Cloning, Expression, Characterization and Immobilization of a Recombinant Carboxylesterase from the Halophilic Archaeon, Halobacterium salinarum NCR-1.

Only a few halophilic archaea producing carboxylesterases have been reported. The limited research on biocatalytic characteristics of archaeal esterases is primarily due to their very low production in native organisms. A gene encoding carboxylesterase from Halobacterium salinarum NRC-1 was cloned and successfully expressed in Haloferax volcanii. The recombinant carboxylesterase (rHsEst) was purified by affinity chromatography with a yield of 81%, and its molecular weight was estimated by SDS-PAGE (33 kDa). The best kinetic parameters of rHsEst were achieved using p-nitrophenyl valerate as substrate (KM = 78 µM, kcat = 0.67 s-1). rHsEst exhibited great stability to most metal ions tested and some solvents (diethyl ether, n-hexane, n-heptane). Purified rHsEst was effectively immobilized using Celite 545. Esterase activities of rHsEst were confirmed by substrate specificity studies. The presence of a serine residue in rHsEst active site was revealed through inhibition with PMSF. The pH for optimal activity of free rHsEst was 8, while for immobilized rHsEst, maximal activity was at a pH range between 8 to 10. Immobilization of rHsEst increased its thermostability, halophilicity and protection against inhibitors such as EDTA, BME and PMSF. Remarkably, immobilized rHsEst was stable and active in NaCl concentrations as high as 5M. These biochemical characteristics of immobilized rHsEst reveal its potential as a biocatalyst for industrial applications.

Surface-Displayed Mannanolytic and Chitinolytic Enzymes Using Peptidoglycan Binding LysM Domains.

Using Lactiplantibacillus plantarum as a food-grade carrier to create non-GMO whole-cell biocatalysts is gaining popularity. This work evaluates the immobilization yield of a chitosanase (CsnA, 30 kDa) from Bacillus subtilis and a mannanase (ManB, 40 kDa) from B. licheniformis on the surface of L. plantarum WCFS1 using either a single LysM domain derived from the extracellular transglycosylase Lp_3014 or a double LysM domain derived from the muropeptidase Lp_2162. ManB and CsnA were fused with the LysM domains of Lp_3014 or Lp_2162, produced in Escherichia coli and anchored to the cell surface of L. plantarum. The localization of the recombinant proteins on the bacterial cell surface was successfully confirmed by Western blot and flow cytometry analysis. The highest immobilization yields (44-48%) and activities of mannanase and chitosanase on the displaying cell surface (812 and 508 U/g of dry cell weight, respectively) were obtained when using the double LysM domain of Lp_2162 as an anchor. The presence of manno-oligosaccharides or chito-oligosaccharides in the reaction mixtures containing appropriate substrates and ManB or CsnA-displaying cells was determined by high-performance anion exchange chromatography. This study indicated that non-GMO Lactiplantibacillus chitosanase- and mannanase-displaying cells could be used to produce potentially prebiotic oligosaccharides.

A Whole-Process Visible Strategy for the Preparation of Rhizomucor miehei Lipase with Escherichia coli Secretion Expression System and the Immobilization.

