Enzymatic Degradation Kinetics Research Articles

Enzymatic degradability of diclofenac ozonation products: A mechanistic analysis

The treatment of waterborne micropollutants, such as diclofenac, presents a significant challenge to wastewater treatment plants due to their incomplete removal by conventional methods. Ozonation is an effective technique for the degradation of micropollutants. However, incomplete oxidation can lead to the formation of ecotoxic by-products that require a subsequent post-treatment step. In this study, we analyze the susceptibility of micropollutant ozonation products to enzymatic digestion with laccase from Trametes versicolor to evaluate the potential of enzymatic treatment as a post-ozonation step. The omnipresent micropollutant diclofenac is used as an example, and the enzymatic degradation kinetics of all 14 detected ozonation products are analyzed by high-performance liquid chromatography coupled with high-resolution mass spectrometry (HPLC-HRMS) and tandem mass spectrometry (MS2). The analysis shows that most of the ozonation products are responsive to chemo-enzymatic treatment but show considerable variation in enzymatic degradation kinetics and efficiencies. Mechanistic investigation of representative transformation products reveals that the hydroxylated aromatic nature of the ozonation products matches the substrate spectrum, facilitating their rapid recognition as substrates by laccase. However, after initiation by laccase, the subsequent chemical pathway of the enzymatically formed radicals determines the global degradability observed in the enzymatic process. Substrates capable of forming stable molecular oxidation products inhibit complete detoxification by oligomerization. This emphasizes that it is not the enzymatic uptake of the substrates but the channelling of the reaction of the substrate radicals towards the oligomerization of the substrate radicals that is the key step in the further development of an enzymatic treatment step for wastewater applications.

Comparison of Hyaluronidase-Mediated Degradation Kinetics of Commercially Available Hyaluronic Acid Fillers In Vitro.

The ability to degrade hyaluronic acid (HA)-based fillers with hyaluronidase allows for better management of adverse effects and reversal of suboptimal treatment outcomes. The aim of this study was to compare the enzymatic degradation kinetics of 16 commercially available HA-based fillers, representing 6 manufacturing technologies. In this nonclinical study, a recently developed in vitro multidose hyaluronidase administration protocol was used to induce degradation of HA-based fillers, enabling real-time evaluation of viscoelastic properties under near-static conditions. Each filler was exposed to repeated doses of hyaluronidase at intervals of 5 minutes to reach the degradation threshold of G' ≤ 30 Pa. Noticeable differences in degradation characteristics were observed based on the design and technology of different filler classes. Vycross fillers were the most difficult to degrade and the Cohesive Polydensified Matrix filler was the least difficult to degrade. Preserved Network Technology products demonstrated proportional increases in gel degradation time and enzyme volume required for degradation across the individual resilient hyaluronic acid (RHA) products and indication categories. No obvious relationship was observed between gel degradation characteristics and the individual parameters of HA concentration, HA chain length, or the degree of modification of each filler when analyzed separately; however, a general correlation was identified with certain physicochemical properties. Manufacturing technology was the most important factor influencing the reversibility of an HA product. An understanding of the differential degradation profiles of commercially available fillers will allow clinicians to select products that offer a higher margin of safety due to their preferential reversibility.

Enzymatic degradation of poly(ethylene terephthalate) (PET): Identifying the cause of the hypersensitive enzyme kinetic response to increased PET crystallinity

Plastic pollution poses a significant environmental challenge, with poly(ethylene terephthalate) (PET) being a major contributor due to its extensive use in single use applications such as plastic bottles and other packaging material. Enzymatic degradation of PET offers a promising solution for PET recycling, but the enzyme kinetics in relation to the degree of crystallinity (XC) of the PET substrate are poorly understood. In this study, we investigated the hypersensitive enzyme kinetic response on PET at XC from ∼8.5–12% at 50 °C using the benchmark PET hydrolysing enzyme LCCICCG. We observed a substantial reduction in the maximal enzymatic reaction rate (invVmax) with increasing XC, corresponding to a 3-fold reduction in invVmax when the XC of PET increased from 8.6% to 12.2%. The kinetic analysis revealed that the level of the Mobile Amorphous Fraction (XMAF) was a better descriptor for the enzymatic degradation rate response than XC (or (100%-XC)). By continuous monitoring of the enzymatic reaction progress, we quantified the lag phase prolongation in addition to the steady-state kinetic rates (vss) of the reactions and found that the duration of the lag phase of a reaction could be predicted from the vss and XC by multiple linear regression modeling. The linear correlation between the duration of the lag phase and the vss of the enzymatic PET degradation affirmed that the LCCICCG worked via a random/endo-type enzymatic attack pattern. The longer lag phase at increased XC of PET is proposed to be due to increased substrate entanglement density as well as unproductive enzyme binding to the crystalline regions of PET. The findings enhance our understanding of PET enzymatic degradation kinetics and its dependence on substrate composition, i.e., XMAF and XC.

