Enzymatic Degradation of Parvifloside

Parvifloside (1), a new furostanol pentaglycoside, was isolated from the fresh rhizomes of Dioscorea parviflora C. T. Ting. On the basis of spectroscopic and chemical methods, its structure was elucidated as (25R)-26-O-β-glucopyranosyl-furost-5-en-3β,22ξ,26-triol 3-O-β-D-glucopyranosyl (1→3)-β-D-glucopyranosyl (1→4)-[α-L-rhamnopyranosyl (1→2)]-β-D-glucopyranoside. Six prosapogenins (2-7)were obtained from the enzymatic degradation of 1by cellulase, but only 3 and 4 were obtained by β-glucosidase. The structures of all compounds were determined by spectroscopic data. The activity of the isolated compounds on deformation of mycelia germinated from Pyricularia oaryzae P-2b conidia was evaluated.

Effect of Chitosan-Cysteine Conjugate on Enzymatic Degradation and Hypoglycemic effect of Insulin

Aim To evaluate the inhibitory effect of chitosan-cysteine conjugate onenzymatic degradation and hypogly-cemic enhancement effect of insulin. Methods Chitosan-cysteineconjugate was synthesized. The protective effect of the conjugate against degradation of insulin byα-chymotrypsin and trypsin was evaluated in vitro. Insulin enteric- microspheres were prepared byusing O_1 /Q_2 emulsion solvent evaporation method. The hypoglycemic enhancement effect of theconjugate was studied by oral administration of insulin solution or enteric-microspheres to rats.Results The thiol group content of the synthesized conjugate was about 200 μmol·g^(-1) polymer,which showed a strong protective effect on insulin from enzymatic degradation in vitro. Almost allthe insulin incubated in a-chymotrypsin solution or trypsin solution without chitosan-cysteineconjugate was degraded entirely within 1 h and 5 h respectively, whereas above 75% of insulinremained in the same content of the enzymatic solution containing 4 mg·mL^(-1) conjugate. The drugloading of insulin enteric-microspheres was about 7% . In vivo experiment, chitosan-cysteineconjugate (85 μg·kg^(-1)) prolonged the hypoglycemic time of insulin solution orenteric-microspheres when administered simultaneously with the absorption enhancer SNAC. ConclusionChitosan-cysteine conjugate has a marked inhibitory effect on the enzymatic degradation of insulinin vitro, and it displays a significant hypoglycemic enhancement effect on insulin oral formulationin vivo.

Enzymatic Degradation of Organochlorine Insecticide Chlordane by Phlebia brevispora

[ Objective ] This study aimed to clarify the mechanism of enzymatic degradation of organochlorine insecticide chlordane by the white rot fungus Pldebia brevispora. [ Method ] The degradation characteristics of chlordane were determined by the crude enzyme extracted from P. brevispora strain by pure culture, ultrasonic fragmentation and centrifuge separation. [ Result] About 33.2% and 10.4% of chlordane were degraded respectively by intracellular enzyme and extracellular enzyme within 20 min. At pH 5.0 and 3℃, the crude enzyme showed the greatest degradation activity （49.9%）, with its Michaelis-Mentn＇s constant （Km ） and maximum degradation rate （ Vmax ） of 3.49 and 8.38 ttmol/min, respectively. [ Conclusion] Extracellular enzyme can degrade chlordane through dehydrogenation or dehydrochlorination. Intracellular enzyme plays a major role in chlordane degradation. It can transform chlordane to some hydroxylated metabolites through hydroxylation or substitution of chlorine atom by hydroxyl group.

Insights into the enzymatic degradation of DNA expedited by typical perfluoroalkyl acids

