Enzyme-catalyzed Method Research Articles

Decreasing acid value of fatty acid ethyl ester products using complex enzymes.

Recently, enzymatic method has been used to prepare biodiesel using various oils. But the high acid value of the biodiesel product using enzyme as a catalyst has been one issue. In this work, an attempt to reduce the acid value of fatty acid ethyl ester (FAEE) product to satisfy the specified requirement (AV ≤ 0.5mgKOH/g), a complex enzyme-catalyzed method was used for the ethanolysis of Semen Abutili seed oil (SASO) (AV = 5.5 ± 0.3mgKOH/g). The effects of various variables (constituents of complex enzyme, type and addition of water removal agent, time, temperature, enzyme addition load, substrate ratio) on the enzymatic reaction were investigated. The optimal reaction conditions were: 1% addition of liquid lipase Eversa® Transform 2.0% and 0.8% of enzyme dry powder CALB, reaction temperature 35°C, alcohol-oil ratio 9:1 (mol/mol), 0.8g/g of 4A-MS and reaction time 24h. Under the optimal reaction conditions, the FAEE yield was 90.8% ± 1.5% and its acid value was decreased from 12.0 ± 0.2mgKOH/g to 0.39 ± 0.10mgKOH/g. In further evaluating the feasibility of preparing FAEE from SASO, the FAEE products obtained under the optimal reaction conditions were purified and evaluated with reference to the ASTM D6751 standard for the main physicochemical indexes. The results obtained were in accordance with the requirements except for the oxidative stability.

Transesterification of phosphatidylcholine with DHA-rich algal oil using immobilized Candida antarctica lipase B to produce DHA-phosphatidylcholine

Docosahexaenoic acid (DHA) enriched with phospholipids (PLs) (DHA-PLs) is a type of structured PL with good physicochemical and nutritional properties. Compared to PLs and DHA, DHA-PLs has higher bioavailability and structural stability and many nutritional benefits. To improve the enzymatic synthesis of DHA-PLs, this study investigated the preparation of phosphatidylcholine (PC) enriched with DHA (DHA-PC) via enzymatic transesterification of algal oil, which is rich in DHA-triglycerides, using immobilized Candida antarctica lipase B (CALB). The optimized reaction system incorporated 31.2% DHA into the acyl chain of PC and converted 43.6% PC to DHA-PC within 72 h at 50 °C, 1:8 PC: algal oil mass ratio, 25% enzyme load (based on total substrate mass), and 0.02 g/mL molecular sieve concentration. Consequently, the side reactions of PC hydrolysis were effectively suppressed and products with high PC content (74.8%) were produced. Molecular structure analysis showed that exogenous DHA was specifically incorporated into the sn-1 site of the PC by immobilized CALB. Furthermore, the evaluation of reusability with eight cycles showed that the immobilized CALB had good operational stability in the present reaction system. Collectively, this study demonstrated the applicability of immobilized CALB as a biocatalyst for synthesizing DHA-PC and provided an improved enzyme-catalyzed method for future DHA-PL synthesis.

High mechanical property and antibacterial poly (3-hydroxybutyrate-co-3-hydroxyvalerate)/functional enzymatically-synthesized cellulose biodegradable composite

Biodegradable materials with antibacterial properties are highly promising. A novel antimicrobial nanocellulose (ECP) was synthesized in one-step by enzyme-catalyzed method to improve the mechanical and antimicrobial properties of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(HB-co-HV)]. The biodegradable nanocomposites were prepared by melt blending and the performance analysis results show that the nanocomposites display enhanced mechanical performances and antibacterial activities. Compared with the neat P(HB-co-HV), the P(HB-co-HV) doped with 0.5 wt%-ECP shows the highest mechanical properties with yield strength/elongation at break of 29.3 MPa, 7.63 %, respectively, an increase of 38 %/59 %, and a clear inhibition zone against Staphylococcus aureus (S. aureus) of approximately 3.0 mm. As a heterogeneous nucleation agent, ECP optimizes nucleation, and the interfacial interaction between phenol group and matrix promotes the compatibility and dispersion of ECP, resulting in superior mechanical properties of ECP-based composites. The P(HB-co-HV)/ECP nanocomposites have great potential in biomedical materials especially for the bone defect filling material.

