Combined Use Of Enzymes Research Articles

Optimizing the Integration of Microwave Processing and Enzymatic Extraction to Produce Polyphenol-Rich Extracts from Olive Pomace.

The integration of green technologies such as microwave- and enzyme-assisted extraction (MEAE) has been shown to improve the extraction efficiency of bioactive compounds while reducing processing time and costs. MEAE using tannase alone (MEAE-Tan), or in combination with cellulase and pectinase (MEAE-Tan-Cel-Pec), was optimized to produce enriched phenolic and antioxidant extracts from olive pomace. The individual and integrated impact of enzyme concentration, temperature, and pomace/water ratio were determined using a central composite rotatable design. Optimal extraction conditions for MEAE-Tan (60 °C, 15 min, 2.34% of enzyme (w/w), and 1:15 pomace/water ratio) and MEAE-Tan-Cel-Pec (46 °C, 15 min, 2% of enzymes (w/w), in the proportion of 1:1:1, and 1:20 pomace/water ratio) resulted in extracts containing 7110.6 and 2938.25 mg GAE/kg, respectively. The antioxidant activity of the extracts was correlated with phenolic acid release, which was enzyme-dependent, as determined with HPLC-DAD analysis. Enzyme selection had a significant impact on the phenolic profile of extracts, with tannase releasing high concentrations of chlorogenic acid and the combined use of enzymes releasing high concentrations of hydroxytyrosol and chlorogenic and ferulic acids. The novelty of this study relies on the integration and optimization of two green technologies (microwave- and enzyme-assisted extraction) to improve the extraction efficiency of bioactive phenolics from olive pomace while reducing processing time and costs. While these techniques have been evaluated isolated, the benefits of using both processing strategies simultaneously remain largely unexplored. This study demonstrates the effectiveness of the integration and processing optimization of two environmentally friendly technologies as a promising alternative to treat agro-industrial byproducts.

Optimization of Ultrasonic-Assisted Enzymatic Extraction of Freeze-Dried Sea Buckthorn (Hippophae rhamnoides L.) Berry Oil Using Response Surface Methodology

The ultrasound-assisted extraction (UAE) of oil has received immense importance nowadays because of the enormous benefits the process offers. However, the literature evaluating this process is scarce for sea buckthorn oil. Furthermore, to date, to the best of our knowledge, a study evaluating the combined use of enzymes and UAE for this oil is lacking. In this study, oil from freeze-dried sea buckthorn berries was extracted using ultrasound-assisted enzymatic extraction (UAEE) and the effect of variables (time, enzyme concentration, and solvent to sample ratio) was evaluated on oil yield and its physiochemical properties (acid value, peroxide value, iodine value, density, and color). The optimum conditions were determined using the response surface methodology. The optimum conditions established were 5.08 mL/g, 14.65 min, and 3.13 U/g for a solvent to sample ratio, ultra-sonication time, and enzyme units, respectively. The oil yield was 18.32%. Physicochemical parameters were found better in UAEE oil than in the Soxhlet-extracted oil. Gas chromatography detected relatively higher levels of fatty acids, including palmitic, palmitoleic, and oleic acids in UAEE oil. The optimum conditions were also verified for adequacy by validation and results were matched with predicted values with 0.8 to 1.5 error %, which states that the model can be utilized to predict oil yield percentage.

Combination of carbohydrases and essential oils improve dietary performance of feedlot steers on a high-energy diet

Essential oils and enzymes are alternatives to feed additives for ruminants that aim to replace the use of ionophores and improve animal performance, but their mechanisms of action are different. Therefore, the present study aimed to verify if there is a synergistic effect in the combined use of enzymes carbohydrates and essential oils on the performance, ingestive behavior and carcass traits of steers fed a high-energy diet. During the finishing period of 78 days, 40 steers were assigned to four treatments: CON- control; ENZ- enzymatic complex; EO- essential oil blend; ENZ+EO - enzymatic complex combined with essential oil blend. Regardless of the feedlot periods, the ENZ+EO treatment caused a reduction in the dry matter intake (12.48%) compared to the control. The ENZ+EO treatment resulted in the lowest mean fecal output and, consequently, the highest dry matter digestibility (DMD) and starch digestibility (SD), compared to the other treatments. Animals that received EO and ENZ+EO in the diet spent more time in feeding. As for the number of times animals visited the feeding trough, the highest values were presented by the animals in the EO, ENZ and ENZ+EO treatments. For the carcass parameters, only the subcutaneous fat thickness on the rib was significantly different between treatments, with the highest values obtained by adding EO and ENZ+EO (8.80 and 8.10 mm respectively). Thus, the combination of carbohydrate enzymes and essential oils proved to be synergistically beneficial in relation to better use of nutrients and productive performance of feedlot steers.

