Shoreline Drying of Microseira (Lyngbya) wollei Biomass Can Lead to the Release and Formation of Toxic Saxitoxin Analogues to the Water Column.

The harmful, filamentous cyanobacteria Microseira (Lyngbya) wollei produces several toxic analogues of saxitoxin (Lyngbya wollei toxins 1-6, or LWTs 1-6), grows in shallow water, and can deposit significant biomass on nearby shorelines. Here, we show that the LWTs are stable in the biomass during subsequent drying but that the process facilitates the later release of LWTs upon return to the water column. Under basic conditions, LWTs hydrolyzed to generate products that were significantly more neurotoxic than the initial toxins. Aqueous LWTs were subjected to conditions of covarying temperature and pH, and their degradation rates and products were determined at each condition. LWTs 1, 5, and 6 degraded faster at pH ≥ 8 at all temperatures. Their degradation products, which included decarbamoyl saxitoxin and LWT 4, were consistent with a base-catalyzed hydrolysis mechanism and represented a net increase in total biomass toxicity normalized against the equivalent toxicity of saxitoxin. The corresponding pre-exponential terms and activation energies for hydrolysis were obtained for pH 6-10 over the temperature range 10-40 °C. A locally weighted scatterplot smoothing (LOWESS) regression was developed to predict the loss of parent toxins and subsequent products in the water column under conditions corresponding to those commonly encountered in cyanobacterial blooms.

Evolutionary origins of pectin methylesterase genes associated with novel aspects of angiosperm pollen tube walls.

The early evolution of angiosperms was marked by a number of innovations of the reproductive cycle including an accelerated fertilization process involving faster transport of sperm to the egg via a pollen tube. Fast pollen tube growth rates in angiosperms are accompanied by a hard shank-soft tip pollen tube morphology. A critical actor in that morphology is the wall-embedded enzyme pectin methylesterase (PME), which in type II PMEs is accompanied by a co-transcribed inhibitor, PMEI. PMEs convert the esterified pectic tip wall to a stiffer state in the subapical flank by pectin de-esterification. It is hypothesized that rapid and precise targeting of PME activity was gained with the origin of type II genes, which are derived and have only expanded since the origin of vascular plants. Pollen-active PMEs have yet to be reported in early-divergent angiosperms or gymnosperms. Gene expression studies in Nymphaea odorata found transcripts from four type II VGD1-like and 16 type I AtPPME1-like homologs that were more abundant in pollen and pollen tubes than in vegetative tissues. The near full-length coding sequence of one type II PME (NoPMEII-1) included at least one PMEI domain. The identification of possible VGD1 homologs in an early-diverging angiosperm suggests that the refined control of PMEs that mediate de-esterification of pectins near pollen tube tips is a conserved feature across angiosperms. The recruitment of type II PMEs into a pollen tube elongation role in angiosperms may represent a key evolutionary step in the development of faster growing pollen tubes.

Fabrication of blueberry anthocyanins-rich gels based on the apricot polysaccharides with different esterification degrees.

To enhance the stability of anthocyanins under conditions such as light, temperature, and pH, an apricot polysaccharide hydrogel for anthocyanins encapsulation was prepared in this study. Apricot polysaccharides with different DEs were prepared by an alkaline de-esterification method. A gel was prepared by mixing the apricot polysaccharides with CaCl2 to encapsulate the anthocyanins; the encapsulation efficiency reached 69.52 ± 0.31 %. Additionally, the gel exhibited favorable hardness (144.17 ± 2.33 g) and chewiness (64.13 ± 1.53 g). Fourier transform infrared (FTIR) and X-ray diffractometer (XRD) spectra confirmed that the formation of the hydrogel primarily relied on electrostatic interactions and hydrogen bonding. Compared with free anthocyanins, it was also found that the gel-encapsulated anthocyanins had a higher retention rate (RR) under different temperatures and light.

Comparison on emulsifying and emulgelling properties of low methoxyl pectin with varied degree of methoxylation from different de-esterification methods.

