Enzymatic Degradation Process Research Articles

Mycoremediation of Remazol Brilliant Blue R in greywater by a novel local strain of Aspergillus iizukae 605EAN: optimisation and mechanism study

ABSTRACT The decolourisation of Remazol Brilliant Blue R (RBBR) by oxidative enzymes [Laccase (Lac), Manganese peroxidase (MnP), Lignin peroxidase (LiP)], from Aspergillus iizukae EAN605 was optimised as a function of initial substrate concentrations (30−100 mg L−1), incubation period (5–15 days) and pH (5–7). The best operating parameters to achieve the highest removal of RBBR were 57.15 mg 100 mL−1 of RBBR, pH 6 and after 8.55 days where the maximum removal was 78.34% vs. 80.00% (R 2 = 77.9%), Lac 0.22 vs. 0.28 µ mg−1, MnP 0.24 vs. 0.27 µ mg−1 and LiP 14.22 vs. 15.12 µ mg−1 of actual and predicted results, respectively. The surface analysis of A. iizukae EAN605 by field emission scanning electron microscopy suggested that the removal process takes place on the surface of the fungal mycelium cell wall as a result of the presence of Lac enzyme in the inner fungal membrane. Moreover, the phytotoxicity test confirms the absence of the dye toxicity after enzymatic degradation process which indicates the efficiency of A. iizukae EAN605 to minimise the dyes' risk to less than the toxicity level. These findings suggest that A. iizukae EAN605 has a potential for the mycoremediation of dyes in the greywater.

Synthesis, self-assembly, biodegradation and drug delivery of polyurethane copolymers from bio-based poly(1,3-propylene succinate)

A series of environmentally-friendly bio-based poly(1,3-propylene succinate) diols (bio-PPS) with different average molecular weights (Mn) were synthesized from bio-based 1,3-propanediol (bio-PDO) and bio-based succinic acid (bio-SA). With the employment of bio-PPS as a hydrophobic block and poly(ethylene glycol) (PEG) as a hydrophilic segment, the well-defined amphiphilic triblock and multi-block polyurethane (PU) copolymers with the urethane linkages between the blocks were synthesized and characterized. The chemical structures and molecular weights of these block PU copolymers were confirmed by proton nuclear magnetic resonance (1H NMR), Fourier transform infrared (FT-IR) spectrum, differential scanning calorimetry (DSC), and gel permeation chromatography (GPC). With the Mn of bio-PPS blocks increasing from 1.40 to 8.93 kg/mol, the degree of hydrogen bonding in the PUs reduced significantly, and the glass transition temperature increased from −42.8 to −35.4 °C and from −40.1 to −33.1 °C for triblock and multi-block copolymers, respectively. Transmission electron microscopy (TEM) images revealed that both triblock and multi-block PU copolymers could spontaneously self-assemble into near-spherical core-shell micelles in aqueous solution. With the increasing bio-PPS content in the copolymers, the sizes of triblock micelles from dynamic light scattering (DLS) measurement varied from 17 to 64 nm, and the critical micelle concentration (CMC) changed from 7.22 to 0.77 mg/L. For the multi-block series, the micelle sizes increased from 24 to 71 nm and the CMC values decreased from 2.85 to 1.79 mg/L. The enzymatic degradation of the micelles for 8 weeks under the physiological environment revealed that hydrophilic PEG segments could be detached from the main chain of triblock micelles, and the degradation mainly occurred at the ester group of bio-PPS blocks in the multi-block ones. Micelles with lower bio-PPS content and multi-block architecture degraded faster during the enzymatic degradation process. A controlled release profile was observed for the doxorubicin (DOX)-loaded micelles, more than 40% and 50% DOX were released after 75 h from triblock and multi-block micelles, respectively. Moreover, the addition of lipase could accelerate the DOX release from the micelles. Cytotoxicity test proved that the PU micelles were nontoxic, while the DOX loaded micelles showed concentration-dependent cytotoxicity to HeLa cells, suggesting its potential application in the drug delivery system.