BACKGROUND: Rhizomucor miehei (RM) lipase is a regioselective lipase widely used in food, pharmaceutical and biofuel industries. However, the high cost and low purity of the commercial RM lipase limit its industrial applications. Therefore, it is necessary to develop cost-effective strategies for large-scale preparation of this lipase. The present study explored the high-level expression of RM lipase using superfolder green fluorescent protein (sfGFP)-mediated Escherichia coli secretion system. RESULTS: The sfGFP(-15) mutant was fused to the C-terminus of RM lipase to mediate its secretion expression. The yield of the fusion protein reached approximately 5.1 g/L with high-density fermentation in 5-L fermentors. Unlike conventional secretion expression methods, only a small portion of the target protein was secreted into the cell culture while majority of the fusion protein was still remained in the cytoplasm. However, in contrast to intracellular expression, the target protein in the cytoplasm could be transported efficiently to the supernatant through a simple washing step with equal volume of phosphate saline (PBS), without causing cell disruption. Hence, the approach facilitated the downstream purification step of the recombinant RM lipase. Moreover, contamination or decline of the engineered strain and degradation or deactivation of the target enzyme can be detected efficiently because they exhibited bright green fluorescence. Next, the target protein was immobilized with anion-exchange and macropore resins. Diethylaminoethyl sepharose (DEAE), a weak-basic anion-exchange resin, exhibited the highest bind capacity but inhibited the activity of RM lipase dramatically. On the contrary, RM lipase fixed with macropore resin D101 demonstrated the highest specific activity. Although immobilization with D101 didn't improve the activity of the enzyme, the thermostability of the immobilized enzyme elevated significantly. The immobilized RM lipase retained approximately 90% of its activity after 3-h incubation at 80 °C. Therefore, D101 was chosen as the supporting material of the target protein. CONCLUSION: The present study established a highly efficient strategy for large-scale preparation of RM lipase. This innovative technique not only provides high-purity RM lipase at a low cost but also has great potential as a platform for the preparation of lipases in the future.

Production of recombinant glycosidases fused with Usp45 and SpaX to avoid the purification and immobilization stages.

The elucidation of the physicochemical properties of glycosidases is essential for their subsequent technological application, which may include saccharide hydrolysis processes and oligosaccharide synthesis. As the application of cloning, purification and enzymatic immobilization methods can be time consuming and require a heavy financial investment, this study has validated the recombinant production of the set of Lacticaseibacillus rhamnosus fucosidases fused with Usp45 and SpaX anchored to the cell wall of Lacticaseibacillus cremoris subsp cremoris MG1363, with the aim of avoiding the purification and stabilization steps. The cell debris harboring the anchored AlfA, AlfB and AlfC fucosidases showed activity against p-nitrophenyl α-L-fucopyranoside of 6.11 ±â€¯0.36, 5.81 ±â€¯0.29 and 9.90 ±â€¯0.58 U/mL, respectively, and exhibited better thermal stability at 50 °C than the same enzymes in their soluble state. Furthermore, the anchored AlfC fucosidase transfucosylated different acceptor sugars, achieving fucose equivalent concentrations of 0.94 ±â€¯0.09 mg/mL, 4.11 ±â€¯0.21 mg/mL, and 4.08 ±â€¯0.15 mg/mL of fucosylgalatose, fucosylglucose and fucosylsucrose, respectively.

Highly Precise and Sensitive Polymerase Chain Reaction Using Mesoporous Silica-Immobilized Enzymes.

A highly precise and sensitive technology that enables DNA amplification/detection from minimal amounts of nucleic acid is expected to find applicability in genetic testing involving small amounts of samples. The use of a free enzyme in conventional DNA amplification techniques, such as the polymerase chain reaction (PCR), frequently causes side reactions (i.e., nonspecific DNA amplification) when ≤103 substrate DNA molecules are present, thereby preventing selective amplification of the target DNA. To address this issue, we have developed a novel DNA amplification system, mesoporous silica-enhanced PCR (MSE-PCR), which involves the immobilization of a thermostable DNA polymerase from Thermococcus kodakaraensis (KOD DNA polymerase) into highly ordered nanopores of the mesoporous silica to control the reaction environment around the enzyme. In the MSE-PCR system using immobilized KOD DNA polymerase, such nonspecific DNA amplification was remarkably inhibited under the same conditions. Furthermore, the optimization of mesoporous silica pore sizes enabled selective and efficient DNA amplification from DNA substrates at the single-molecule level, i.e., one ten-thousandth of the amount of substrate DNA required for a DNA amplification reaction using a free enzyme. The results obtained in this study have shown that the nanopores of mesoporous silica can inhibit nonspecific reactions in DNA amplification, thereby considerably improving the specificity and sensitivity of the DNA polymerase reaction.

Expression and immobilization of trypsin-like domain of serine protease from Pseudomonas aeruginosa for improved stability and catalytic activity.

