 U/mL, when expressed using commercial lactose, lactose from whey, and IPTG, respectively. Higher activities could be obtained by changing the protocol of enzyme extraction and, for instance, the enzymatic extract produced with whey presented a catalytic activity of 3.8 U/mL. The specific activity of the enzyme extracts produced using lactose (commercial or residual whey) after enzyme purification was also higher when compared to the enzyme expressed with IPTG. Best results were achieved when enzyme expression was conducted using 4 g/L of residual whey lactose for 11 h. These results proved the efficacy of an alternative and economic protocol for the effective expression of a recombinant L-AI aiming its high-scale production.

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 Km values of 28 and 218 mM for MANAE-N-His-L-AI and MANAE-C-His-L-AI, respectively.

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.

Operational and Thermal Stability Analysis of Thermomyces lanuginosus 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. Graphical Abstract ᅟ.

Engineering the l-Arabinose Isomerase from Enterococcus Faecium for d-Tagatose Synthesis.

l-Arabinose isomerase (EC 5.3.1.4) (l-AI) from Enterococcus faecium DBFIQ E36 was overproduced in Escherichia coli by designing a codon-optimized synthetic araA gene. Using this optimized gene, two N- and C-terminal His-tagged-l-AI proteins were produced. The cloning of the two chimeric genes into regulated expression vectors resulted in the production of high amounts of recombinant N-His-l-AI and C-His-l-AI in soluble and active forms. Both His-tagged enzymes were purified in a single step through metal-affinity chromatography and showed different kinetic and structural characteristics. Analytical ultracentrifugation revealed that C-His-l-AI was preferentially hexameric in solution, whereas N-His-l-AI was mainly monomeric. The specific activity of the N-His-l-AI at acidic pH was higher than that of C-His-l-AI and showed a maximum bioconversion yield of 26% at 50 °C for d-tagatose biosynthesis, with Km and Vmax parameters of 252 mM and 0.092 U mg-1, respectively. However, C-His-l-AI was more active and stable at alkaline pH than N-His-l-AI. N-His-l-AI follows a Michaelis-Menten kinetic, whereas C-His-l-AI fitted to a sigmoidal saturation curve.

Chemical improvement of chitosan-modified beads for the immobilization of Enterococcus faecium DBFIQ E36 L-arabinose isomerase through multipoint covalent attachment approach.

D-tagatose is produced from D-galactose by the enzyme L-arabinose isomerase (L-AI) in a commercially viable bioprocess. An active and stable biocatalyst was obtained by modifying chitosan gel structure through reaction with TNBS, D-fructose or DMF, among others. This led to a significant improvement in L-AI immobilization via multipoint covalent attachment approach. Synthetized derivatives were compared with commercial supports such as Eupergit(®) C250L and glyoxal-agarose. The best chitosan derivative for L-AI immobilization was achieved by reacting 4 % (w/v) D-fructose with 3 % (w/v) chitosan at 50 °C for 4 h. When compared to the free enzyme, the glutaraldehyde-activated chitosan biocatalyst showed an apparent activity of 88.4 U g (gel) (-1) with a 211-fold stabilization factor while the glyoxal-agarose biocatalyst gave an apparent activity of 161.8 U g (gel) (-1) with an 85-fold stabilization factor. Hence, chitosan derivatives were comparable to commercial resins, thus becoming a viable low-cost strategy to obtain high active L-AI insolubilized derivatives.