Perfluoroalkyl acids(PFAAs)are considered forever chemicals,gaining increasing attention for their hazardous impacts.However,the ecological effects of PFAAs remain unclear.Environmental DNA(eDNA),as the environmental gene pool,is often collected for evaluating the ecotoxicological effects of pollutants.In this study,we found that all PFAAs investigated,including perfluorohexanoic acid,perfluorooctanoic acid,perfluorononanoic acid,and perfluorooctane sulfonate,even at low concentrations(0.02 and 0.05 mg/L),expedited the enzymatic degradation of DNA in a nonlinear dose–effect relationship,with DNA degradation fragment sizes being lower than 1,000 bp and 200 bp after 15 and 30 min of degradation,respectively.This phenomenon was attributed to the binding interaction between PFAAs and AT bases in DNA via groove binding.van der Waals force(especially dispersion force)and hydrogen bonding are the main binding forces.DNA binding with PFAAs led to decreased base stacking and right-handed helicity,resulting in loose DNA structure exposing more digestion sites for degrading enzymes,and accelerating the enzymatic degradation of DNA.The global ecological risk evaluation results indicated that PFAA contamination could cause medium and high molecular ecological risk in 497 samples from 11 contamination-hot countries(such as the USA,Canada,and China).The findings of this study show new insights into the influence of PFAAs on the environmental fates of biomacromolecules and reveal the hidden molecular ecological effects of PFAAs in the environment.

Influence of residual chirality on the conformation and enzymatic degradation of glycopolypeptide based biomaterials

Glycopolypeptides as analogs of glycoproteins or glycosaminoglycans represent attractive building blocks for the construction of biomimetic biomaterials.However,the effects of amino acid chirality on the conformation and enzymatic degradation of glycopolypeptides are often overlooked.Here,we synthesized and characterized a range of glycopolypeptides composed of galactosylated poly(γ-propargylglutamate)s containing L-and/or D-glutamate residues.Glycopolypeptides containing pure Lglutamate residues were predominantlyα-helical,and the helicity increased over the degree of polymerization of the polypeptide backbones(24 to 44).The glycopolypeptide with pure D-glutamate residues adopted a mirroredα-helical conformation,whilst apparent random coil conformation was observed for the glycopolypeptide with equally mixed enantiomeric residues.The enzymatic degradation rates of the glycopolypeptides were markedly reduced following the introduction of D-glutamate residues into backbones.Galactoside pendants on these glycopolypeptides maintained their binding to peanut agglutinin.These structureproperty relationships provide new insight for the design of biomimetic biomaterials containing glycopolypeptides.

pH-responsive Micelles from a Blend of PEG-b-PLA and PLA-b-PDPA Block Copolymers: Core Protection Against Enzymatic Degradation

pH-responsive micelles with a biodegradable PLA core and a mixed PEG/PDPA shell were prepared by self-assembly of poly（ethylene glycol）-b-poly（lactic acid） （PEG-b-PLA） and poly（2-（diisopropylamino）ethyl methacrylate）-b-poly（lactic acid） （PDPA-b- PLA）. The micellization status with different pH and the enzyme degradation behavior were characterized by IH-NMR spectroscopy, dynamic light scattering measurement and zeta potential test. The pH turning point of PDPA block was determined to be in the range of 5.5-7.0. While the pH was above 7.0, the PDPA block collapsed onto the PLA core and could protect the PLA core from invasion of enzyme, as a result, the micelle exhibited a resistance to the enzymatic degradation.

Influence of Teflon Substrate on Crystallization and Enzymatic Degradation of Polymorphic Poly(butylene adipate)

Oriented and non-oriented Teflon films, which were found to have the same crystalline structure, but different surface morphologies, were used to sandwich poly（butylene adipate） （PBA） films during isothermal crystallization. It was found that both the Teflon surface structure and the PBA polymorphic structure are the determining factors to induce epitaxial crystallization. The oriented Teflon film was able to induce epitaxial crystallization of PBA α crystal, while the non-oriented Teflon did not induce any epitaxial crystallization of PBA. Epitaxial crystallization did not occurred for PBA β crystals between neither the oriented nor the non-oriented Teflon films. The enzymatic degradation rate of PBA films was not determined by the epitaxial crystallization, in fact it was still dependent on the polymorphic crystal structure of PBA. The morphological changes of PBA films after enzymatic degradation confirmed again that the epitaxial crystallization only occurred for the PBA film with α crystal structure which was produced by being sandwiched between oriented Teflon films, and it happened only on the surface of PBA films.