Low methoxyl pectin (LMP) with different degree of methoxylation (DM, 40-50 %, 20-30 % and 5-10 %) were prepared from commercially available citrus pectin using high hydrostatic pressure assisted enzymatic (HHP-pectin) and traditional alkaline (A-pectin) de-esterification method. The results showed that both de-esterification methods and DM exhibited LMPs with varied physicochemical, structural, and functional properties. As the DM decreased, LMP showed a decrease in molecular weight (Mw), while an increase in negative charges and rhamnogalacturonan I (RG-I) ratio, accompanied with better emulsion stability, emulsion gel strength and water-holding properties. Relative to A-pectin, HHP-pectin had higher Mw and lower RG-I side chain ratio, contributing to its better thermal stability, apparent viscosity, and emulgelling properties. HHP-pectin with lower DM (5-10 %) showed superior thickening, emulsifying and emulgelling properties, while that with higher DM (40-45 %) had superior thermal stability, which provided alternative for de-esterification and targeted structural modification of pectin.

Physicochemical properties of pectin-Fe(III) gained by HG-type hawthorn with different esterification degree.

In this study, the complexation ability of HG-type hawthorn pectin with trivalent iron ions after de-esterification was investigated. The moderate esterification reaction could significantly increase the iron content in HG-type hawthorn pectin. Fourier transform infrared spectroscopy (FT-IR) and X-ray photoelectron spectroscopy (XPS) experiments proved that -OH and -COOH in the pectin acted as a bridge connecting Fe3+ leading to the formation of ß-FeOOH structure, and the trivalent iron ions were successfully complexed into the HG-type hawthorn pectin. In addition, infrared and ultraviolet spectroscopic scans, particle size, and potentiometric measurements were carried out to demonstrate the complexation coordination mechanism of hawthorn pectin with Fe3+, and there were differences in the complexation effect of HG-type hawthorn pectin with different degrees of esterification. The gelling properties of HG-type hawthorn pectin were subsequently verified by in vitro gastrointestinal tract simulation experiments to aid the smooth passage of ferric ions through the gastric juices and reduce irritation. The success of the experiments demonstrated that HG-type hawthorn pectin is an excellent raw material for metal complexation, and the degree of esterification is one of the important factors affecting its complexation effect, which proves its potential application value as an iron supplement.

Presence of free gallic acid and gallate moieties reduces auto-oxidative browning of epicatechin (EC) and epicatechin gallate (ECg).

Auto-oxidation of flavan-3-ols leads to browning and consequently loss of product quality during storage of ready-to-drink (RTD) green tea. The mechanisms and products of auto-oxidation of galloylated catechins, the major flavan-3-ols in green tea, are still largely unknown. Therefore, we investigated auto-oxidation of epicatechin gallate (ECg) in aqueous model systems. Oxidation products tentatively identified based on MS included δ- or γ-type dehydrodicatechins (DhC2s) as the main contributors to browning. Additionally, various colourless products were detected, including epicatechin (EC) and gallic acid (GA) from degalloylation, ether-linked ε-type DhC2s, and 6 new coupling products of ECg and GA possessing a lactone interflavanic linkage. Supported by density function theory (DFT) calculations, we provide a mechanistic explanation on how presence of gallate moieties (D-ring) and GA affect the reaction pathway. Overall, presence of gallate moieties and GA resulted in a different product profile and less intense auto-oxidative browning of ECg compared to EC.

Rheological Behavior of Pectin Gels Obtained from Araçá (Psidium cattleianum Sabine) Fruits: Influence of DM, Pectin and Calcium Concentrations.

