Cryoprotective effect of soluble soybean polysaccharides and enzymatic hydrolysates on the myofibrillar protein of Nemipterus virgatus surimi.

Cryoprotective effect and potential mechanism of soluble soybean polysaccharides (SSPS) and enzymatic hydrolysates on surimi was investigated. After hydrolysis, the molecular weight of SSPS significantly decreased, and the hydrolysates prepared by endo-polygalacturonase (EPG-SSPS) was the lowest (154 kDa). Infrared spectrum analysis revealed that enzymatic hydrolysis didn't alter the functional groups of SSPS, but it did augment the exposure to hydroxyl groups. Surimi containing 5 % EPG-SSPS had the lowest freezable water after 20 days of frozen storage. Furthermore, the 5 % EPG-SSPS group manifested the highest metrics in total sulfhydryl (8.0 × 10-5 mol/g), active sulfhydryl content (6.7 × 10-5 mol/g), Ca2+-ATPase activity, and exhibited the lowest level in carbonyl content, surface hydrophobicity (153 µg). Notably, the 5 % EPG-SSPS maintained the stability of protein structure. Conclusively, SSPS enzymatic hydrolysate using endo-polygalacturonase imparted superior cryoprotective effect on the myofibrillar protein of surimi, and the mechanism might be a decrease in molecular weight and exposure of hydroxyl groups.

Supplementation of citrus pectin with whole-cell pectinase PG5 on Pichia pastoris promotes recovery of colitis and enhances intestinal barrier function in DSS-treated mice.

A whole-cell biocatalyst was developed by genetically engineering pectinase PG5 onto the cell surface of Pichia pastoris using Gcw12 as the anchoring protein. Whole-cell PG5 eliminated the need for enzyme extraction and purification, while also exhibiting enhanced thermal stability, pH stability, and resistance to proteases in vitro compared to free PG5. Magnetic resonance mass spectrometry analysis revealed that whole-cell PG5 efficiently degraded citrus pectin, resulting in the production of a mixture of pectin oligosaccharides. The primary components of the mixture were trigalacturonic acid, followed by digalacturonic acid and tetragalacturonic acid. Supplementation of citrus pectin with whole-cell PG5 resulted in a more pronounced protective effect compared to free PG5 in alleviating colitis symptoms and promoting the integrity of the colonic epithelial barrier in a mouse model of dextran sulfate sodium-induced colitis. Hence, this study demonstrates the potential of utilizing whole-cell pectinase as an effective biocatalyst to promote intestinal homeostasis in vivo.

A Smart Pesticide-Controlled Release Platform with Dual Stimuli-Responsive Functions for Enhanced Treatment of Plant Black Shank.

Realizing controllable input of botanical pesticides is conducive to improving pesticide utilization, reducing pesticide residues, and avoiding environmental pollution but is extremely challenging. Herein, we constructed a smart pesticide-controlled release platform (namely, SCRP) for enhanced treatment of tobacco black shank based on encapsulating honokiol (HON) with mesoporous hollow structured silica nanospheres covered with pectin and chitosan oligosaccharide (COS). The SCRP has a loading capacity of 12.64% for HON and could effectively protect HON from photolysis. Owing to the pH- and pectinase-sensitive property of the pectin, the SCRP could smartly release HON in response to a low pH or a rich pectinase environment in the black shank-affected area. Consequently, the SCRP effectively inhibits the infection of P. nicotianae on tobacco with a controlled rate for tobacco black shank of up to 87.50%, which is mainly due to the SCRP's capability in accumulating ROS, changing cell membrane permeability, and affecting energy metabolism. In addition, SCRP is biocompatible, and the COS layer enables SCRP to show a significant growth-promoting effect on tobacco. These results indicate that the development of a stimuli-responsive controlled pesticide release system for plant disease control is of great potential and value for practical agriculture production.

Changes in the Main Physicochemical Properties and Electrochemical Fingerprints in the Production of Sea Buckthorn Juice by Pectinase Treatment.