In situ evaluation of cell cultivation on dynamically changed poly(ε-caprolactone) film caused by enzymatic degradation

In situ evaluation of cell cultivation on degrading poly(ε-caprolactone) (PCL) films was studied.New culture surroundings were constructed for cell growth by using PCL films as substrates and adding Pseudomonas cepacia lipase to accelerate biodegradation of PCL films.MTT experiments for 10 h indicated the low cytotoxicity of lipase solution with concentration up to 0.2 mg/mL for MG-63 cells growth on PCL films.With the optimized lipase concentration and degradation time,we studied cell growth behavior on dynamically changed PCL films by adding lipase to the culture surroundings.MTT,fluorescence microscopy and scanning electron microscopy (SEM) were used to evaluate cell viability,proliferation and morphologies.It was found that cell viability and proliferation were not affected by the added lipase solution negatively.In contrast,cells cultured on degrading PCL films showed good growth behavior with clear fusiform shape and pseudopods.Importantly,the enzymatic degradation of PCL films with cells attachment showed distinctive morphology compared to the degradation in lipase solution without cells.The simultaneous cell growth and PCL film degradation were well discussed in this work,which may better understand the interaction between cell growth and polymer degradation.

Beyond the gluten-free diet:Innovations in celiac disease therapeutics

Celiac disease(CD)is an autoimmune disorder exacerbated by the ingestion of gluten in genetically susceptible individuals,leading to intestinal inflammation and damage.This chronic disease affects approximately 1%of the world’s po-pulation and is a growing health challenge due to its increasing prevalence.The development of CD is a complex interaction between genetic predispositions and environmental factors,especially gluten,culminating in a dysregulated immune response.The only effective treatment at present is a strict,lifelong gluten-free diet.However,adherence to this diet is challenging and often incomplete,so research into alternative therapies has intensified.Recent advances in under-standing the molecular and immunological aspects of CD have spearheaded the development of novel pharmacologic strategies that should provide more effec-tive and manageable treatment options.This review examines the latest inno-vations in CD therapies.The focus is on drugs in advanced clinical phases and targeting specific signaling pathways critical to the disease pathogenesis.We dis-cuss both quantitative strategies such as enzymatic degradation of gluten,and qualitative approaches including immunomodulation and induction of gluten tolerance.Innovative treatments currently under investigation include transglu-taminase inhibitors,which prevent the modification of gluten peptides,and nano-particle-based therapies to recalibrate the immune response.These new therapies not only promise to improve patient outcomes but are also expected to improve quality of life by reducing the burden of dietary restrictions.The integration of these new therapies could revolutionize the treatment of CD and shift the para-digm from strict dietary restrictions to a more flexible and patient-friendly thera-peutic approach.This review provides a comprehensive overview of the future prospects of CD treatment and emphasizes the importance of continued research and multidisciplinary collab-oration to integrate these advances into standard clinical practice.

Enhanced digestion inhibition and mucus penetration of F127-modified self-nanoemulsions for improved oral delivery

Self-nanoemulsifying systems(SNEs) have excellent ability to improve the solubility ofpoorly water-soluble drugs(PWSD). However, SNEs are likely to be degraded in gastroin-testinal(GIT) when their surface is recognized by lipase/co-lipase enzyme complex, result-ing in rapid release and precipitation of encapsulated drugs. The precipitates are then cap-tured and removed by intestinal mucus, reducing the delivery efficacy of SNEs. Herein, theamphiphilic polymer Pluronic? F127 was incorporated into long and short-chain triglyc-erides(LCT, SCT) based SNEs to diminish the recognition and therefore minimized theirdegradation by enzymes and clearance by mucus. The SNEs were characterized in termsof particle size, zeta potential and stability. Ex vivo multiple particles tracking studies wereperformed by adding particle solution into fresh rat mucus. Cellular uptake of SNEs wereconducted by using E12 cells, the absorption and distribution in small intestine were alsostudied after oral administration in male Sprague-Dawley(SD) rats. The in vitro digestionrate of SNEs were found to be in following order SCT-SNE > SCT-F127-SNE > LCT-SNE > LCT-F127-SNE. Moreover, the LCT-F127-SNE was found to be most effective in enhancing cellularuptake, resulting in 3.5-fold, 2.1-fold and 1.7-fold higher than that of SCT-SNE, LCT-SNE andSCT-F127-SNE, respectively. After incubating the SNE with E12 cells, the LCT-F127-SNE ex-hibited the highest amount regarding both mucus penetration and cellular uptake, with anuptake amount number(via bicinchoninic acid(BCA) analysis) of 3.5-fold, 2.1-fold and 1.7-fold higher than that of SCT-SNE, LCT-SNE and SCT-F127-SNE, respectively. The in vivo results revealed that orally administered LCT-F127-SNE could significantly increase the bioavailability of Cyclosporine A(CsA), which was approximately 2.43-fold, 1.33-fold and 1.80-fold higher than that of SCT-SNE, SCT-F127-SNE and LCT-SNE, respectively. We address in this work that F127-modified SNEs have potentials to improve oral drug absorption by significantly reducing gastrointestinal enzymatic degradation and simultaneously enhancing mucus penetration.