In this work, purified pectins from Araçá fruits (Psidium cattleianum Sabine) were obtained and characterized after partial demethylation. On each prepared sample, the carboxylic yield was obtained by titration, the degree of methylation (DM) by 1H-NMR, and the molecular weight distribution by steric exclusion chromatography (SEC). Then, the gelation ability in the presence of calcium counterions was investigated and related to DM (59-0%); the pectin concentration (2-10 g L-1); and the CaCl2 concentration (0.1-1 mol L-1) used for dialysis. The critical pectin concentration for homogeneous gelation was above 2 g L-1 when formed against 1 mol L-1 CaCl2. The elastic modulus (G') increased with pectin concentration following the relationship G'~C2.8 in agreement with rigid physical gel network predictions. The purified samples APP and APP-A with DM ≥ 40% in the same conditions released heterogeneous systems formed of large aggregates. Gels formed against lower concentrations of CaCl2 down to 0.1 mol L-1 had a higher degree of swelling, indicating electrostatic repulsions between charged chains, thus, counterbalancing the Ca2+ cross-linkage. Compression/traction experiments demonstrated that an irreversible change in the gel structure occurred during small compression with an enhancement of the G' modulus.

Physicochemical characteristics and functional properties of high methoxyl pectin with different degree of esterification.

Moderate alkali de-esterification can change the physicochemical characteristics and thus the functional properties of high methoxyl pectin (HMP). The results revealed that de-esterification could increase negative charges (Zeta potential from -21 to -31 mV), decrease molecular weight (from 448 to 136 kDa) and apparent viscosity of HMP. Homogalacturonan (HG) content decreased (from 62% to 49%) while rhamnogalacturonan Ⅰ (RG-Ⅰ) content increased (from 32% to 46%) after de-esterification. The group characteristics of HMP with different degree of esterification (DE) were similar and no obvious impact was made on degree of crystallinity by alkali de-esterification. A conformation transition of HMP molecule implied by Congo red test were occurred as the DE decreased. With the decrease of DE, the molecular structure of HMP became shorter and smaller, and the entanglement was weaker. The de-esterification caused slight decrease of thermal stability. Alkali de-esterification would weaken the gel property and the emulsifying ability of HMP.

Effect of different levels of esterification and blockiness of pectin on the functional behaviour of pea protein isolate-pectin complexes.

This research examines changes to the functional (solubility, emulsifying and foaming) properties of pea protein isolate when complexed with commercial citrus pectin of different structural attributes. Specifically, a high methoxy (P90; degree of esterification: 90.0%; degree of blockiness: 64.5%; galacturonic acid content 11.4%) and low methoxy (P29; degree of esterification: 28.6%; degree of blockiness: 31.1%; galacturonic acid: 70%) pectin at their optimum mixing ratios with pea protein isolate (4:1 pea protein isolate to P90; 10:1 pea protein isolate to P29) were assessed at the pHs associated with critical structure forming events during the complexation process (soluble complexation (pHc), pH 6.7 and 6.1; insoluble complex formation (pHϕ1), pH 4.0 and 5.0; maximum complexation (pHopt), pH 3.5 and 3.8; dissolution of complexes, pH 2.4 and 2.1; for admixtures of pea protein isolate-P90 and pea protein isolate-P29, respectively). Pea protein isolate solubility was improved from 41 to 73% by the presence of P90 at pH 6.0 and was also moderately increased at pH 4.0 and pH 5.0 by P90 and P29, respectively. The emulsion stability of both pea protein isolate-pectin complexes was higher than the homogeneous pea protein isolate at all critical pHs except pHopt as well as pHc for pea protein isolate-P29 only. P90, with the higher level blockiness and esterification, displayed better foaming properties at the maximal complexation pH when complexed with pea protein isolate than pea protein isolate-P29 or pea protein isolate alone. However at pHϕ2, pea protein isolate-P29 admixtures produced foams with 100% stability, increasing pea protein isolate foam stability by 85%. The enhanced functionality of pea protein isolate-pectin complexes based on the type of pectin used at critical pHs indicates they may be useful biopolymer ingredients in plant protein applications.

Conformational Behavior, Topographical Features, and Antioxidant Activity of Partly De-Esterified Arabinoxylans.