Enhanced effects of curcumin encapsulated in polycaprolactone-grafted oligocarrageenan nanomicelles, a novel nanoparticle drug delivery system

One of the most effective strategies to enhance the bioavailability and the therapeutic effect of hydrophobic drugs is the use of nanocarriers. We have used κ-carrageenan extracted from Kappaphycus alvarezii to produce oligocarrageenan via an enzymatic degradation process. Polycaprolactone (PCL) chains were grafted onto the oligocarrageenans using a protection/deprotection technique yielding polycaprolactone-grafted oligocarrageenan. The resulting amphiphilic copolymers formed spherical nanomicelles with a mean size of 187 ± 21 nm. Hydrophobic drugs such as curcumin were efficiently encapsulated in the micelles and released within 24–72 h in solution. The micelles were non-cytotoxic and facilitated the uptake of curcumin by endothelial EA-hy926 cells. They also increased the anti-inflammatory effect of curcumin in TNF-alpha-induced inflammation experiments. Finally, in vivo experiments supported a lack of toxicity in zebrafish and thus the potential use of polycaprolactone-grafted oligocarrageenan to improve the delivery of hydrophobic compounds to different organs, including liver, lung and brain as shown in mice.

Selective enzymatic degradation and porous morphology of poly(butylene succinate)/poly(lactic acid) blends

Poly(butylene succinate) (PBS) and poly(lactic acid) (PLA) were melt-blended in different proportions and selectively degraded by cutinase and proteinase K, respectively. The selective enzymatic degradation process was systematically investigated. The degraded PBS/PLA blends were analyzed via scanning electron microscopy, Fourier-transform infrared spectroscopy, powder X-ray diffraction, and differential scanning calorimetry. The results of the weight loss of PBS/PLA blends degraded by cutinase and proteinase K suggested that PLA hindered the cutinase-catalyzed degradation of PBS, whereas the addition of PBS in the blends accelerated the degradation of PLA within a specific PBS/PLA ratio. The change in crystallinity after degradation was closely related to the different way of degradation. The characterization of PBS/PLA blends after degradation showed that selective enzymatic degradation could not completely degrade PBS or PLA component. After degradation, the pores formed by proteinase K were more uniform and larger than those formed by cutinase. This work provides a new insight into the selective enzymatic degradation processes of the porous materials, which will be used in tissue engineering or oil-water separation in the future.

Direct yellow degradation by combined Fenton-enzymatic process

Abstract The removal of direct yellow 106 (DY106, C.I.40300) by a combined process of Fenton oxidation and enzymatic degradation in a sequential or mixed batch reactor is discussed. Experiments were first conducted with the enzymatic and chemical oxidation processes separately in order to determine the effects of various parameters such as pH, ferrous ion, hydrogen peroxide, dye and enzyme concentrations on the overall yield and kinetics of both processes. Decolourization was followed by UV-visible spectroscopy and high performance liquid chromatography (HPLC). Results showed that 89.5 % of DY106 were removed by enzymatic treatment after only 2 min, while 10 min of contact time were necessary to eliminate more than 98 % of 50 mg/L by Fenton’s process. A high performance was achieved under optimized conditions by the mixed combined process with time reduction down to 5 min. The study was also conducted to evaluate the efficiency of combined Fenton’s reaction as a pre-treatment and post treatment process combined with C-peroxidase at different ferrous ions concentrations. The optimal doses of Fe2+ were 2.5 mM, 1m M and 0.8 mM for Fenton, Fenton-enzymatic sequential and mixed processes. Intermediate products absorbing in UV range were detected for single Fenton or enzymatic treatment but were eliminated in all combined enzymatic-Fenton processes. Phytotoxicity tests showed that no toxicity was detected after treatment by combined process.

A Study of the Viscosity Characteristics of Chitosan Solutions in the Presence of Organic Cosolvents

The effect of organic cosolvents (ethanol and isopropanol) on the viscosity characteristics of acetic acid aqueous solutions of chitosan is studied. Sorption and deformation–strength properties, as well as resistance to the process of enzymatic degradation of materials obtained from solutions, are determined. It is shown that the introduction of cosolvents into the chitosan–1% acetic acid system is accompanied by a consistent decrease in the intrinsic viscosity of polymers. It is found that, in the presence of cosolvents, at concentrations below the crossover concentration, chitosan molecules exist as systems of interacting (aggregated) macromolecules rather than isolated macromolecular coils. A change in the conformation of chitosan and the type of macromolecular aggregates in acetic acid solutions in the presence of cosolvents leads to a number of consequences: polymer units become less accessible to an enzyme preparation, the rate of enzymatic degradation decreases, and mechanical characteristics of film materials change.