Eco-friendly and mildly modification of wood cell walls with heat treated wood extracts to improve wood decay resistance

Wood protection using plant-derived extracts has shown some promise but the extracts are often easily leached making them less attractive for long-term wood protection. The potential of extracts from heat treated wood sawdust as a natural substitute for wood preservatives was explored, and a mild enzyme-catalyzed method was used to improve its leaching resistance. Laccase was used to catalyze grafting reactions of extracts from heat treated spruce (Picea asperata Mast.) and larch (Larix gmelinii (Rupr.) Kuzen.). The resulting extracts were screened for antifungal activity and then impregnated into poplar blocks (Populus tomentosa Carr.). Fourier Transform Infrared Spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) were used to characterize extracts reactions. Gas chromatography-mass spectrometry (GC-MS) was used to analyze the grafted compounds. Acetone extracts from both heat treated spruce and larch exhibited good activity against the test fungi. Heat treated spruce and larch acetone extracts grafted on modified poplar and subjected to leaching showed better activity against the white-rot fungus, Trametes versicolor (L. ex Fr.) Quél. than the brown-rot fungus, Gloeophyllum trabeum (Pers.: Fr.) Murr. FTIR and XPS results indicated that heat-treated spruce and larch extracts were grafted on poplar by laccase catalysis. Squalene, 3-hydroxy-4-methoxybenzoic acid, and n-hexadecanoic acid were among those compounds suggested to be grafted. The results suggest that laccase-mediated pre-treatment of plant extracts has the potential to increase resistance to leaching. Trials with additional plant-extracts are recommended.

Enzymatic synthesis of hydrophilic phytosterol polyol esters and assessment of their bioaccessibility and uptake using an in vitro digestion/Caco-2 cell model

A novel enzyme-catalyzed method was developed for the synthesis of phytosterol polyol esters from β-sitosterol and polyols (sorbitol, mannitol and xylitol) by two-step transesterification using divinyl adipate (DVA) as a link. A high conversion (exceeding 94%) of β-sitosterol with a vinyl group was achieved, in the presence of Candida rugosa lipase (CRL), at low temperature (35 °C) within 30 min. Subsequently, the maximum conversion of phytosterol polyol esters (>94%) was obtained using alkaline protease from Bacillus subtilis at 65 °C. Phytosterol polyol esters had enhanced thermal stability (up to an above 355 °C) and excellent water solubility (4.6–7.9 mM at 35 °C). Moreover, obvious increases in the bioaccessibility (41.5–63.6%) and intestinal uptake (5.2–6.5%) were observed using a simulated gastrointestinal digestion/Caco-2 cell model. These results highlighted the key role of hydrophilic structural modifications on physicochemical properties and absorption of phytosterols.

An improved method to determine the hydroxypropyl content in modified starches by 1H NMR

Hydroxypropyl (HP) starches are used as thickeners and stabilizers in foods. The properties of the etherified starches are dependent on the degree of hydroxypropylation. The objective of this study was to develop an improved acid hydrolysis method to degrade and solubilize HP starches for determining HP content in modified starches by 1H NMR. The effects of acid (DCl) concentration and temperature on hydrolysis and solubilization of hydroxypropylated starches were examined. The optimal conditions proposed to hydrolyze HP starches were 10% starch solid content, 3.5% (wt%) DCl in D2O as the solvent, sodium acetate as an internal standard, and heating at 90 °C for 1 h. Optionally, 6% (v/v) TFA-d may be added as the water-peak shifting reagent. Six hydroxypropylated starches and two cross-linked and hydroxypropylated starches were hydrolyzed with this improved acid method, and the results of the HP content were in agreement with those from an enzyme-catalyzed method.