Enzymatic hydrolysis using pectinase is critical for producing high-yield and quality sea buckthorn juice. This study determined the optimal temperature, time, and enzyme dosage combinations to guide manufacturers. A temperature of 60 °C, hydrolysis time of 3 h, and 0.3% enzyme dosage gave 64.1% juice yield-25% higher than without enzymes. Furthermore, monitoring physicochemical properties reveals enzyme impacts on composition. Higher dosages increase soluble solids up to 15% and soluble fiber content by 35% through cell wall breakdown. However, excessive amounts over 0.3% decrease yields. Pectin concentration also declines dose-dependently, falling by 91% at 0.4%, improving juice stability but needing modulation to retain viscosity. Electrochemical fingerprinting successfully differentiates process conditions, offering a rapid quality control tool. Its potential for commercial inline use during enzymatic treatment requires exploration. Overall, connecting optimized parameters to measured effects provides actionable insights for manufacturers to boost yields, determine enzyme impacts on nutrition/functionality, and introduce novel process analytical technology. Further investigations of health properties using these conditions could expand sea buckthorn juice functionality.

Analytical characterization of flavan-3-ol-rich apple juices produced with the innovative spiral filter press technology.

The retention of flavan-3-ols and other phenolic compounds during apple juice production at pilot plant scale (200 kg, cv. Boskoop) was investigated. An oxygen-excluding spiral filter press and a conventional decanter were used with and without pectinase mash treatment. Phenolic compounds were comprehensively identified and quantitated by RP-UHPLC and HILIC, both coupled to DAD-FLD and DAD-ESI(-)-QTOF-HR-MS/MS. These techniques combined with using a NIST cocoa flavan-3-ol standard allowed for the first time an individual quantification of flavan-3-ol fractions (DP 1-7) in apple juices. Spiral filter-pressed juices were exposed to less oxidation and exhibited four times higher total phenolic compound levels than decanter-made juices (1016 vs. 262 mg/L). Apple juices derived from pectinase-treated mashes had lower total phenolic compound levels than their non-treated counterparts. However, those made by spiral filter press still retained significantly higher concentrations (780 vs. 104 mg/L). Flavan-3-ols were especially well retained by spiral filter press processing, reaching unprecedentedly high concentrations of up to 713 mg/L. A 280 mL serving of non-treated spiral filter-pressed juice would therefore suffice to provide the daily intake of 200 mg flavan-3-ols, equaling the dose of cocoa flavan-3-ols associated with an authorized European health claim for healthy blood flow.

Production and immobilization of pectinases from Penicillium crustosum in magnetic core-shell nanostructures for juice clarification.

Global market of food enzymes is held by pectinases, mostly sourced from filamentous fungi via submerged fermentation. Given the one-time use nature of enzymes to clarify juices and wines, there is a crucial need to explore alternatives for enzyme immobilization, enabling their reuse in food applications. In this research, an isolated fungal strain (Penicillium crustosum OR889307) was evaluated as a new potential pectinase producer in submerged fermentation. Additionally, the enzyme was immobilized in magnetic core-shell nanostructures for juice clarification. Findings revealed that Penicillium crustosum exhibited enzymatic activities higher than other Penicillium species, and pectinase production was enhanced with lemon peel as a cosubstrate in submerged fermentation. The enzyme production (548.93 U/mL) was optimized by response surface methodology, determining the optimal conditions at 35 °C and pH 6.0. Subsequently, the enzyme was covalently immobilized on synthesized magnetic core-shell nanoparticles. The immobilized enzyme exhibited superior stability at higher temperatures (50 °C) and acidic conditions (pH 4.5). Finally, the immobilized pectinases decreased 30 % the orange juice turbidity and maintained 84 % of the enzymatic activity after five consecutive cycles. In conclusion, Penicillium crustosum is a proven pectinase producer and these enzymes immobilized on functionalized nanoparticles improve the stability and reusability of pectinase for juice clarification.

Purification, biochemical characterization, and biotechnological applications of a multifunctional enzyme from the Thermoascus aurantiacus PI3S3 strain.