Understanding the dynamics of keratin weakening and hydrolysis by proteases.

Keratin is the structural protein in hair, nails, feathers and horns. Keratin is recalcitrant, highly disulfide bonded and is generally inaccessible to common proteases. Only certain types of proteases, called keratinases, are able to cleave the peptide bonds within the keratin structure. Due to this outstanding activity, keratinases have potential application in industries such as livestock, cosmetics and pharmaceuticals. Yet, the process of enzymatic keratin degradation is poorly understood, affecting the development of industrial enzyme formulations that may require full or only partial modification or weakening. Here we investigate the dynamics of keratin weakening and hydrolysis, showing that the decrease in hair mechanical strength is associated with cuticle removal and damage to the cortex and complete breakdown is dependent on reducing agents. Proteases with keratinolytic activity were selected and applied to hair with degradation examined by mechanical, biochemical and microscopic techniques. The extent of keratin degradation was highly enhanced by the presence of reducing agents, principally sodium thioglycolate, exceeding 90% degradation within 16 h of enzymatic treatment. Application was extended to feathers showing that the findings are relevant to improving the use of keratinases in a variety of industries. Overall, the outcomes provide valuable insights into the keratin degradation process by enzymes for the optimization of cosmetic and pharmaceutical products and for livestock waste recycling among other important applications.

Highly branched linear-comb random copolyesters of ε-caprolactone and δ-valerolactone: Isodimorphism, mechanical properties and enzymatic degradation behavior

In this work, a series of highly branched linear-comb random copolyesters of ε-caprolactone (CL) and δ-valerolactone (VL) with high molecular weight and narrow distribution in various compositions were successfully synthesized. The composition-dependent microstructure, thermal and mechanical properties, and enzymatic degradation behavior of the resulting copolyesters were investigated systematically. The cocrystallization behavior was observed for the highly branched linear-comb random CL/VL copolyesters over the entire composition range, and a pseudo-eutectic point of isodimorphism occurred at about 50 mol% VL incorporated in copolymer. The obtained copolymers displayed lower Young's modulus, yield strength, and breaking strength, but higher elongation at break. Furthermore, the copolymers presented much higher degradation rate during enzymatic degradation process in comparison with parent homopolymers. More importantly, it was found that both mechanical properties and degradation behavior showed a similar pseudo-eutectic behavior of isodimorphism. Consequently, the correlation among of isodimorphism, mechanical properties, and degradation behavior depending on composition is highly significant, and the physical properties and degradation rate of the isodimorphic copolyesters could be continuously tailored by the feedstocks.

Umbelliferone instability during an analysis involving its extraction process

Umbelliferone (7-hydroxycoumarin) is one of the most popular compounds of the coumarins family. This compound receives the attention of scientists due to its diverse bioactivities in a number of applications in various therapeutic fields. An interesting aspect of umbelliferone is its structural lability. The enzymatic degradation process of umbelliferone to its hydroxylated (esculetin), glucosylated (skimmin), and methylated (herniarin) derivatives is already known from the literature. In this paper, we describe the possibility of umbelliferone transformation to other derivatives. We found that eight compounds were formed from umbelliferone during its simulated extraction under reflux performed in different conditions (different heating times and solvents used). Six of them (4,7-dihydroxy-3,4-dihydro-2H-chromen-2-one, 3,7-dihydroxy-3,4-dihydro-2H-chromen-2-one, methyl (2E)-3-(2,4-dihydroxyphenyl)prop-2-enoate, ethyl (2E)-3-(2,4-dihydroxyphenyl)prop-2-enoate, (2E)-3-[2-(acetyloxy)-4-hydroxyphenyl]prop-2-enoic acid, (2E)-3-(2-amino-4-hydroxyphenyl)prop-2-enoic acid) have not been reported yet. Some of these compounds were also identified in extracts of plant materials containing umbelliferone—chamomile flower and cinnamon bark. Compound separation was carried out using the HPLC apparatus. All compounds were identified based on their MS fragmentation paths. The presented results are useful for food producers and consumers, as umbelliferone transformation products can be formed during food product storage, their preparation or processing.Graphical abstract