Protein engineering and enzyme immobilization strategies have produced numerous biocatalysts for modern industrial applications. In this study, we have also used these two strategies for improving the operational stability and catalytic efficiency of serine protease from Pseudomonas aeruginosa. The enzyme serine protease was truncated to separate its trypsin-like domain from the PDZ1 and PDZ2 domains. The truncated trypsin-like domain was expressed in Escherichia coli BL21, and its catalytic activity and thermostability were estimated. Later this trypsin-like domain was immobilized with 2% Na-alginate. The immobilized domain showed 10°C increase in optimum temperature compared to its free counterpart. Kinetic studies showed two-folds increased Vmax of the immobilized domain. Likewise, the Km value of this domain was 11.5 folds lower compared to the free trypsin-like domain. The catalytic efficiency (Kcat /Km ) of the immobilized enzyme also elevated to 311 folds. Additionally, the immobilized trypsin-like domain remained active in the presence of surfactants (Triton-X 100, SDS, and Tween-40) and metal ions (Mg2+ , Ca2+ , Na+ , and Zn2+ ). It also efficiently removes gelatin layer from X-ray film and hair from sheepskin. Thus, the immobilized trypsin-like domain of serine protease, with increased thermostability and catalytic efficiency, is operationally more stable than the soluble truncated trypsin-like domain.

Expression of a ß-glucosidase from Trichoderma reesei in Escherichia coli using a synthetic optimized gene and stability improvements by immobilization using magnetite nano-support.

The enzymatic conversion of lignocellulosic biomass to fermentable sugars is determined by the enzymatic activity of cellulases; consequently, improving enzymatic activity has attracted great interest in the scientific community. Cocktails of commercial cellulase often have low ß-glucosidase content, leading to the accumulation of cellobiose. This accumulation inhibits the activity of the cellulolytic complex and can be used to determine the enzymatic efficiency of commercial cellulase cocktails. Here, a novel codon optimized ß-glucosidase gene (B-glusy) from Trichoderma reesei QM6a was cloned and expressed in three strains of Escherichia coli (E. coli). The synthetic sequence containing an open reading frame (ORF) of 1491 bp was used to encode a polypeptide of 497 amino acid residues. The ß-glucosidase recombinant protein that was expressed (57 kDa of molecular weight) was purified by Ni agarose affinity chromatography and visualized by SDS-PAGE. The recombinant protein was better expressed in E. coli BL21 (DE3), and its enzymatic activity was higher at neutral pH and 30 °C (22.4 U/mg). Subsequently, the ß-glucosidase was immobilized using magnetite nano-support, after which it maintained >65% of its enzymatic activity from pH 6 to 10, and was more stable than the free enzyme above 40 °C. The maximum immobilization yield had enzyme activity of 97.2%. In conclusion, ß-glucosidase is efficiently expressed in the microbial strain E. coli BL21 (DE3) grown in a simplified culture medium.

Design of a Superpositively Charged Enzyme: Human Carbonic Anhydrase II Variant with Ferritin Encapsulation and Immobilization.

Supercharged proteins exhibit high solubility and other desirable properties, but no engineered superpositively charged enzymes have previously been made. Superpositively charged variants of proteins such as green fluorescent protein have been efficiently encapsulated within Archaeoglobus fulgidus thermophilic ferritin (AfFtn). Encapsulation by supramolecular ferritin can yield systems with a variety of sequestered cargo. To advance applications in enzymology and green chemistry, we sought a general method for supercharging an enzyme that retains activity and is compatible with AfFtn encapsulation. The zinc metalloenzyme human carbonic anhydrase II (hCAII) is an attractive encapsulation target based on its hydrolytic activity and physiologic conversion of carbon dioxide to bicarbonate. A computationally designed variant of hCAII contains positively charged residues substituted at 19 sites on the protein's surface, resulting in a shift of the putative net charge from -1 to +21. This designed hCAII(+21) exhibits encapsulation within AfFtn without the need for fusion partners or additional reagents. The hCAII(+21) variant retains esterase activity comparable to the wild type and spontaneously templates the assembly of AfFtn 24mers around itself. The AfFtn-hCAII(+21) host-guest complex exhibits both greater activity and thermal stability when compared to hCAII(+21). Upon immobilization on a solid support, AfFtn-hCAII(+21) retains enzymatic activity and exhibits an enhancement of activity at elevated temperatures.