This study aimed to investigate the effect of arabinoxylans (AX) partial de-esterification with feruloyl esterase on the polysaccharide conformational behavior, topographical features, and antioxidant activity. After enzyme treatment, the ferulic acid (FA) content in AX was reduced from 7.30 to 5.48 µg FA/mg polysaccharide, and the molecule registered a small reduction in radius of gyration (RG), hydrodynamic radius (Rh), characteristic ratio (C∞), and persistence length (q). A slight decrease in α and a small increase in K constants in the Mark-Houwink-Sakurada equation for partially de-esterified AX (FAX) suggested a reduction in molecule structural rigidity and a more expanded coil conformation, respectively, in relation to AX. Fourier transform infrared spectroscopy spectra of AX and FAX presented a pattern characteristic for this polysaccharide. Atomic force microscopy topographic analysis of FAX showed a more regular surface without larger hollows in relation to AX. The antioxidant activity of FAX, compared to AX, was reduced by 30 and 41% using both 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS+) and 1,1-diphenyl-2-picryl-hydrazyl (DPPH) methods, respectively. These results suggest that feruloyl esterase treatment of AX could offer a strategy to tailor AX chains conformation, morphological features, and antioxidant activity, impacting the development of advanced biomaterials for biomedical and pharmaceutical applications.

Preparation of Clindamycin Hydrochloride Loaded De-Esterified Low-Methoxyl Mango Peel Pectin Film Used as a Topical Drug Delivery System.

In this study, we aimed to develop a low-mexthoxyl pectin (LMP) from mango peel pectin through a de-esterification method for use as a film forming agent. The prepared de-esterified pectin (DP) was compared to commercial LMP (cLMP) which possessed a 29% degree of esterification (DE). Mango peel pectin was extracted from ripe Nam Dokmai mango peel using the microwave-assisted extraction method. Pectin derived from the mango peel was classified as a high mexthoxyl pectin (79% DE) with 75% of galacturonic acid (GalA) content. A de-esterification experiment was designed by central composite design to plot the surface response curve. Our prepared DP was classified as LMP (DE 29.40%) with 69% GalA. In addition, the Fourier-transform infrared spectrophotometer (FTIR) spectra of the DP were similar to cLMP and the pectin backbone was not changed by the de-esterification process. Strikingly, the cLMP and DP films showed non-significant differences between their physical properties (p > 0.05) with respect to the puncture strength (13.72 N/mm2 and 11.13 N/mm2 for the cLMP and DP films, respectively), percent elongation (2.75% and 2.52% for the cLMP and DP films, respectively), and Young's modulus (67.69 N/mm2 and 61.79 N/mm2 for the cLMP and DP films, respectively). The de-esterified pectin containing clindamycin HCl (DPC) and low-methoxyl pectin containing clindamycin HCl (cLMPC) films demonstrated 93.47% and 98.79% of drug loading content. The mechanical properties of the cLMPC and DPC films were improved possibly due to their crystal structures and a plasticizing effect of clindamycin HCl loaded into the films. The DPC film exhibited a drug release profile similar to that of the cLMPC film. Our anti-bacterial test of the films found that the cLMPC film showed 41.11 and 76.30 mm inhibitory clear zones against Staphylococcus aureus and Cutibacterium acnes, respectively. The DPC film showed 40.78 and 74.04 mm clear zones against S. aureus and C. acnes, respectively. The antibacterial activities of the cLMPC and DPC films were not significantly different from a commercial clindamycin solution. The results of this study suggest that mango peel pectin can be de-esterified and utilized as an LMP and the de-esterified pectin has the potential for use as a film forming agent, similar to cLMP. In addition, the remarkable use of de-esterified mango peel pectin to prepare films, as shown by our study, holds a great promise as an alternative material for anti-bacterial purposes.

Carboxylesterase, a de-esterification enzyme, catalyzes the degradation of chlorimuron-ethyl in Rhodococcus erythropolis D310-1.