Tunable enzymatic biodegradation of poly(butylene succinate): biobased coatings and self-degradable films

Biodegradation of polyesters driven by enzymes is considered as one of the most effective way of degradation of these materials, as a way to control plastics accumulation in the environment. In this study, we present two different strategies to tune the enzymatic degradation of PBS films triggered by a lipase from Pseudomonas cepacia. Firstly, the kinetics of enzymatic degradation of PBS films was regulated by applying multilayer coats of polysaccharide alginate and chitosan (Alg/Chi) films. Secondly, self-degradable PBS films were prepared by embedding lipase-filled alginate particles. For comparison purposes, a detailed enzymatic degradation study of neat PBS films exposed to a lipase from P. cepacia in solution was made to determine the main experimental parameters influencing their degradation in solution. The results showed that an increase in enzyme concentration increased the degradation extent and rate of neat PBS films. At a fixed enzyme concentration, stirring of the solution containing the enzyme and the PBS also increased the biodegradation rate. In the case of the PBS films coated with a different number of Alg/Chi layers by spray-assisted LbL and subjected to enzymatic degradation experiments in solution, the extent of degradation was found to be dependent on the number of protective coating layers. Therefore, the Alg/Chi biobased coating constitutes an effective barrier to the diffusion of the lipase, thus proving its effectiveness in modulating the enzymatic activity as a function of coating thickness. In the case of self-degradable PBS containing lipase-embedded alginate beads (employed to protect the enzyme during high-temperature processing), only limited biodegradation was observed as the amount of encapsulated enzyme employed was too small. Nonetheless, these results are promising, as the enzymatic activity –indicative of the degradation capacity of the enzyme– determined for all these samples was about 2 orders of magnitude lower than that of previous assays.

Mechanism and kinetics of enzymatic degradation of polyester microparticles using a shrinking particle-shrinking core model.

Generalized shrinking particle (SPM) and shrinking core (SCM) models were developed to the kinetics of heterogenous enzymatic degradation of polymer microparticles in a continuous microflow system. This enzymatic degradation was performed in a microfluidic device designed to both physically separate and immobilize the microparticles. Then time-resolved measurements were made using image processing of the physical changes of the particles during degradation. The kinetics of enzyme-polymer intermediate formation, enzymatic bond cleavage, and enzyme diffusion through the layer of degraded substrate (SCM only) were mathematically derived to predict the time-resolved degradation of the substrate. The proposed models were tested against the degradation of 15-25 μm particles of polycaprolactone (PCL) and poly (butylene adipate-co-terephthalate) (PBAT) by cutinase enzyme from Humicola insolens. Degradation of PCL microparticles followed the SPM model and its kinetics were found to be zero-order, while the SCM model applied to PBAT microparticles showed first-order kinetics. Further, the degradation of polybutylene succinate (PBS), and poly butylene-sebacate-co-terephthalate (PBSeT) microparticles demonstrated wide applicability of the method. The use of image processing simplifies the required analysis by eliminating the need to remove aliquots or concentrate effluent for additional analytical characterization.

Elucidations on the Performance and Reversibility of Treatment with Hyaluronic Acid Based Dermal Fillers: In vivo and in vitro Approaches.