Engineering of a chitosanase fused to a carbohydrate-binding module for continuous production of desirable chitooligosaccharides.

Chitooligosaccharides (CHOS) with multiple biological activities are usually produced through enzymatic hydrolysis of chitosan or chitin. However, purification and recycling of the enzyme have largely limited the advancement of CHOS bioproduction. Here, we engineered a novel enzyme by fusing the native chitosanase Csn75 with a carbohydrate-binding module (CBM) that can specifically bind to curdlan. The recombinase Csn75-CBM was successfully expressed by Pichia pastoris and allowed one-step purification and immobilization in the chitosanase immobilized curdlan packed-bed reactor (CICPR), where a maximum adsorption capacity of 39.59 mg enzyme/g curdlan was achieved. CHOS with degrees of polymerization of 2-5 (a hydrolysis yield of 97.75%), 3-6 (75.45%), and 3-7 (73.2%) were continuously produced by adjusting the ratio of enzyme and chitosan or the flow rate of chitosan. Moreover, the CICPR exhibited good stability and reusability after several cycles. The recombinase Csn75-CBM has greatly improved the efficiency of the bioproduction of CHOS.

Construction, Investigation and Application of TEV Protease Variants with Improved Oxidative Stability.

Tobacco etch virus protease (TEVp) is a useful tool for removing fusion tags, but wild-type TEVp is less stable under oxidized redox state. In this work, we introduced and combined C19S, C110S and C130S into TEVp variants containing T17S, L56V, N68D, I77V and S135G to improve protein solubility, and S219V to inhibit self-proteolysis. The solubility and cleavage activity of the constructed variants in Escherichia coli strains including BL21(DE3), BL21(DE3)pLys, Rossetta(DE3) and Origami(DE3) under the same induction conditions were analyzed and compared. The desirable soluble amounts, activity, and oxidative stability were identified to be reluctantly favored in the TEVp. Unlike C19S, C110S and C130S hardly impacted on decreasing protein solubility in the BL21(DE3), but they contributed to improved tolerance to the oxidative redox state in vivo and in vitro. After two fusion proteins were cleaved by purified TEVp protein containing double mutations under the oxidized redox state, the refolded disulfide-rich bovine enterokinase catalytic domain or maize peroxidase with enhanced yields were released from the regenerated amorphous cellulose via affinity absorption of the cellulose-binding module as the affinity tag.

Cost-Effective Production of L-DOPA by Tyrosinase-Immobilized Polyhydroxyalkanoate Nanogranules in Engineered Halomonas bluephagenesis TD01.

3,4-dihydroxyphenyl-L-alanine (L-DOPA) is a preferred drug for Parkinson's disease, with an increasing demand worldwide that mainly relies on costly and environmentally problematic chemical synthesis. Yet, biological L-DOPA production is unfeasible at the industrial scale due to its low L-DOPA yield and high production cost. In this study, low-cost Halomonas bluephagenesis TD01 was engineered to produce tyrosinase TyrVs-immobilized polyhydroxyalkanoate (PHA) nanogranules in vivo, with the improved PHA content and increased immobilization efficiency of TyrVs accounting for 6.85% on the surface of PHA. A higher L-DOPA-forming monophenolase activity of 518.87 U/g PHA granules and an L-DOPA concentration of 974.36 mg/L in 3 h catalysis were achieved, compared to those of E. coli. Together with the result of L-DOPA production directly by cell lysates containing PHA-TyrVs nanogranules, our study demonstrated the robust and cost-effective production of L-DOPA by H. bluephagenesis, further contributing to its low-cost industrial production based on next-generation industrial biotechnology (NGIB).

Combination of the mutations for improving activity of TEV protease in inclusion bodies.