The Earliest Discovery of the Role of Magnesium Ions on Stabilizing the Tertiary Structure of the Transfer RNA and Its Biological Significance —A Short Memoir

In early of 1960s, I was a graduate student studying on tRNA biochemistry. In the course of the research, the magnesium ions stabilized the tertiary structure of tRNA, resulting in its resistance to enzymatic degradation was discovered independently. The experiment of deaminated (denatured) tRNA obtained from native tRNA was designed and conducted and further proved the validity of this finding. It was found that magnesium ions could stabilize the tertiary structure of the natrive tRNA but could not stabilize structure of the deaminated tRNA. In term of the methodology, this stabilization technique has been widely applied in sequencing analysis of RNA and has greatly promoted the progress in the study of primary structure of RNA. More importantly, the stabilization of the tertiary structure of RNA by magnesium ions plays a key role both in the processing of messenger RNAs and the ribozyme activity. After our first article in Chinese was published in 1963, a paper of Nishimura & Novelli came into our note. The received date of their paper was March 22 of 1963, only 4 days earlier than that of our first paper. Thus, we and Nishimura & Novelli made almost at the same time the earliest discovery of the role of magnesium ions on stabilizing the tertiary structure of the transfer RNA and thus resulted in resistance of tRNA degradation by enzymes. However, this discovery was not initially appreciated for a period of time but was finally “visualized” and proved by X-ray crystal structure of yeast phenylalanine tRNA, which has provided more accurate information on the geometry of the magnesium-binding sites in tRNA.

THE UNIFORMITY OF DISTRIBUTION OF SUBSTITUENTS ALONG THE CHAIN OF CARBOXYMETHYL CELLULOSE SYNTHESIZED IN TWO-PHASE MEDIUM C_6H_6——C_2H_5OH

Based on the property that carboxymethylcellulose (CMC ) can be de-graded to anhydroglucose residues by cellulase, the rate constant (K) of enzymatic degra-dation of CMC synthesized in the benzene-ethanol medium has been determined. Further-morel experimental equation K = 2.71× 10^(-2)(DS)^(-1.2) reflecting the relationship betweenK and the degree of substitution (DS) is correlated and used to describe chemical mi-crostructure uniformity of the distribution of substituents along chains effectively. Chainstructure parameters of enzymatic degradation products of CMC-the number of chainbreaks and the percentage of glucose released have been also measured. Average length ofsubstituted and unsubstituted chain segments are calculated simultaneously. Through thestudy of static and dynamic procedures of enzymatic degradation, the method to charac-terize the distribution of substituents of CMC along the chain has been improved.

Chitosan-g-PAA Hydrogels for Colon-Specific Drug Delivery: Preparation, Swelling Behavior and in vitro Degradability

A series of cross-linked hydrogels for colon-specific drug delivery were synthesized by graft copolymerization of Chitosan and acrylic acid using N, N＇-methylene-bis-（acrylamide） as a cross-linker. Their swelling behavior in different pH buffer solutions and colonic enzymatic degradability were studied. The obtained results show that these hydrogels have good pH sensitivity which can avoid drug release in stomach, and their swelling kinetics in stimulant intestinal environment follow second-order swelling kinetics equation. The factors influencing the swelling kinetics include the degree of cross-linking and the composition, which may control no release or a little amount release of drug inside the hydrogels in the small intestine by tailoring these factors. The gels are degradable by colonic enzymes and there is a correlativity between the degradation of networks and the swelling degree of the gels, which may trigger the release of drug in the colon. The hydrogels show a great potential for their application in oral colon-specific drug delivery system.