Analysis of D-dimer laboratory findings and clinical evaluation

D-dimer is a product of the enzymatic degradation of the fibrinogen-fibrin molecule, and its existence is demonstrable in circulation. The test based limits may be considered as normal values. It was first thought to be a product of coagulation, then a product of lysis. High-concentration D-dimer in blood detected in thromboembolic diseases is considered to be of diagnostic value. In cases where thromboembolism was ruled out despite elevated titres but heparin (LMWH) or CLOPIDOGREL was given as a cautionary measure, we found that D-dimer values remained elevated. This finding means that in vivo coagulation is not a precondition to D-dimer formation. Analysis of such cases uncovers liver or kidney disease in the background, but old age may also be a factor. Often elevated ferritin levels were observed 'in parallel' with elevated D-dimer values. These findings lead us to presume an enzymatic degradation process of 'elderly' protein molecules, which is universally applicable. Orv Hetil. 2017; 158(50): 1971-1976.

Application of Electron Paramagnetic Resonance and Solid-state 13C Nuclear Magnetic Resonance of Cross-polarization/Magic Angle Spinning to Study Enzymatic Degradation of Silk Fabrics.

The enzymatic degradation of silk by protease XIV has been investigated by using scanning electron microscopy (SEM), Fourier transfer infrared spectroscopy (FTIR), solid-state 13C nuclear magnetic resonance of cross-polarization/magic angle spinning (13C CP/MAS solid state NMR) and electron paramagnetic resonance (EPR). Micro-morphology of protease XIV aged samples showed that microfilaments were stripped out from the surface of silk fibers. The results of FTIR and 13C CP/MAS solid-state NMR indicated that the enzymatic degradation process could be divided into two stages. The EPR spectra indicated that the enzymatic degradation process was related to the free radical with the g-factor value of 2.0043. We also proposed that at the first degradation stage, the free radicals were apt to lose activities due to the loose structure of the non-crystalline region; at the second degradation stage, the free radicals produced in the crystalline region tended to be stored.

A facile strategy for fabricating PCL/PEG block copolymer with excellent enzymatic degradation

Poly(ω-caprolactone) (PCL) has been the focus in many research initiatives, especially regarding various ways though which to introduce reactive sites to the polyester chains. In this work, hydroxyl groups were covalently introduced into the chain of PCL by reacting ester groups with 6-amino-1-hexanol. Poly(ethylene glycol) (PEG) was crosslinked to the PCL chain through esterification, by using N,N’-carbonyldiimidazole (CDI) as catalyst. The prepared PCL-PEG copolymers were characterized by various analytical techniques such as Fourier transform infrared spectroscopy (FT-IR), 1H NMR and X-ray photoelectron spectroscopy (XPS). Static water contact angles measurement indicated that hydrophilicity of the copolymer films modified by PEG has improved considerably. The copolymer films were prepared by casting the polymer solution onto a stainless plate. The degradation of the copolymer samples, in a phosphate buffer solution containing a type of Lipase (Aspergillus Oryzae), was observed at different time intervals. The results of weight loss ratio, during the degradation process, indicated that blocking PEG chains onto the PCL chains can effectively accelerate the process of enzymatic degradation of the resulting copolymer. The changes in molecular weight of the copolymer films were investigated by Gel Permeation Chromatography (GPC) and the copolymers were characterized by 1H NMR during degradation. Surface morphology of PCL and PCL-PEG polymers, before and after degradation, was observed by Scanning electron microscopy (SEM), and emphasized that the enzymatic degradation of PCL-PEG copolymers occurred faster by comparison with PCL, starting from the surface layer of the films.

Recent technologies in insulin delivery systems /Yeni teknolojilerle insulin uygulama yollari

Studies for different ways of insulin delivery have started since the first discovery of insulin. However, desired biological effect of non-parenteral routes has not been achieved, yet. Unfortunately the use of insulin has been limited to parenteral routes due to enzymatic degradation process by mucosal peptidase, mucosal barrier resulting in insufficient absorption and poor permeability throughout the intestinal mucosa. The parenteral route of insulin has been used in different ways including intramuscular, subcutaneous, intravenous and intraperitoneal ways. However, the parenteral route has side effects involving patient's incompliance due to fear of injection, local discomfort including bleeding at injection site, injection pain, lipohypertrophy as well as some disadvantages such as glycemic fluctuation. Alternative routes of non-invasive insulin delivery including oral, nasal, buccal, ophtalmic, rectal, vaginal and transdermal systems have been performed eventhough successful results have not been achived due to the abovementioned barriers. Additionally, recently approved tecnology of insulin delivery through pulmoner route has also been one of the methods aimed at replacing parenteral route. However, recent pulmonary technology of insulin delivery requires higher doses and frequent applications. Furthermore, inhaler insulin is not applicable to smokers and is not used in patients with pulmonary diseases or infections. Today, desired clinical efficacy and safety on the use of non-invasive routes of insulin have not yet been achieved and studies are continuing with newly developed technologies.