The filamentous Thermoascus aurantiacus fungus characterized by its thermophilic nature, is recognized as an exceptional producer of various enzymes with biotechnological applications. This study aimed to explore biotechnological applications using polygalacturonase (PG) derived from the Thermoascus aurantiacus PI3S3 strain. PG production was achieved through submerged fermentation and subsequent purification via ion-exchange chromatography and gel filtration methods. The crude extract exhibited a diverse spectrum of enzymatic activities including amylase, cellulase, invertase, pectinase, and xylanase. Notably, it demonstrated the ability to hydrolyze sugarcane bagasse biomass, corn residue, and animal feed. The purified PG had a molecular mass of 36 kDa, with optimal activity observed at pH 4.5 and 70 °C. The activation energy (Ea) was calculated as 0.513 kJ mol-1, highlighting activation in the presence of Ca2+. Additionally, it displayed apparent Km, Vmax, and Kcat values of at 0.19 mg mL-1, 273.10 U mL-1, and 168.52 s-1, respectively, for hydrolyzing polygalacturonic acid. This multifunctional PG exhibited activities such as denim biopolishing, apple juice clarification, and demonstrated both endo- and exo-polygalacturonase activities. Furthermore, it displayed versatility by hydrolyzing polygalacturonic acid, carboxymethylcellulose, and xylan. The T. aurantiacus PI3S3 multifunctional polygalacturonase showed heightened activity under acidic pH, elevated temperatures, and in the presence of calcium. Its multifunctional nature distinguished it from other PGs, significantly expanding its potential for diverse biotechnological applications.

A plant mechanism of hijacking pathogen virulence factors to trigger innate immunity.

Polygalacturonase-inhibiting proteins (PGIPs) interact with pathogen-derived polygalacturonases to inhibit their virulence-associated plant cell wall-degrading activity but stimulate immunity-inducing oligogalacturonide production. Here we show that interaction between Phaseolus vulgaris PGIP2 (PvPGIP2) and Fusarium phyllophilum polygalacturonase (FpPG) enhances substrate binding, resulting in inhibition of the enzyme activity of FpPG. This interaction promotes FpPG-catalyzed production of long-chain immunoactive oligogalacturonides, while diminishing immunosuppressive short oligogalacturonides. PvPGIP2 binding creates a substrate binding site on PvPGIP2-FpPG, forming a new polygalacturonase with boosted substrate binding activity and altered substrate preference. Structure-based engineering converts a putative PGIP that initially lacks FpPG-binding activity into an effective FpPG-interacting protein. These findings unveil a mechanism for plants to transform pathogen virulence activity into a defense trigger and provide proof of principle for engineering PGIPs with broader specificity.

MD simulation to comprehend polygalacturonase inactivation mechanism during thermal and non-thermal effects of infrared processing.

Food quality is greatly impacted by traditional heat methods for polygalacturonase (PG) inactivation; therefore, it's imperative to develop a novel infrared (IR) inactivation approach and identify its mechanism. Utilizing molecular dynamics (MD) simulation, this study verified the PG's activity, structure, active sites, and substrate channel under the single thermal and non-thermal effects of IR. PG activity was significantly reduced by IR, and structure was unfolded by increasing random coils (65.62 %) and decreasing ß-sheets (29.11 %). MD data indicated that the relative locations of PG's active sites were altered by both IR effects, and the enzyme-substrate channel was shortened (10.53 % at 18 µm and 15.79 % at 80 °C). The thermal effect of IR on the inactivation of PG was significantly more pronounced than its non-thermal effect. This study unveiled the mechanism by which the infrared disrupted PG's activity, active sites, and substrate channels; thus, it expanded the infrared technique's efficacy in enzyme control.

Thermoalkalophilic polygalacturonase from a novel Glutamicibacter sp.: Bioprospecting, strain improvement, statistical optimization and applications.