The aim of this study was to investigate the performance and the reversibility of different classes of Hyaluronic Acid (HA) dermal fillers. We analysed 4 HA based fillers, belonging to 3 different chemical classes of products, commonly used in the field of wrinkles correction: linear HA 8 mg/mL (Viscoderm 0.8), thermically stabilized hybrid complexes of high and low molecular weight HA molecules at a concentration of 32 mg/mL and 45 mg/mL respectively (Profhilo and Profhilo Structura) and cross-linked HA 25 mg/mL (Aliaxin GP). The products were tested by a well-established animal model. The generated implants were analyzed through High-Frequency Ultrasound technology. Then, reversibility of the treatment was evaluated by enzymatic degradation kinetics studies, characterised by a combined approach of Carbazole assay and HP-SEC/TDA method. Implants generated by linear HA 8 mg/mL remained detectable by ultrasound acquisition for 4 weeks, whereas those generated by injection of HA hybrid complex 32 mg/mL were detectable for 10 weeks. HA hybrid complex 45 mg/mL and cross-linked HA 25 mg/mL were detectable for 29 and at least 33 weeks, respectively. Enzymatic degradation kinetics studies demonstrated that the HA content in HA hybrid complex 45 mg/mL was almost completely depolymerized and homogeneous after 3 h of treatment. For cross-linked HA 25 mg/mL, 24 h of incubation are needed to obtain the same degree of depolymerization. The study confirmed the ability of the experimental model to predict the behaviour of HA based dermal fillers in vivo and showed the innovative aspects of HA hybrid complex 45 mg/mL, that combines the high-safety profile, in terms of reversibility of the treatment, of the linear HA-based products with the durability of a high degree cross-linked gels, paving the way to the chance to be used for a wide range of applications in the field of aesthetic medicine.

3D Printing of Cellulase-Laden Cellulose Nanofiber/Chitosan Hydrogel Composites: Towards Tissue Engineering Functional Biomaterials with Enzyme-Mediated Biodegradation.

The 3D printing of a multifunctional hydrogel biomaterial with bioactivity for tissue engineering, good mechanical properties and a biodegradability mediated by free and encapsulated cellulase was proposed. Bioinks of cellulase-laden and cellulose nanofiber filled chitosan viscous suspensions were used to 3D print enzymatic biodegradable and biocompatible cellulose nanofiber (CNF) reinforced chitosan (CHI) hydrogels. The study of the kinetics of CNF enzymatic degradation was studied in situ in fibroblast cell culture. To preserve enzyme stability as well as to guarantee its sustained release, the cellulase was preliminarily encapsulated in chitosan–caseinate nanoparticles, which were further incorporated in the CNF/CHI viscous suspension before the 3D printing of the ink. The incorporation of the enzyme within the CHI/CNF hydrogel contributed to control the decrease of the CNF mechanical reinforcement in the long term while keeping the cell growth-promoting property of chitosan. The hydrolysis kinetics of cellulose in the 3D printed scaffolds showed a slow but sustained degradation of the CNFs with enzyme, with approximately 65% and 55% relative activities still obtained after 14 days of incubation for the encapsulated and free enzyme, respectively. The 3D printed composite hydrogels showed excellent cytocompatibility supporting fibroblast cell attachment, proliferation and growth. Ultimately, the concomitant cell growth and biodegradation of CNFs within the 3D printed CHI/CNF scaffolds highlights the remarkable potential of CHI/CNF composites in the design of tissue models for the development of 3D constructs with tailored in vitro/in vivo degradability for biomedical applications.

Exploring ex vivo peptideolysis of thymopentin and lipid-based nanocarriers towards oral formulations

The oral delivery of medicines is the most popular route of administration for patients. However, thymopentin (TP5) is only available in the market in forms for parenteral administration. In large part, this is because of extensive peptidolytic degradation in the gastrointestinal tract (GIT), which decreases the amount of TP5 available for absorption. This study aims to understand the extent of TP5 peptideolysis and determine effective inhibitors and suitable lipid-based nanocarriers to aid in the development of an effective oral delivery formulation. Enzymatic degradation kinetics of TP5 was investigated in the presence or absence of mucosal and luminal components extracted from various parts of the rat intestine, including the duodenum, jejunum, ileum, and colon. Inhibition of TP5 enzymatic peptidolysis was screened in the presence or absence of EDTA, trypsin and chymotrypsin inhibitors from soybean (SBTCI), and bestatin. TP5 with SBTCI was loaded into lipid-based nanocarriers, including microemulsions, niosomes and solid lipid nanoparticles. These TP5-loaded nanocarriers were investigated through characterization of morphology, particle size, zeta potential, entrapment efficacy (EE%), and ex vivo rat intestinal degradation studies to select a lead formulation for a future oral drug delivery study. The degradation kinetics of TP5 followed pseudo-first-order kinetics, and the biological metabolism of TP5 was displayed in the presence of luminal contents, indicating that TP5 is sensitive to luminal enzymes. Notably, a considerable decrease in TP5 peptidolysis was found in the presence of SBTCI, bestatin, and EDTA. TP5 and SBTCI were loaded into three lipid-based delivery systems, displaying superior protection under ex vivo intestinal luminal contents and mucosal homogenates for 6 h compared with the pure drug solution. These findings suggest that using select inhibitors and lipid-based nanocarriers can decrease peptide degradation and may improve oral bioavailability of TP5 following oral administration.