Gastric carboxylesterases of the edible crab Cancer pagurus (Crustacea, Decapoda) can hydrolyze biodegradable plastics

A promising strategy to counteract the progressing plastic pollution of the environment can involve the replacement of persistent plastics with biodegradable materials.Biodegradable polymers are enzymatically degradable by various hydrolytic enzymes.However,these materials can reach the environment in the same way as conventional plastics.Therefore,they are accessible to terrestrial,freshwater,and marine biota.Once ingested by marine organisms,highly active enzymes in their digestive tracts may break down biodegradable compounds.We incubated microparticles of five different biodegradable plastics,based on polylactictic acid(PLA),polybutylene succinate(PBS),polybutylene adipate terephthalate(PBAT)and polyhydroxybutyrate-co-valerate(PHBV),in-vitro with the gastric fluid of the edible crab Cancer pagurus and evaluated the hydrolysis rates by pH Stat titration.A plastic blend of PLA with PBAT showed the highest hydrolysis rate.The enzymes in the gastric fluid of crabs were separated by anion exchange chromatography.Fractions with carboxylesterase activity were identified using fluorescent methylumbelliferyl(MUF)-derivatives.Pooled fractions with high carboxylesterase activity also hydrolyzed a PLA/PBAT plastic blend.Carboxylesterases showed molecular masses of 40–45 kDa as determined by native gel electrophoresis(SDS-PAGE).Our study demonstrated that digestive carboxylesterases in the gastric fluid of C.pagurus exhibit a high potential for hydrolyzing certain biodegradable plastics.Since esterases are common in the digestive tract of organisms,it seems likely that other invertebrates possess the ability to hydrolyze biodegradable plastics.

Interactions of extracellular DNA with aromatized biochar and protection against degradation by DNaseⅠ

With increasing environmental application,biochar(BC)will inevitably interact with and impact environmental behaviors of widely distributed extracellular DNA(eDNA),which however still remains to be studied.Herein,the adsorption/desorption and the degradation by nucleases of eDNA on three aromatized BCs pyrolyzed at 700℃were firstly investigated.The results show that the eDNA was irreversibly adsorbed by aromatized BCs and the pseudo-second-order and Freundlich models accurately described the adsorption process.Increasing solution ionic strength or decreasing pH below 5.0 significantly increased the eDNA adsorption on BCs.However,increasing pH from 5.0 to 10.0 faintly decreased eDNA adsorption.Electrostatic interaction,Ca ion bridge interaction,andπ-πinteraction between eDNA and BC could dominate the eDNA adsorption,while ligand exchange and hydrophobic interactions were minor contributors.The presence of BCs provided a certain protection to eDNA against degradation by DNase I.BC-bound eDNA could be partly degraded by nuclease,while BC-bound nuclease completely lost its degradability.These findings are of fundamental significance for the potential application of biochar in eDNA dissemination management and evaluating the environmental fate of eDNA.

Polypeptide analysis for nanopore-based protein identification

Presently,proteins are identified by cleaving them with proteases,measuring the mass to charge ratio of the fragments with a mass spectrometer,and matching the fragments to segments within known proteins in databases.We earlier demonstrated that a nanometer-scale pore formed by aerolysin(AeL)can discriminate between,and therefore identify,three similar size proteins from their trypsin-cleaved polypeptide fragments.With this nanopore-protease method,the protein’s identity is instead determined from characteristic ionic current blockade patterns caused by the polypeptide fragments that enter the nanopore.The results also suggested that not all of the theoretically expected cleavage products partition into the pore.To better understand the mechanism by which polypeptide fragments are captured,and how different polypeptides reduce the pore’s ionic current,we studied the effects of 11 identical length polypeptides with different net charges and charge distributions.We show that under certain experimental conditions,negative,positive,and neutral polypeptides are driven into the AeL pore by the same applied voltage polarity.The capture rate and dwell time of polypeptides in the pore depend strongly on the ionic strength,the magnitude of the applied voltage,and the net charge and charge distribution of the polypeptides.The dwell time distribution depends nonmonotonically on the applied voltage(regardless of the polymer’s net charge),and its maximum value depends on the polypeptide net charge and charge distribution.The maximum dwell time for different polypeptides does not occur at the same applied voltage amplitude,which conceivably might complicate the detection and discrimination of some polypeptide fragments.Although additional experiments,computer simulations,and artificial intelligence research are needed to better understand how to optimize the partitioning of enzymatically cleaved fragments into the AeL nanopore,the method is still capable of accurately identifying proteins.