A Weibull statistics-based lignocellulose saccharification model and a built-in parameter accurately predict lignocellulose hydrolysis performance.

Renewable energy from lignocellulosic biomass has been deemed an alternative to depleting fossil fuels. In order to improve this technology, we aim to develop robust mathematical models for the enzymatic lignocellulose degradation process. By analyzing 96 groups of previously published and newly obtained lignocellulose saccharification results and fitting them to Weibull distribution, we discovered Weibull statistics can accurately predict lignocellulose saccharification data, regardless of the type of substrates, enzymes and saccharification conditions. A mathematical model for enzymatic lignocellulose degradation was subsequently constructed based on Weibull statistics. Further analysis of the mathematical structure of the model and experimental saccharification data showed the significance of the two parameters in this model. In particular, the λ value, defined the characteristic time, represents the overall performance of the saccharification system. This suggestion was further supported by statistical analysis of experimental saccharification data and analysis of the glucose production levels when λ and n values change. In conclusion, the constructed Weibull statistics-based model can accurately predict lignocellulose hydrolysis behavior and we can use the λ parameter to assess the overall performance of enzymatic lignocellulose degradation. Advantages and potential applications of the model and the λ value in saccharification performance assessment were discussed.

Efficient enzymatic degradation process for hydrolysis activity of the Carrageenan from red algae in marine biomass

Carrageenan is a generic name for a family of polysaccharides obtained from certain species of red algae. New methods to produce useful cost-efficiently materials from red algae are needed to convert enzymatic processes into fermentable sugars. In this study, we constructed chimeric genes cCgkA and cCglA containing the catalytic domain of κ-carrageenase CgkA and λ-carrageenase CglA from Pseudoalteromonas carrageenovora fused with a dockerin domain. Recombinant strains expressing the chimeric carrageenase resulted in a halo formation on the carrageenan plate by alcian blue staining. The recombinant cCgkA and cCglA were assembled with scaffoldin miniCbpA via cohesin and dockerin interaction. Carbohydrate binding module (CBM) in scaffoldin was used as a tag for cellulose affinity purification using cellulose as a support. The hydrolysis process was monitored by the amount of reducing sugar released from carrageenan. Interestingly, these results indicated that miniCbpA, cCgkA and cCglA assembled into a complex and that the dockerin-fused enzymes on the scaffoldin had synergistic activity in the degradation of carrageenan. The observed enhancement of activity by carrageenolytic complex was 3.1-fold-higher compared with the corresponding enzymes alone. Thus, the assemblies of advancement of active enzyme complexes will facilitate the commercial production of useful products from red algae biomass which represents inexpensive and sustainable feed-stocks.

Xyloglucan–Cellulose Nanocrystal Multilayered Films: Effect of Film Architecture on Enzymatic Hydrolysis

Understanding the hydrolysis process of lignocellulosic substrates remains a challenge in the biotechnology field. We aimed here at investigating the effect of substrate architecture on the enzymatic degradation process using two different multilayered model films composed of cellulose nanocrystals (CNCs) and xyloglucan (XG) chains. They were built by a spin-assisted layer-by-layer (LbL) approach and consisted either of (i) an alternation of CNC and XG layers or of (ii) layers of mixed (CNC/XG) complexes alternated with polycation layers. Neutron reflectivity (NR) was used to determine the architecture and composition of these films and to characterize their swelling in aqueous solution. The films displayed different [XG]/[CNC] ratios and swelling behavior. Enzymatic degradation of films was then performed and investigated by quartz crystal microbalance with dissipation monitoring (QCM-D). We demonstrated that some architectural features of the substrate, such as polysaccharide accessibility, porosity, and cross-links, influenced the enzymatic degradation.