Changes in content of antioxidants and hydrolytic stability of black rice bran after heat‐ and enzymatic stabilizations and degradation kinetics during storage

This research aimed to compare heat and enzymatic stabilization methods in terms of hydrolytic stability and changes in antioxidant compounds of rice brans during ambient storage. Raw non-pigmented and black rice bran (RNB and RBB) showed high lipase activity (269.0 and 241.9 U/kg, respectively). Heat-stabilized non-pigmented and black rice brans (HNB and HBB) and enzymatically stabilized non-pigmented and black rice brans (ENB and EBB) had much lower lipase activity (25.8 to 32.8 U/kg). Lipase activity was not increased in the stabilized samples during 8-week storage and had high hydrolytic stability. Compared with raw rice brans, tocols, and anthocyanin contents in HNB and HBB decreased significantly, but γ-oryzanol increased significantly, while all antioxidant compounds in ENB and EBB increased significantly. Antioxidant contents of rice bran decreased during storage following zero- and first-order reaction kinetics. EBB showed the lowest k and the highest t1/2 values, indicating more stability of these antioxidants.

Enzymatic degradation behaviors and kinetics of bio-degradable jute/poly (lactic acid) (PLA) composites

The jute fiber reinforced poly (lactic acid) (PLA) composite renders a complete bio-degradable characteristic with relative remarkable mechanical properties, while it is strongly sensitive to the enzyme environments. To elaborate the enzymatic degradation mechanism of the jute/PLA composites, this study performed a 35 days degradation in an enzyme solution containing protease K, cellulase, ligninase, and pectinase. The changes of water absorption, crystallinity, molecular weight, morphologies, chemical compositions during enzymatic degradation were experimentally evaluated. We divided the total degradation process into three stages according to the primary degradation targets: the macromolecular chains of PLA, the fiber-matrix interfaces, and the jute fiber. The degradation kinetics was proposed based on the Michaelis–Menten kinetics approach. The elucidation of enzymatic degradation mechanisms of jute/PLA composite is meaningful for improving the application prospects of the bio-degradable green materials.

Methacrylate-Modified Gold Nanoparticles Enable Non-Invasive Monitoring of Photocrosslinked Hydrogel Scaffolds.

Photocrosslinked hydrogels, such as methacrylate-modified gelatin (gelMA) and hyaluronic acid (HAMA), are widely utilized as tissue engineering scaffolds and/or drug delivery vehicles, but lack a suitable means for non-invasive, longitudinal monitoring of surgical placement, biodegradation, and drug release. Therefore, we developed a novel photopolymerizable X-ray contrast agent, methacrylate-modified gold nanoparticles (AuMA NPs), to enable covalent-linking to methacrylate-modified hydrogels (gelMA and HAMA) in one-step during photocrosslinking and non-invasive monitoring by X-ray micro-computed tomography (micro-CT). Hydrogels exhibited a linear increase in X-ray attenuation with increased Au NP concentration to enable quantitative imaging by contrast-enhanced micro-CT. The enzymatic and hydrolytic degradation kinetics of gelMA-Au NP hydrogels were longitudinally monitored by micro-CT for up to one month in vitro, yielding results that were consistent with concurrent measurements by optical spectroscopy and gravimetric analysis. Importantly, AuMA NPs did not disrupt the hydrogel network, rheology, mechanical properties, and hydrolytic stability compared with gelMA alone. GelMA-Au NP hydrogels were thus able to be bioprinted into well-defined three-dimensional architectures supporting endothelial cell viability and growth. Overall, AuMA NPs enabled the preparation of both conventional photopolymerized hydrogels and bioprinted scaffolds with tunable X-ray contrast for noninvasive, longitudinal monitoring of placement, degradation, and NP release by micro-CT.