Tobacco etch virus protease (TEVp) is an enzymatic reagent to remove fusion tag, but additional purification steps are required for removing the TEVp after cleavage reaction is finished. Use of carrier-free and dependent TEVp immobilizates can eliminate protease contamination. In this work, we identified that, among the four constructed missense variants, the insoluble variant with the highest activity was correspondent with the soluble one tested formerly. The activities of the insoluble 15 codon variants were assayed and the variant with highest activity was selected. The K45F and/or E106G mutations have been reported on slightly improving protein stability of the wild-type TEVp, but only E106G mutation enhanced soluble production and activity of the selected TEVp variant, and it increased soluble amounts of two codon variants with the impaired folding. The decreased activity and use efficiency of the optimized TEVp variant in inclusion bodies was balanced by the determined high level production, lower leaking amounts of the protein, the enhanced resistance to the limited proteolysis mediated by protease K and trypsin, and the increased inhibition of auto-cleavage, as comparison to those of the immobilized soluble one. Thus, the TEVp construct is a potential alternate for simplifying protein purification protocols after tag-removal.

Quantitative measurements of free and immobilized RgDAAO Michaelis-Menten constant using an electrochemical assay reveal the impact of covalent cross-linking on substrate specificity.

Challenges facing enzyme-based electrochemical sensors include substrate specificity, batch to batch reproducibility, and lack of quantitative metrics related to the effect of enzyme immobilization. We present a quick, simple, and general approach for measuring the effect of immobilization and cross-linking on enzyme activity and substrate specificity. The method can be generalized for electrochemical biosensors using an enzyme that releases hydrogen peroxide during its catalytic cycle. Using as proof of concept RgDAAO-based electrochemical biosensors, we found that the Michaelis-Menten constant (Km) decreases post immobilization, hinting at alterations in the enzyme kinetic properties and thus substrate specificity. We confirm the decrease in Km electrochemically by characterizing the substrate specificity of the immobilized RgDAAO using chronoamperometry. Our results demonstrate that enzyme immobilization affects enzyme substrate specificity and this must be carefully evaluated during biosensor development.

Surface charge engineering of Thermomyces lanuginosus lipase improves enzymatic activity and biodiesel synthesis.

OBJECTIVES: This study was aimed at engineering charged residues on the surface of Thermomyces lanuginosus lipase (TLL) to obtain TLL variant with elevated performance for industrial applications. RESULTS: Site-directed mutagenesis of eight charged amino acids on the TLL surface were conducted and substitutions on the negatively charged residues D111, D158, D165, and E239 were identified with elevated specific activities and biodiesel yields. Synergistic effect was not discovered in the double mutants, D111E/D165E and D165E/E239R, when compared with the corresponding single mutants. One TLL mutant, D165E, was identified with increased specific activity (456.60 U/mg), catalytic efficiency (kcat/Km: 44.14 s-1 mM-1), the highest biodiesel conversion yield (93.56%), and comparable thermostability with that of the TLL. CONCLUSIONS: Our study highlighted the importance of surface charge engineering in improving TLL activity and biodiesel production, and the resulting TLL mutant, D165E, is a promising candidate for biodiesel industry.

Development of biotinylated and magnetic bead-immobilized enzymes for efficient glyco-engineering and isolation of antibodies.

The chemoenzymatic remodeled monoclonal antidodies with well-defined glycan structure at the Fc domain display improved biological activities, such as ADCC and ADCP, and are more likely to yield a better safety profile by eliminating the non-human glycans derived from CHO cell culture. We covalently immobilize wild type endoglycosidase S (EndoS), fucosidase, and EndoS2 mutant on magnetic beads through a linker to efficiently generate homogeneous antibody glycoforms without additional purification step to remove endoglycosidase and fucosidase. We also used the biotinylated wild type EndoS2 and EndoS2 mutant in combination with covalently immobilized fucosidase on magnetic beads to allow the sequential removal of endoglycosidases and fucosidase for efficient glyco-engineering and isolation of antibodies without purifying deglycosylated antibody intermediate. Notably, the relatively expensive fucosidase can be recovered to reduce the cost, and the strong affinity of streptavidin to biotin would complete the isolation of biotinylated enzymes. We used Trastuzumab as a model to demonstrate both approaches were reliable for the large-scale production and isolation of antibodies without the residual contamination of endoglycosidase to avoid deglycosylation over storage time.