The current research discusses a multidimensional bioprocess development, that includes bioprospecting, strain improvement, media optimisation, and applications of the extracted enzyme. A potent alkalophilic polygalacturonase (PG) producing bacterial strain was isolated and identified as a novel Glutamicibacter sp. Furthermore, strain improvement by UV and chemical mutagenesis not only improved the enzyme (PGmut) production but also enhanced its temperature optima from 37 °C to 50 °C. The use of solid substrate fermentation, followed bystatistical optimisation through PB and RSM, substantially increasedPGmut production. A 10-fold increase in enzyme production (632 U/gm) was observed when sugarcane bagasse with a pH of 10.5, 66.8 % moisture, and an inoculum size of 10.15 % was used. The model's accuracy was supported by p-value (p < 0.0001), and an R2 of 0.9940. A pilot-scale experiment, demonstrated ≈ 62,229 U/100 gm PG activity. Additionally, the enzyme's efficacy in demucilization of coffee beans, and bioscouring of jute fibre indicated that it is a valuable biocatalyst.

An overview of microbial enzymatic approaches for pectin degradation.

Pectin, a complex natural macromolecule present in primary cell walls, exhibits high structural diversity. Pectin is composed of a main chain, which contains a high amount of partly methyl-esterified galacturonic acid (GalA), and numerous types of side chains that contain almost 17 different monosaccharides and over 20 different linkages. Due to this peculiar structure, pectin exhibits special physicochemical properties and a variety of bioactivities. For example, pectin exhibits strong bioactivity only in a low molecular weight range. Many different degrading enzymes, including hydrolases, lyases and esterases, are needed to depolymerize pectin due to its structural complexity. Pectin degradation involves polygalacturonases/rhamnogalacturonases and pectate/pectin lyases, which attack the linkages in the backbone via hydrolytic and ß-elimination modes, respectively. Pectin methyl/acetyl esterases involved in the de-esterification of pectin also play crucial roles. Many α-L-rhamnohydrolases, unsaturated rhamnogalacturonyl hydrolases, arabinanases and galactanases also contribute to heterogeneous pectin degradation. Although numerous microbial pectin-degrading enzymes have been described, the mechanisms involved in the coordinated degradation of pectin through these enzymes remain unclear. In recent years, the degradation of pectin by Bacteroides has received increasing attention, as Bacteroides species contain a unique genetic structure, polysaccharide utilization loci (PULs). The specific PULs of pectin degradation in Bacteroides species are a new field to study pectin metabolism in gut microbiota. This paper reviews the scientific information available on pectin structural characteristics, pectin-degrading enzymes, and PULs for the specific degradation of pectin.

Fluorescence and pectinase double-triggered chitosan/pectin/calcium propionate/curcumin-ß-cyclodextrin complex film for pork freshness monitoring and maintenance.

An intelligent and active food packaging film based on chitosan (CS), pectin (P), calcium propionate (CP), and curcumin-ß-cyclodextrin complex (Cur-ß-CD) was prepared. The CS/P/CP/Cur-ß-CD film exhibited improved hydrophobicity (74.78 ± 0.53°), water vapor (4.55 ± 0.16 × 10-11 g·(m·s·Pa)-1), and oxygen (1.50 ± 0.06 × 10-12 g·(m·s·Pa)-1) barrier properties, as well as antioxidant (72.34 ± 3.79 % for DPPH and 86.05 ± 0.14 % for ABTS) and antibacterial (79.41 ± 2.89 % for E. coli and 83.82 ± 3.96 % for S. aureus) activities. The release of CP and Cur could be triggered by pectinase, with their cumulative release reaching 92.62 ± 1.20 % and 42.24 ± 1.15 %, respectively. The CS/P/CP/Cur-ß-CD film showed delayed alterations in surface color, pH value, total volatile bases nitrogen, total viable counts, thiobarbituric acid reactive substance, hardness, and springiness of pork. Additionally, the fluorescence intensity of the film gradually decreased. In conclusion, we have developed a pH-responsive film with pectinase-triggered release function, providing a new concept for the design of multi-signal responsive intelligent food packaging.

Innovative biocatalyst synthesis of pectinolytic enzymes by cross-linking strategy: Potentially immobilised pectinases for the production of pectic-oligosaccharides from pectin.