Hemoglobin – source of reactive oxygen species

Erythrocytes are especially vulnerable to reactive oxygen species because of their direct role in oxygen transport. Moreover, hemoglobin contains iron ions (Fe²⁺), which catalyze both the Fenton reaction and lipid peroxidation. Reactive oxygen species in erythrocytes are also generated through nonenzymatic and enzymatic processes of heme degradation. The nonenzymatic process of heme degradation is initiated by e.g. hydrogen peroxide, whereas the process of enzymatic degradation is under the influence of heme oxygenase. In both cases biliverdin, carbon monoxide (CO) and iron ions (Fe²⁺) are generated. These products of heme degradation can initialize the oxidative processes within erythrocytes, but at low concentrations exhibit cytoprotective properties.

Statistical optimization of enzymatic degradation process for oil palm empty fruit bunch (OPEFB) in rotary drum bioreactor using crude cellulase produced from Aspergillus niger EFB1

Oil palm empty fruit bunch (OPEFB) was pretreated with 2% (v/v) HNO3 and degraded by Aspergillus niger EFB1 crude cellulase. Through 2 Level Factorial Design (2LFD), it was found that OPEFB concentration, temperature, incubation time, concentration of Tween 80 and agitation speed have significant effect in reducing sugar production. A standard Response Surface Methodology (RSM) design known as Central Composite Design (CCD) was used to optimize the enzymatic degradation condition of OPEFB in rotary drum bioreactor. Reducing sugar level of 1.183g/L was obtained with the following optimized degradation conditions: 1.95% (w/v) OPEFB, 0.5% (v/v) Tween 80, 55°C, 87.5rpm in the incubation period of 3 days and 16h. The optimal degradation condition improved reducing sugar production by 1.07 fold compared to that before optimization in shake flasks culture. The optimization strategy of enzymatic degradation of OPEFB inside rotary drum bioreactor led to increase in glucose, xylose, arabinose, galactose and mannose production by 3, 2.5, 1.64, 19.37 and 22.52 fold, respectively. The improvement in reducing sugar and polyoses production were comparable with the reduction in OPEFB cellulose and hemicellulose content by 89.32% and 48.17% respectively after enzymatic degradation in optimized condition.

Glycan Sequence‐Dependent Nod2 Activation Investigated by Using a Chemically Synthesized Bacterial Peptidoglycan Fragment Library

Nucleotide oligomerization domain-containing protein 2 (Nod2), an innate immune receptor, recognizes bacterial cell-wall peptidoglycan (PGN), the minimum ligand of which is muramyl dipeptide (MDP). Enzymatic digestion of PGN appears to be important for Nod2 recognition. PGN is degraded by muramidase or glucosamidase through a process that produces two types of glycan sequence; glycans containing GlcNAcβ(1→4)MurNAc or MurNAcβ(1→4)GlcNAc. In this report, a range of disaccharide or tetrasaccharide fragments of each sequence were chemically synthesized, and their activities in stimulating human Nod2 (hNod2) were investigated. The results reveal that hNod2 recognitions is dependent on the glycan sequence, as demonstrated by comparing the activities of glycans with the same peptide moieties. (MurNAcβ(1→4)GlcNAc)(2) -containing structures exhibited stronger activity than those containing (GlcNAcβ(1→4)MurNAc)(2) . The results suggest that differences in the enzymatic degradation process affect the host's immunomodulation process.

Biodegradation Study of Microcrystalline Chitosan and Microcrystalline Chitosan/β-TCP Complex Composites

Bone repair or regeneration is a common and complicated clinical problem in orthopedic surgery. The importance of natural polymers, such as microcrystalline chitosan, and minerals such as HAp and β-TCP, has grown significantly over the last two decades due to their renewable and biodegradable source, increasing the knowledge and functionality of composites in technological and biomedical applications. This study compares the biodegradation process, bioactivity, structure, morphology, and mechanical properties of microcrystalline chitosan and microcrystalline chitosan/β-TCP complex; the latter according to the new method of preparation. The complex showed a homogeneous network structure with regular pores, good bioactivity, even after 60 days of conducting the hydrolytic and enzymatic degradation process, showing a bacteriostatic and bactericidal activity. The complex indicates that it could be used successfully as a base for implants and scaffolds production in orthopedic surgery.