High-level extracellular production and immobilisation of methyl parathion hydrolase from Plesiomonas sp. M6 expressed in Pichia pastoris.

Methyl parathion hydrolase (MPH) hydrolyses methyl parathion efficiently and specifically. Herein, we produced MPH from Plesiomonas sp. M6 using a Pichia pastoris multi-copy expression system. The original signal peptide sequence of the target gene was removed, and a modified coding sequence was synthesised. Multi-copy expression plasmids containing MPH were constructed using pHBM905BDM, and used to generate recombinant strains containing 1, 2, 3 or 4 copies of the MPH gene. The results showed that a higher target gene copy number increased the production of recombinant MPH (MPH-R), as anticipated. The expression level of the recombinant strain containing four copies of the MPH gene was increased to 1.9 U/ml using 500 ml shake flasks, and the specific activity was 15.8 U/mg. High-density fermentation further increased the target protein yield to 18.4 U/ml. Several metal ions were tested as additives, and Ni2+, Co2+ and Mg2+ at a concentration of 1 mM enhanced MPH-R activity by 196%, 201% and 154%, respectively. Enzyme immobilisation was then applied to overcome the difficulties in recovery, recycling and long-term stability associated with the free enzyme. Immobilised MPH-R exhibited significantly enhanced thermal and long-term stability, as well as broad pH adaptability. In the presence of inhibitors and chelating agents such as sodium dodecyl sulphate (SDS), immobilised MPH-R displayed 2-fold higher activity than free MPH-R, demonstrating its potential for industrial application.

Engineered human CYP2C9 and its main polymorphic variants for bioelectrochemical measurements of catalytic response.

Polymorphism is an important aspect in drug metabolism responsible for different individual response to drug dosage, often leading to adverse drug reactions. Here human CYP2C9 as well as its polymorphic variants CYP2C9*2 and CYP2C9*3 present in approximately 35% of the Caucasian population have been engineered by linking their gene to the one of D. vulgaris flavodoxin (FLD) that acts as regulator of the electron flow from the electrode surface to the haem. The redox properties of the immobilised proteins were investigated by cyclic voltammetry and electrocatalysis was measured in presence of the largely used anticoagulant drug S-warfarin, marker substrate for CYP2C9. Immobilisation of the CYP2C9-FLD, CYP2C9*2-FLD and CYP2C9*3-FLD on DDAB modified glassy carbon electrodes showed well defined redox couples on the oxygen-free cyclic voltammograms and mid-point potentials of all enzymes were calculated. Electrocatalysis in presence of substrate and quantification of the product formed showed lower catalytic activities for the CYP2C9*3-FLD (2.73 ± 1.07 min-1) and CYP2C9*2-FLD (12.42 ± 2.17 min-1) compared to the wild type CYP2C9-FLD (18.23 ± 1.29 min-1). These differences in activity among the CYP2C9 variants are in line with the reported literature data, and this set the basis for the use of the bio-electrode for the measurement of the different catalytic responses towards drugs very relevant in therapy.

Directed Enzyme Evolution and Encapsulation in Peptide Nanospheres of Quorum Quenching Lactonase as an Antibacterial Treatment against Plant Pathogen.