Pectinases are outstanding multienzymes, which have the potential to produce new emerging pectic-oligosaccharides (POS) via enzymatic hydrolysis of pectin. However, free pectinase is unable to undergo repeated reaction for the production of POS. This study proposed a sustainable biocatalyst of pectinases known as cross-linked pectinase aggregates (CLPA). Pectinase from Aspergillus aculeatus was successfully precipitated using 2 mg/mL pectinase and 60 % acetone for 20 min at 20 °C, which remained 36.3 % of its initial activity. The prepared CLPA showed the highest activity recovery (85.0 %), under the optimised conditions (0.3 % (v/v) starch and glutaraldehyde mixture (St/Ga), 1.5: 1 of St/Ga, 25 °C, 1.5 h). Furthermore, pectin-degrading enzymes from various sources were used to produce different CLPA. The alteration of pectinase secondary structure gave high stability in acidic condition (pH 4), thermostability, deactivation energy and half-life, and improved storage stability at 4 °C for 30 days. Similarly to their free counterpart, the CLPA exhibited comparable enzymatic reaction kinetics and could be reused eight times with approximately 20 % of its initial activity. The developed CLPA does not only efficaciously produced POS from pectin as their free form, but also exhibited better operational stability and reusability, making it more suitable for POS production.

Pharmamedia and pectinase production by Bacillus subtilis Mz-12P.

Bacterial isolates collected from the environment were screened for pectinolytic activity, and a strain with the highest activity was selected and identified as Bacillus subtilis Mz-12. The presence of pectin hydrolase, lyase, and esterase activities was confirmed. Pectinase was purified and characterized. Enzyme production was optimized with respect to temperature, pH, and growth medium. Enzyme stability and activity were characterized under different temperatures and pH values. The results showed that this strain was capable of producing high yields of pectinase in commercial medium (Pharmamedia) 24.6 U/mL compared to other media. The purified pectinase of 22.3 kDa produced was constitutive in nature. The isolated enzyme from this strain displayed a wide range of temperature and pH stability, with the optimal activity observed at pH 9.0 and 50°C. These results indicate that the B. subtilis Mz-12 strain is a favorable candidate for industrial enzyme production. The use of Pharmamedia is reported for first time for pectinase production.

Valorization of wheat straw into paper by ultrafiltered enzymatic bleaching approach.

In this study, the potential of ultrafiltered xylano-pectinolytic enzymatic bleaching approach was investigated, for manufacturing wheat straw-based paper. The enzymatic step was found to be most effective, with xylanase-pectinase dose of 4-1.7 IU/g pulp and time period of 180 min. The absorption spectra of the pulp free filtrate samples obtained after treatment of the pulp with ultrafiltered enzymes showed the removal of more impurities, in comparison to the treatment with crude enzymes. Microscopic analysis also showed the removal of lignin impurities in enzymatically bleached pulp samples. This bleaching approach using enzymes resulted in 27% reduction in ClO2 dose. Ultrafiltered enzymes treated pulp samples also showed improved quality-related parameters, and Gurley porosity, burst index, breaking length, double fold, tear index, and viscosity increased by 19.05, 13.70, 8.18, 29.27, 4.41, and 13.27%, respectively. The lignin content, TDS, TSS, BOD and COD values also decreased in the effluent samples obtained after enzymatic bleaching plus 73% chemical bleaching dose. The BOD and COD values of the effluent samples improved by 23.01 and 23.66%, respectively. Thus, indicating the potential of ultrafiltered xylano-pectinolytic enzymes in reducing pollution during bleaching of wheat straw. This is the first study, mentioning the efficacy of ultrafiltered enzymes in the bleaching of wheat straw-based paper with better optical-strength-related properties and effluent characteristics.

Toward a zero waste approach: Utilization of sugarcane bagasse for dye removal and multienzymes production.