Microfabricated potentiometric sensor for personalized methacholine challenge tests during the COVID-19 pandemic

The methacholine challenge test is considered to be the gold standard bronchoprovocation test used to diagnose asthma, and this test is always performed in pulmonary function labs or doctors’ offices. Methacholine (MCH) acts by inducing airway tightening/bronchoconstriction, and more importantly, MCH is hydrolyzed by cholinesterase enzyme (ChE). Recently, the American Thoracic Society raised concerns about pulmonary function testing during the COVID-19 pandemic due to recently reported correlation between cholinesterase and COVID-19 pneumonia severity/mortality, and it was shown that cholinesterase levels are reduced in the acute phase of severe COVID-19 pneumonia. This work describes the microfabrication of potentiometric sensors using copper as the substrate and chemically polymerized graphene nanocomposites as the transducing layer for tracking the kinetics of MCH enzymatic degradation in real blood samples. The in-vitro estimation of the characteristic parameters of the MCH metabolism [Michaelis–Menten constant (Km) and reaction velocity (Vmax)] were found to be 241.041 μM and 56.8 μM/min, respectively. The proposed sensor is designed to be used as a companion diagnostic device that can (i) answer questions about patient eligibility to perform methacholine challenge tests, (ii) individualize/personalize medical dosing of methacholine, (iii) provide portable and inexpensive devices allowing automated readouts without the need for operator intervention (iv) recommend therapeutic interventions including intensive care during early stages and reflecting the disease state of COVID-19 pneumonia. We hope that this methacholine electrochemical sensor will help in assaying ChE activity in a “timely” manner and predict the severity and prognosis of COVID-19 to improve treatment outcomes and decrease mortality.

Common Reference-Based Tandem Mass Tag Multiplexing for the Relative Quantification of Peptides: Design and Application to Degradome Analysis of Diphtheria Toxoid.

Currently, animal tests are being used to confirm the potency and lack of toxicity of toxoid vaccines. In a consistency approach, animal tests could be replaced if production consistency (compared to known good products) can be proven in a panel of in vitro assays. By mimicking the in vivo antigen processing in a simplified in vitro approach, it may be possible to distinguish aberrant products from good products. To demonstrate this, heat-exposed diphtheria toxoid was subjected to partial digestion by cathepsin S (an endoprotease involved in antigen processing), and the peptide formation/degradation kinetics were mapped for various heated toxoids. To overcome the limitations associated with the very large number of samples, we used common reference-based tandem mass tag (TMT) labeling. Instead of using one label per condition with direct comparison between the set of labels, we compared multiple labeled samples to a common reference (a pooled sample containing an aliquot of each condition). In this method, the number of samples is not limited by the number of unique TMT labels. This TMT multiplexing strategy allows for a 15-fold reduction of analysis time while retaining the reliability advantage of TMT labeling over label-free quantification. The formation of the most important peptides could be followed over time and compared among several conditions. The changes in enzymatic degradation kinetics of diphtheria toxoid revealed several suitable candidate peptides for use in a quality control assay that can distinguish structurally aberrant diphtheria toxoid from compliant toxoids.

An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films

Increased interest in poly(ethylene terephthalate) (PET)-degrading enzymes (PETases) have generated efforts to find mutants with improved catalytic activity and thermostability. Here, we present a simple and fast method to determine relative enzyme kinetics through bulk absorbance measurements of released products over time. A thermostable variant of PETase from Ideonella sakaiensis was engineered (R280A S121E D186H N233C S282C) with a denaturation temperature of 69.4 ± 0.3 °C. This was used to assess the method’s ability to determine relative enzyme kinetics across variants and reveal structure–function relationships. Measurements at 24 and 72 h at 400 nM of enzyme suggest that the mutations improved catalytic rates 5- to 7-fold. On the contrary, kinetic analyses of the thermostable variant and wild-type reveal different reaction trajectories despite similar maximum catalytic rates, resulting in higher product accumulation from the thermostable variant over time. The results of the assay support the necessity for kinetic measurements to determine relationships between sequence and function for IsPETase and other PET hydrolases.