Protein Labeling via a Specific Lysine-Isopeptide Bond Using the Pilin Polymerizing Sortase from Corynebacterium diphtheriae.

Proteins that are site-specifically modified with peptides and chemicals can be used as novel therapeutics, imaging tools, diagnostic reagents and materials. However, there are few enzyme-catalyzed methods currently available to selectively conjugate peptides to internal sites within proteins. Here we show that a pilus-specific sortase enzyme from Corynebacterium diphtheriae (CdSrtA) can be used to attach a peptide to a protein via a specific lysine-isopeptide bond. Using rational mutagenesis we created CdSrtA3M, a highly activated cysteine transpeptidase that catalyzes in vitro isopeptide bond formation. CdSrtA3M mediates bioconjugation to a specific lysine residue within a fused domain derived from the corynebacterial SpaA protein. Peptide modification yields greater than >95% can be achieved. We demonstrate that CdSrtA3M can be used in concert with the Staphylococcus aureus SrtA enzyme, enabling dual, orthogonal protein labeling via lysine-isopeptide and backbone-peptide bonds.

Peptide Synthesis through Cell-Free Expression of Fusion Proteins Incorporating Modified Amino Acids as Latent Cleavage Sites for Peptide Release.

Chlorinated analogues of Leu and Ile are incorporated during cell-free expression of peptides fused to protein, by exploiting the promiscuity of the natural biosynthetic machinery. They then act as sites for clean and efficient release of the peptides simply by brief heat treatment. Dehydro analogues of Leu and Ile are similarly incorporated as latent sites for peptide release through treatment with iodine under cold conditions. These protocols complement enzyme-catalyzed methods and have been used to prepare calcitonin, gastrin-releasing peptide, cholecystokinin-7, and prolactin-releasing peptide prohormones, as well as analogues substituted with unusual amino acids, thus illustrating their practical utility as alternatives to more traditional chemical peptide synthesis.

Fast in situ generated ɛ-polylysine-poly (ethylene glycol) hydrogels as tissue adhesives and hemostatic materials using an enzyme-catalyzed method.

In this study, novel bio-inspired in situ hydrogels as tissue adhesives and hemostatic materials were designed and prepared based on ɛ-polylysine-grafted poly(ethylene glycol) and tyramine via enzymatic cross-linking. The enzymatic cross-linked method enabled fast gelation within seconds, which facilitated its therapeutic applications. By changing the cross-linking conditions, the storage modulus of the hydrogels could be tunable and the mechanical strength influenced the tissue adhesiveness of the hydrogels. Besides, the hydrogels showed fine network structures with appropriate pore sizes, which were thought to be a contributing factor to the strong adhesiveness. Benefiting from the strong mechanical properties and fine network structures, the ɛ-polylysine-grafted poly(ethylene glycol) and tyramine hydrogels exhibited superior wound-healing and hemostatic ability compared to conventional and commercially available medical materials. Moreover, indirect cytotoxicity assessment indicated that the ɛ-polylysine-grafted poly(ethylene glycol) and tyramine hydrogels were nontoxic to the L929 cell. These results demonstrated that the enzymatic cross-linked in situ ɛ-polylysine hydrogels hold high potential for tissue sealants and hemostatic materials.

A Performance Evaluation of Glycolipid Biosurfactant Synthesized by the Enzyme-catalyzed Method

The practice of enhanced oil recovery (EOR) technique shows that the study and application of biosurfactant flooding system have a vast potential for future development. The authors challenge the traditional idea that the oil displacement surfactant must have ultra low interfacial tension. By changing the wettability of reservoir rock as main target, the glycolipid biosurfactant compounded system was developed by enzyme-catalyzed method in laboratory, and a series of experiments had been done combining with reservoir physical measurement. The properties of the active system were characterized by interfacial characters, disbonded, seepage characteristics, antibiotic property, and oil displacement efficiency.