Global production of sugarcane bagasse (SB) by sugar industries exceeds more than 100 tons per annum. SB is rich in lignin and polysaccharide and hence can serve as a low-cost energy and carbon source for the growth of industrially important microorganism. However, various other applications of SB have also been investigated. In this study, SB was used as an adsorbent to remove an azo dye, malachite green. Subsequently, the dye-adsorbed SB was fermented by Trametes pubescens MB 89 for the production of laccase enzyme. The fungal pretreated SB was further utilized as a substrate for the simultaneous production of multiple plant cell wall degrading enzymes including, cellulase, xylanase, pectinase, and amylase by thermophilic bacterial strains. Results showed that 0.1% SB removed 97.04% malachite green at 30°C after 30 min from a solution containing 66 ppm of the dye. Fermentation of the dye-adsorbed SB by T. pubescens MB 89 yielded 667.203 IU mL-1 laccase. Moreover, Brevibacillus borstelensis UE10 produced 38.41 and 18.6 IU mL-1 ß-glucosidase and pectinase, respectively, by using fungal-pretreated SB. Cultivation of B. borstelensis UE27 in the medium containing the same substrate yielded 32.14 IU mL-1 of endoglucanase and 27.23 IU mL-1 of ß-glucosidase. Likewise, Neobacillus sedimentimangrovi UE25 could produce a mix of ß-glucosidase (37.24 IU mL-1 ), xylanase (18.65 IU mL-1 ) and endoglucanase (26.65 IU mL-1 ). Hence, this study led to the development of a method through which dye-containing textile effluent can be treated by SB along with the production of industrially important enzymes.

Pectin polysaccharide contribution to oleosome extraction after wet milling of rapeseed.

Oleosomes are lipid composites providing energy storage in oilseeds. They possess a unique structure, comprised of a triglyceride core stabilized by a phospholipid-protein membrane, and they have shown potential to be used as ingredients in several food applications. Intact oleosomes are extracted by an aqueous process which includes soaking, milling, and gravitational separation. However, the details of the complexes formed between oleosomes, proteins and pectin polysaccharides during this extraction are not known. It was hypothesized that pectins play an important role during the oleosome separation, and different proteins will be complexed on the surface of the oleosomes, depending on the pH of extraction. Rapeseed extracts were treated with and without pectinase (Pectinex Ultra SP-L) and extracted at pH 5.7 or 8.5, as this will affect electrostatic complexation. Acidic conditions led to co-extraction of storage proteins, structured as dense oleosome emulsions, stabilized by a network of proteins and polysaccharides. Pectinase intensified this effect, highlighting pectic polysaccharides' role in bridging interactions among proteins and oleosomes under acidic conditions. The presence of this dense interstitial layer around the oleosomes protected them from coalescence during extraction. Conversely, under alkaline conditions, the extraction process yielded more purified oleosomes characterized by a larger particle size, most likely due to coalescence. Nevertheless, pectinase addition at pH 8.5 mitigated coalescence tendencies. These results contribute to a better understanding of the details of the colloidal complexes formed during extraction and can be used to modulate the composition of the extracted fractions, with significant consequences not only for yields and purity but also for the functional properties of the ingredients produced.

Potential of psychrotolerant rhizobacteria for the growth promotion of wheat (Triticum aestivum L.).

Wheat is the second most important staple crop grown and consumed worldwide. Temperature fluctuations especially the cold stress during the winter season reduces wheat growth and grain yield. Psychrotolerant plant growth-promoting rhizobacteria (PGPR) may improve plant stress-tolerance in addition to serve as biofertilizer. The present study aimed to isolate and identify PGPR, with the potential to tolerate cold stress for subsequent use in supporting wheat growth under cold stress. Ten psychrotolerant bacteria were isolated from the wheat rhizosphere at 4 °C and tested for their ability to grow at wide range of temperature ranging from -8 °C to 36 °C and multiple plant beneficial traits. All bacteria were able to grow at 4 °C to 32 °C temperature range and solubilized phosphorus except WR23 at 4 °C, whereas all the bacteria solubilized phosphorus at 28 °C. Seven bacteria produced indole-3-acetic acid at 4 °C, whereas all produced indole-3-acetic acid at 28 °C. Seven bacteria showed the ability to fix nitrogen at 4 °C, while all the bacteria fixed nitrogen at 28 °C. Only one bacterium showed the potential to produce cellulase at 4 °C, whereas four bacteria showed the potential to produce cellulase at 28 °C. Seven bacteria produced pectinase at 4 °C, while one bacterium produced pectinase at 28 °C. Only one bacterium solubilized the zinc at 4 °C, whereas six bacteria solubilized the zinc at 28 °C using ZnO as the primary zinc source. Five bacteria solubilized the zinc at 4 °C, while seven bacteria solubilized the zinc at 28 °C using ZnCO3 as the primary zinc source. All the bacteria produced biofilm at 4 °C and 28 °C. In general, we noticed behavior of higher production of plant growth-promoting substances at 28 °C, except pectinase assay. Overall, in vitro testing confirms that microbes perform their inherent properties efficiently at optimum temperatures rather than the low temperatures due to high metabolic rate. Five potential rhizobacteria were selected based on the in vitro testing and evaluated for plant growth-promoting potential on wheat under controlled conditions. WR22 and WR24 significantly improved wheat growth, specifically increasing plant dry weight by 42% and 58%, respectively. 16S rRNA sequence analysis of WR22 showed 99.78% similarity with Cupriavidus campinensis and WR24 showed 99.9% similarity with Enterobacter ludwigii. This is the first report highlighting the association of C. campinensis and E. ludwigii with wheat rhizosphere. These bacteria can serve as potential candidates for biofertilizer to mitigate the chilling effect and improve wheat production after field-testing.