A Companion Diagnostic for Personalizing Mivacurium at the Point-of-Care

Mivacurium chloride is envisioned as the “gold-adjunct” in anesthesia and emergency medicine. People across the world are administered mivacurium in emergency situations as clinicians prefer it among the frequently used neuromuscular blockers thanks to its rapid action and short duration. Mivacurium is promptly degraded and deactivated by plasma cholinesterase enzyme (PChE), however during emergency situations, some individuals were found to be unable to metabolize mivacurium shortly after its injection, resulting in prolonged paralysis and potentially lethal respiratory apnea. The most clinically significant cause of this residual paralysis and apnea is the reduced cholinesterase ability to effectively hydrolyze mivacurium. Hitherto, monitoring of mivacurium enzymatic degradation has been remaining a long-standing challenge for analytical chemists as well as anesthesiologists. Firstly, it undergoes rapid hydrolysis by the PChE-enzyme 8 min post-injection, therefore, frequent sampling all through the first minutes after mivacurium administration is mandatory. Secondly, its isolation from biological specimens is extremely difficult due to its chemical properties which involves both hydrophilic and lipophilic characteristics. These challenges impede the development of a universal protocol to be established for screening of PChE-activity towards mivacurium and which clearly identifies patients susceptible to prolonged paralysis. In this study, we designed a point-of-care (PoC) potentiometric sensor that can perform tracking of mivacurium enzymatic degradation kinetics with high accuracy and rapidly. Real serum samples were utilized for in vitro estimation of the rate of mivacurium metabolism, where values of 227 μmol l−1 and 62 μM min−1 were obtained for the characteristic parameters of the enzymatic reaction, Km and Vmax, respectively. Correlation of the results obtained using the proposed approach and earlier LC-MS/MS study is shown.

Modified acellular porcine corneal matrix in deep lamellar transplantation of rabbit cornea.

This study presents to develop a modified acellular porcine corneal matrix (MAPCM) to maintain high transparency, stability and biocompatibility as a rabbit deep cornea replacement using 1-ethyl-3–(3-dimethylaminopropyl)-carbodiimide crosslinking and a mild decellularization technique. Scaffolds are translucent and remain higher amount of glycosaminoglycans after decellularization than acellular porcine corneal matrix (APCM). Enzymatic degradation kinetics and mechanical properties of scaffolds are regulated by 1-ethyl-3–(3-dimethylaminopropyl)-carbodiimide -crosslinking density. The porous structure and ultrastructure of collagenous lamellae are maintained, and the pore size of MAPCM crosslinked with 0.5% (w/v) 1-ethyl-3–(3-dimethylaminopropyl)-carbodiimide is 13.26 ± 1.65 µm, similar to that of normal porcine cornea. The transmittance of MAPCM gets 79.1 ± 0.45 to 92.7 ± 1.4% in the visible light range. Results from a CCK-8 assay indicate that MAPCM gets higher cell proliferation rate of rabbit corneal stroma cells than APCM. Since collagen fibres structural integrity and regularity of MAPCM are retained after crosslinking, the opacity and stability of MAPCM are better than those of APCM within 4 weeks of animal implantation. In addition, there is no indication of an immune response or neovascularization in or around the transplanted disc. These results reveal that MAPCM may be a more suitable scaffold for corneal substitute construction.

Comparative studies of pure cultures and a consortium of white-rot fungi to degrade a binary mixture of dyes by solid-state fermentation and performance at different scales

White-rot fungi have been widely used in pure culture and consortia for treating recalcitrant compounds, such as synthetic dyes, since they are able to cleave and mineralize lignin using ligninolytic enzymes. Moreover, solid-state fermentation stands out in the treatment of dyes due to its low environmental impact. In this study we evaluated the degradation of allura red and tartrazine adsorbed onto corncob by either pure cultures or binary consortia of Trametes versicolor and Pleurotus pulmonarius. We performed a comparative study in terms of culture conditions optimization, degradation, activity of ligninolytic enzymes kinetics, and cytotoxicity assessment to unravel the differences between using individual cultures or consortium of white-rot fungi. 94% of the dye mixture was degraded in pure cultures, but only 64.3% was broken-down by the consortium. Enzymatic activity and degradation kinetics showed that T. versicolor maximum laccase activity corresponded to the plateau phase of degradation whilst for P. pulmonarius and consortium no match between both profiles was found. Also, T. versicolor extracts were not cytotoxic to CHO–K1 cell line. The process was therefore magnified using T. versicolor in pure culture. The degradation efficiency was maintained in all the sizes, allowing an increase of 20 times the amount of treated dyes.