Microbial degradation is considered to be the most acceptable method for degradation of chlorimuron-ethyl, a typical long-term residual sulfonylurea herbicide, but the underlying mechanism at the genetic and biochemical levels is unclear. In this work, the genome sequence of the chlorimuron-ethyl-degrading bacterium Rhodococcus erythropolis D310-1 was completed, and the gene clusters responsible for the degradation of chlorimuron-ethyl in D310-1 were predicted. A carboxylesterase gene, carE, suggested to be responsible for carboxylesterase de-esterification, was cloned from D310-1. CarE was expressed in Escherichia coli BL21 and purified to homogeneity. The active site of the chlorimuron-ethyl-degrading enzyme CarE and the biochemical activities of CarE were elucidated. The results demonstrated that CarE is involved in catalyzing the de-esterification of chlorimuron-ethyl. A carE deletion mutant strain, D310-1ΔcarE, was constructed, and the chlorimuron-ethyl degradation rate in the presence of 100 mg L-1 chlorimuron-ethyl within 120 h decreased from 86.5 % (wild-type strain D310-1) to 58.2 % (mutant strain D310-1ΔcarE). Introduction of the plasmid pNit-carE restored the ability of the mutant strain to utilize chlorimuron-ethyl. This study is the first to demonstrate that carboxylesterase can catalyze the de-esterification reaction of chlorimuron-ethyl and provides new insights into the mechanism underlying the degradation of sulfonylurea herbicides and a theoretical basis for the utilization of enzyme resources.

Effect of enzyme de-esterified pectin on the electrostatic complexation with pea protein isolate under different mixing conditions.

Native high methoxy citrus pectin (NP) was de-esterified by pectin methyl esterase to produce modified pectins [MP (42, 37, and 33)] having different degrees of esterification. Complex coacervation between a pea protein isolate (PPI) and each pectin was investigated as a function of pH (8.0-1.5) and mixing ratio (1:1-30:1, PPI-pectin). Complex formation was found to be optimal for biopolymer-mixing ratios of 8:1, 8:1, 25:1 and 25:1 for PPI complexed with NP, MP42, MP37 and MP33, respectively, at pHs 3.6, 3.5, 3.9 and 3.9. And, the critical pHs associated with complex formation (accessed by turbidity) was found to shift significantly to higher pHs as the degree of esterification of the pectin decreased, whereas the shift in the pH corresponding to their initial interactions was minimal with degree of esterification. Complexation of PPI with NP and MP42 greatly improved the protein solubility.

Deesterification of astaxanthin and intermediate esters from Haematococcus pluvialis subjected to stress.

Haematococcus pluvialis is the richest biological source of astaxanthin under unfavorable growing conditions. Many reports have discussed the optimal astaxanthin extraction methods. Free-astaxanthin could be still hindered by microalgae extracts composition or by prolonged extraction times. In this study we evaluated the effect of enzymolysis and saponification deesterification processes of astaxanthin and its carotenoid precursors under high irradiance and nitrogen deprivation stress time conditions. Results showed that cholesterol esterase facilitated astaxanthin deesterification (975.65 µg mg-1 DW) while saponification positively affected zeaxanthin (1038.68 µg mg-1 DW).

Comparative study on gelling properties of low methoxyl pectin prepared by high hydrostatic pressure-assisted enzymatic, atmospheric enzymatic, and alkaline de-esterification.

The gel behavior of low methoxyl pectin (LMP) de-esterified by high hydrostatic pressure-assisted enzymatic (HHP-pectin, 400 MPa/40 °C/12 min), atmospheric enzymatic (E-pectin, 40 °C/2 h), and alkaline method (A-pectin, pH = 12/25 °C/25 min) was comparatively investigated, with regard to gel strength, water-holding capacity (WHC), rheological properties, and microstructure of pectin gels. Results showed that the de-esterification method had a strong influence on gelling properties of LMPs. The gelation of all LMPs was significantly affected by pectin, Ca2+ concentration and pH of the solution, but enzymatically de-esterified LMPs formed gels with higher gel strength, WHC and better viscoelasticity than those from alkaline de-esterified method. HHP-pectin gels showed even higher gel strength at the same Ca2+ concentrations and better WHC at the same pH, as compared to those from E-pectin. Moreover, the de-esterification time for HHP-pectin was much shorter than that for E-pectin. These findings indicated that HHP could be a potential alternative for high efficient pectin de-esterification, preparing LMPs with better gelling property.

Effect of alkaline de-esterified pectin on the complex coacervation with pea protein isolate under different mixing conditions.