Genome-Wide Analysis of the Polygalacturonase Gene Family Sheds Light on the Characteristics, Evolutionary History, and Putative Function of Akebia trifoliata.

Polygalacturonase (PG) is one of the largest families of hydrolytic enzymes in plants. It is involved in the breakdown of pectin in the plant cell wall and even contributes to peel cracks. Here, we characterize PGs and outline their expression profiles using the available reference genome and transcriptome of Akebia trifoliata. The average length and exon number of the 47 identified AktPGs, unevenly assigned on 14 chromosomes and two unassembled contigs, were 5399 bp and 7, respectively. The phylogenetic tree of 191 PGs, including 47, 57, 51, and 36 from A. trifoliata, Durio zibethinus, Actinidia chinensis, and Vitis vinifera, respectively, showed that AktPGs were distributed in all groups except group G and that 10 AktPGs in group E were older, while the remaining 37 AktPGs were younger. Evolutionarily, all AktPGs generally experienced whole-genome duplication (WGD)/segmental repeats and purifying selection. Additionally, the origin of conserved domain III was possibly associated with a histidine residue (H) substitute in motif 8. The results of both the phylogenetic tree and expression profiling indicated that five AktPGs, especially AktPG25, could be associated with the cracking process. Detailed information and data on the PG family are beneficial for further study of the postharvest biology of A. trifoliata.

Effect of ultrasonic treatment on enzymes: Decoupling the relation between the ultrasonic driven conformational change and enzyme activity.

Considering the growing interest in enzyme-based extraction technology as a safe and eco-friendly extraction technique, along with the relatively high cost associated with enzymatic applications, it became necessary to explore novel strategies aimed to improve enzyme activity. In this study, the impact of ultrasonic treatment on commercial cellulase and pectinase was investigated. As this effect may be influenced by various ultrasonic and enzyme-related parameters, changes in enzyme conformation were explored under optimal and non-optimal enzyme conditions. The intrinsic fluorescence spectrum was utilized as a tool for monitoring these changes. Additionally, the enzyme's catalytic potential was also assessed under the same conditions. Results indicated that the impact of ultrasonic treatment on enzyme conformation primarily depends on the total ultrasonic energy delivered to the system, rather than other ultrasonic parameters such as power, sample volume, treatment time, or duty cycle. The maximum relative decrease in intrinsic fluorescence intensity of Pectinex® Ultra Clear (PUC) and Pectinex® Ultra SPL (PUS) after ultrasonic treatment was approximately 51% and 55%, respectively, while the decrease induced by thermal denaturation was 25% and 30% respectively. Furthermore, a blue shift in the fluorescence spectrum of both pectinases was observed upon sonication for all process conditions indicating a change in enzyme conformation. However, ultrasonic treatment did not result in a significant change in enzyme activity, suggesting that these conformational adjustments may occur in regions other than the active sites. Moreover, ultrasonicated pectinases and cellulases did not exhibit any improvement in their catalytic potential under either optimal or non-optimal conditions.