Enzyme–metal nanocomposites for antibacterial applications

Metal nanoparticles have been used as antibacterial agents widely, and the combined use of enzymes and metal nanoparticles promotes antibacterial activity, achieving a synergistic effect. Additionally, enzymes decrease the amounts of metals and increase biocompatibility, thereby reducing toxicity of metals. However, the efficiency of enzymes is hindered when coupled with metals, which causes deactivation in the function of enzymes. How can a balance be struck between metals and enzymes? Although the antibacterial mechanism of metal nanoparticles is relatively clear, how enzyme–metal nanocomposites work against bacteria is not conclusive. Here, we describe several examples on the synthesis of enzyme–metal nanocomposites via co-immobilization or in situ reduction and summarize how enzyme–metal nanocomposites combat microorganisms.

Capillary electrophoresis with stationary nanogel zones of galactosidase and Erythrina cristagalli lectin for the determination of β(1–3)-linked galactose in glycans

A thermally responsive nanogel is used to create stationary zones of enzyme and lectin in a separation capillary. Once patterned in the capillary, analyte is driven through the zone, where it is converted to a specific product if an enzyme is used or captured if a lectin is used. These stationary zones are easily expelled after the analysis and then re-patterned in the capillary. The nanogel is compatible with enzymes and lectins and improves the stability of galactosidase, enabling more cost-effective use of biological reagents that provide insight into glycan structure. A feature of using stationary zones is that the reaction time can be controlled by the length of the zone, the applied field controlling the analyte mobility, or the use of electrophoretic mixing by switching the polarity of the applied voltage while the analyte is located in the zone. The temperature, applied voltage, and length of the stationary zone, which are factors that enhance the performance of the enzyme, are characterized. The combined use of enzymes and lectins in capillary electrophoresis is a new strategy to advance rapid and automated analyses of glycans using nanoliter volumes of enzymes and lectins. The applicability of this use of stationary zones of enzyme and lectin in capillary electrophoresis is demonstrated with the identification of β(1–3)-linked galactose in N-glycan.

Biosynthesis of polyhydroxyalkanoates co-polymer in E. coli using genes from Pseudomonas and Bacillus

Expression of Pseudomonas aeruginosa genes PHA synthase1 (phaC1) and (R)-specific enoyl CoA hydratase1 (phaJ1) under a lacZ promoter was able to support production of a copolymer of Polyhydroxybutyrate (PHB) and medium chain length polyhydoxyalkanoates (mcl-PHA) in Escherichia coli. In order to improve the yield and quality of PHA, plasmid bearing the above genes was introduced into E. coli JC7623, harboring integrated beta-ketothiolase (phaA) and NADPH dependent-acetoacetyl CoA reductase (phaB) genes from a Bacillus sp. also driven by a lacZ promoter. The recombinant E. coli (JC7623ABC1J1) grown on various fatty acids along with glucose was found to produce 28-34% cellular dry weight of PHA. Gas chromatography and (1)H Nuclear Magnetic Resonance analysis of the polymer confirmed the ability of the strain to produce PHB-co-Hydroxy valerate (HV)-co-mcl-PHA copolymers. The ratio of short chain length (scl) to mcl-PHA varied from 78:22 to 18:82. Addition of acrylic acid, an inhibitor of beta-oxidation resulted in improved production (3-11% increase) of PHA copolymer. The combined use of enzymes from Bacillus sp. and Pseudomonas sp. for the production of scl-co-mcl PHA in E. coli is a novel approach and is being reported for the first time.