High methoxy citrus pectin (UM88) was saponified to produce modified pectin [M(72, 42, and 9)], with different levels of degree of esterification (DE), to investigate the complex coacervation of pea protein isolate (PPI) with pectin [UM88 and M(72, 42, and 9)]. Regardless of the DE value of pectin, the critical pH corresponding to when insoluble complexes form shifted to higher pH as the mixing ratio increased. The maximum amount of coacervates formed at a biopolymer-mixing ratio of 8:1, 8:1, 10:1 and 15:1 for PPI with UM88, M72, M42, and M9, respectively. Maximum interactions for the protein-pectin admixtures occurred between pH 3.70 and 3.85. PPI complexed with modified pectin displayed greater interactions under their optimal mixing conditions compared to the unmodified pectin. The de-esterification of pectin resulted in more rigid and stiffer pectin, which enhanced its interaction with PPI by shifting the critical parameters to a higher value.

Directed Evolution of Sulfonylurea Esterase and Characterization of a Variant with Improved Activity.

Esterase SulE detoxicates a variety of sulfonylurea herbicides through de-esterification. SulE exhibits high activity against thifensulfuron-methyl but low activity against other sulfonylureas. In this study, two variants, m2311 (P80R) and m0569 (P80R and G176A), with improved activity were screened from a mutation library constructed by error-prone PCR. Variant m2311 showed a higher activity against sulfonylureas in comparison variant m0569 and was further investigated. The kcat/ Km value of variant m2311 for metsulfuron-methyl, sulfometuron-methyl, chlorimuron-ethyl, tribenuron-methyl, and ethametsulfuron-methyl increased by 3.20-, 1.72-, 2.94-, 2.26- and 2.96-fold, respectively, in comparison with the wild type. Molecular modeling suggested that the activity improvement of variant m2311 is due to the substitution of Pro80 by arginine, leading to the formation of new hydrogen bonds between the enzyme and substrate. This study facilitates further elucidation of the structure and function of SulE and provides an improved gene resource for the detoxification of sulfonylurea residues and the genetic engineering of sulfonylurea-resistant crops.

Purification and Characterization of a Salt-Dependent Pectin Methylesterase from Carica papaya Fruit Mesocarp-Exocarp Tissue.

Pectin methylesterase (PME) is a ubiquitous cell wall enzyme, which de-esterifies and modifies pectins for food applications. Functional properties of pectin rely on molecular weight and degree of esterification, and thus de-esterification by PME influences the pectin functionality. The main aim of the study is to purify and biochemically characterize PME from the outer mesocarp-exocarp tissue of unripe Carica papaya L. fruit. The ion-exchange and gel-permeation chromatography purified enzyme exhibited a specific activity of 2363.1 ± 92.8 units/mg protein, with a fold purification of 10.6, and final recovery of 9.0%. The PME showed a low apparent mass of 27 kDa by SDS-PAGE. The optimal activity of purified PME was found at pH 7.0, and at 60 °C. The enzyme is fairly stable at 60 °C for 10 min, retaining 60% activity. The optimum activity was found with 0.25 mol/L monovalent salts indicating that this PME is salt-dependent. The Km of PME was 0.22 mg/mL, and the Vmax value was 1289.15 ± 15.9 units/mg. The increase in the calcium sensitivity of the PME-treated pectin indicated a blockwise mode of action. The PME significantly differs from other known plant PMEs in their biochemical properties. Manual inspection and MASCOT searching of generated tryptic peptides confirmed no homology to known papaya PME sequences. The preliminary results indicate that the papaya PME can be potentially utilized to modify pectin functionality at elevated temperature. However, further investigation is required to understand the usefulness of this enzyme for the modification of pectins for various food applications. PRACTICAL APPLICATION: In this work, a small, 27 kDa papaya PME was purified by ion-exchange and gel-permeation chromatography and biochemically characterized. The papaya PME significantly differs from other known plant PMEs in their biochemical properties. The preliminary results like fair thermostability coupled with high temperature optimum indicate that the papaya PME can be potentially utilized to modify pectin functionality at high temperature. Modification of pectin functionality at elevated temperatures is advantageous since it evades the detrimental action of other pectinolytic enzymes.