Relationship between the molecular structure of duckweed starch and its in vitro enzymatic degradation kinetics

Starch molecular structural effects in duckweed (Lemna minor and Landoltia punctata) controlling in vitro enzymatic degradation kinetics was studied. The molecular size distributions of fully-branched starches and the chain length distributions (CLDs) of enzymatically debranched duckweed starches were obtained using size-exclusion-chromatography (SEC). The CLDs of both debranched amylose and amylopectin were fitted with models using biologically-meaningful parameters. While there were no significant correlations between amylose content and starch degradation rate, the total amounts of amylose with shorter chain length negatively correlated with undigested starch content, and the amount of amylopectin long chains negatively correlated with the degradation rate coefficient. This provides new knowledge for the utilization of duckweed starches in bioethanol production.

Degradation of carboxymethylcellulose using ultrasound and β-glucanase: Pathways, kinetics and hydrolysates’ properties

In order to provide an efficient way to degrade carboxymethylcellulose (CMC), three pathways were investigated: enzymolysis, combination of ultrasound pretreatment and enzymolysis, and sonoenzymolysis. Effects of these treatments on enzymatic kinetics, degradation kinetics and properties of degraded CMC were investigated. The degradation degree of CMC was increased by 18.90% and 35.73% with ultrasound pretreatment (at an intensity of 24 W/mL for 30 min) and sonoenzymolysis (at an intensity of 9 W/mL for 50 min), compared with that obtained under the traditional enzymolysis. Analysis of kinetics demonstrated that ultrasound, both pretreatment and combined with β-glucanase, could accelerate CMC degradation. Measurements of rheological properties, molecular weight and structures of CMC hydrolysates revealed that ultrasound broke the glycosidic bond of CMC chains without changing its primary structure. The sonoenzymolysis process was the most efficient method to degrade CMC, with potential to provide a way to obtain CMC with lowest molecular weight or viscosity.

Short-time dental resin biostability and kinetics of enzymatic degradation

Resin biostability is of critical importance to the durability of methacrylate-based dental resin restorations. Current methods for evaluating biostability take considerable time, from weeks to months, and provide no short-time kinetics of resin degradation. The objective of this study is to develop a more sensitive method to assess resin biostability over short-time spans (hours to days) that will enhance our understanding of biostability and its resin chemistry. Ultra-flat resin films of equimolar urethane dimethacrylate (UDMA) and triethylene glycol dimethacrylate (TEGDMA) are produced through photo-curing between two flat surfaces. Next, metal-covered enclaves and bare-resin channels are generated using stencil lithography to create both degradable and protected (internal reference) regions simultaneously in a single specimen. Resins having three different degrees of vinyl conversion (DC) are compared, and changes of surface roughness and step height in the two regions are monitored by atomic force microscopy (AFM) before and after incubated in enzyme solutions and saline controls. Specimen biostability is ranked based on the topological profile changes when viewed in cross-section before and after enzymatic challenges. In addition, a model is proposed to quantify specimen enzymatic degradation. Based on this model, enzymatic degradation is detected as early as 4 h, and a surge of enzymatic degradation is detected between 4 h and 8 h. The correlation between the DC of resin network and the surge in degradation is discussed. In summary, this new method is effective in ranking biostability and quantifying enzymatic degradation while also reducing labor, time and cost, which lends itself well to materials development and evaluation of dental resins. Statement of SignificanceWe report, for the first time, the short-time kinetics of enzymatic degradation of methacrylate dental resins. A nanotechnology based method is developed to accelerate the evaluation of resin biostability. This new method reduces experimental time from weeks to one or two days, which will significantly reduce the costs of labor and enzymes. It also introduces a corresponding parameter (ΔH) and a three-cause model for ranking biostability, which confirms the correlation of chemical structure (DC) and material performance and opens new opportunities for studying the resin biostability and its impact on dental applications. Overall, this is a new tool for evaluating resin biostability and developing new materials.