The need to increase agricultural yield has led to an extensive use of antibiotics against plant pathogens, which has resulted in the emergence of resistant strains. Therefore, there is an increasing demand for new methods, preferably with lower chances of developing resistant strains and a lower risk to the environment or public health. Many Gram-negative bacterial pathogens use quorum sensing, a population-density-dependent regulatory mechanism, to monitor the secretion of N-acyl-homoserine lactones (AHLs) and pathogenicity. Therefore, quorum sensing represents an attractive antivirulence target. AHL lactonases hydrolyze AHLs and have potential antibacterial properties; however, their use is limited by thermal instability and durability, or low activity. Here, we demonstrate that an AHL lactonase from the phosphotriesterase-like lactonase family exhibits high activity with the AHL secreted from the plant pathogen Erwinia amylovora and attenuates infection in planta. Using directed enzyme evolution, we were able to increase the enzyme's temperature resistance (T50, the temperature at which 50% of the activity is retained) by 8 °C. Then, by performing enzyme encapsulation in nanospherical capsules composed of tertbutoxycarbonyl-Phe-Phe-OH peptide, the shelf life was extended for more than 5 weeks. Furthermore, the encapsulated and free mutant were able to significantly inhibit up to 70% blossom's infection in the field, achieving the same efficacy as seen with antibiotics commonly used today to treat the plant pathogen. We conclude that specific AHL lactonase can inhibit E. amylovora infection in the field, as it degrades the AHL secreted by this plant pathogen. The combination of directed enzyme evolution and peptide nanostructure encapsulation significantly improved the thermal resistance and shelf life of the enzyme, respectively, increasing its potential in future development as antibacterial treatment.

Design of epidermal growth factor immobilization on 3D biocompatible scaffolds to promote tissue repair and regeneration.

Exogenous application of human epidermal growth factor (hEGF) stimulates epidermal wound healing. The aim of this study was to develop bioconjugates based on hEGF mimicking the protein in its native state and thus suitable for tissue engineering applications, in particular for treating skin-related disorders as burns. Ribonuclease A (RNase A) was used to investigate a number of different activated-agarose carriers: cyanogen bromide (CNBr)-activated-agarose and glyoxyl-agarose showed to preserve the appropriate orientation of the protein for receptor binding. EGF was immobilized on these carriers and immobilization yield was evaluated (100% and 12%, respectively). A peptide mapping of unbound protein regions was carried out by LC-MS to take evidence of the residues involved in the immobilization and, consequently, the flexibility and surface accessibility of immobilized EGF. To assess cell proliferative activities, 10, 25, 50, and 100 ng/mL of each immobilized EGF sample were seeded on fibroblast cells and incubated for 24, 48 and 72 h. The immobilized growth factor showed significantly high cell proliferative activity at 50 and 100 ng/mL compared to control and soluble EGF. Although both of the immobilized samples show dose-dependency when seeded with high number of fibroblast cells, CNBr-agarose-EGF showed a significantly high activity at 100 ng/mL and 72 h incubation, compared to glyoxyl-agarose-EGF.

Umbelliferone esters with antibacterial activity produced by lipase-mediated biocatalytic pathway.

OBJECTIVE: The present report describes the enzymatic acylation of umbelliferone with different vinyl esters as acyl donors biocatalyzed by the commercial lipase Novozym® 435, and the investigation for their antibacterial activity against ATCC and clinical strains isolated from hospital infection sites. RESULTS: The umbelliferone esters (1-5) were synthesized through the acylation reaction of 7-hydroxy-2H-chromen-2-one with different long chain vinyl esters catalyzed by the lipase Novozym 435. The reaction conditions were: 10% Novozym 435; tetrahydrofuran:acetone (3:1) for the reactions with acetate, propionate and butyrate vinyl esters 50-90% conversion, and (9:1) for decanoate and laurate vinyl esters 10-15% conversion; acyl donor/umbelliferone molar ratio of 10:1 and 60 °C. All the umbelliferone esters were characterized NMR and (HRMS). The antibacterial activity of the products were tested using the broth microdilution method in order to determine the minimum inhibitory concentration (MIC). The results displayed by 7-laurate and 7-decanoate-umbelliferone esters showed the highest antibacterial potential, with 1 mM inhibitory activity for ATCC 33591, a methicillin and oxacillin resistant Staphylococcus aureus strain. They were also able to inhibit gram-negative bacterial strains, such as Pseudomonas aeruginosa (MIC 0.5 mM) and Klebsiella pneumoniae (MIC 1 mM). In addition, 7-laurate- and 7-decanoate-umbelliferone esters were able to inhibit all clinical strains (MIC 1 mM; except 7-laurate-umbelliferone in which MIC 0.5 mM against 55a). CONCLUSIONS: This is the first study performing the biocatalysis of umbelliferone followed by the purification of the products and the antibacterial evaluation.