Utilization of wild Cressa cretica biomass for pectinase production from a halo-thermotolerant bacterium.

Halophytes are the native inhabitants of saline environment. Their biomass can be considered as a potential substrate for the production of microbial enzymes. This study was intended at feasible utilization of a halophytic biomass, Cressia cretica, for pectinase production using a halo- and thermo-tolerant bacterium, Bacillus vallismortis MH 10. The data from fractionation of the C. cretica biomass revealed presence of 17% pectin in this wild biomass. Seven different factors (temperature, agitation, pH, inoculum size, peptone concentration, substrate concentration, and incubation time) affecting pectinase production using C. cretica were assessed through a statistical tool, Plackett-Burman design. Consequently, two significant factors (incubation time and peptone concentration) were optimized using the central composite design. The strain produced 20 IU mL-1 of pectinase after 24 h under optimized conditions. The enzyme production kinetics data also confirmed that 24 h is the most suitable cultivation period for pectinase production. Fourier transform infrared spectroscopy and scanning electron microscopy of C. cretica biomass ascertained utilization of pectin and structural changes after fermentation. The purification of pectinase by using DEAE column yielded specific activity and purification fold of 88.26 IU mg-1 and 3.2, respectively. The purified pectinase had a molecular weight of >65 kDa. This study offers prospects of large-scale production of pectinase by halotolerant strain in the presence of economical and locally grown substrate that makes the enzyme valuable for various industrial operations.

Molecular dynamics simulation of the interaction of a raspberry polygalacturonase (RiPG) with a PG inhibiting protein (RiPGIP) isolated from ripening raspberry (Rubus idaeus cv. Heritage) fruit as a model to understand proteins interaction during fruit softening.

Polygalacturonase (PG) is an important hydrolytic enzyme involved in pectin disassembly and the subsequent textural changes during fruit ripening. Although the interaction of fungal PGs with other proteins has been documented, the interaction of plant PGs with other plant proteins has not yet been studied. In this study, the molecular mechanisms involved in raspberry fruit ripening, particularly the polygalacturonase (RiPG) interaction with polygalacturonase inhibiting protein (RiPGIP) and substrate, were investigated with a structural approach. The 3D model of RiPG2 and RiPGIP3 was built using a comparative modeling strategy and validated using molecular dynamics (MD) simulations. The RiPG2 model structure comprises 11 complete coils of right-handed parallel ß-helix architecture, with an average of 27 amino acid residues per turn. The structural model of the RiPGIP3 displays a typical structure of LRR protein, with the right-handed superhelical fold with an extended parallel ß-sheet. The conformational interaction between the RiPG2 protein and RiPGIP3 showed that RiPGIP3 could bind to the enzyme and thereby leave the active site cleft accessible to the substrate. All this evidence indicates that RiPG2 enzyme could interact with RiPGIP3 protein. It can be a helpful model for evaluating protein-protein interaction as a potential regulator mechanism of hydrolase activity during pectin disassembly in fruit ripening.

A review on pectinase properties, application in juice clarification, and membranes as immobilization support.

Pectic substances cause haziness and high viscosity of fruit juices. Pectinase enzymes are biological compounds that degrade pectic compounds. Nontoxicity and ecofriendly nature make pectinases excellent biocatalysts for juice clarification. However, the poor stability and nonreusability of pectinases trim down the effectiveness of the operation. The immobilization techniques have gained the attention of researchers as it augments the properties of the enzymes. Literature has reported the stability improvement of enzymes like lipase, laccase, hydrogen peroxidase, and cellulase upon immobilization on the membrane. However, only a few research articles divulge pectinase immobilization using a membrane. The catalysis-separation synergy of membrane-reactor has put indelible imprints in industrial applications. Immobilization of pectinase on the membrane can enhance its performance in juice processing. This review delineates the importance of physicochemical and kinematic properties of pectinases relating to the juice processing parameters. It also includes the influence of metal-ion cofactors on enzymes' activity. Considering the support and catalytic-separation facets of the membrane, the prediction of the membrane as support for pectinase immobilization has also been carried out.

Evaluation and enzyme-aided enhancement of anti-photoaging properties of Camellia japonica in UVA-irradiated keratinocytes.

Exposure to ultraviolet (UV) radiation is the main reason behind extrinsic skin aging. Changes due to chronic UV exposure are called photoaging. Natural products are effective ingredients against UV-mediated skin damage. Present study investigated the anti-photoaging properties of Camellia japonica flowers which possess various bioactivities. To enrich the extracts of C. japonica flowers, pectinase and beta-glucosidase treatment was employed. Anti-photoaging effect was screened using the changes in MMP-1 and collagen levels in UVA-irradiated human HaCaT keratinocytes. The crude extract of C. japonica flowers (CE) was shown to decrease the UVA-induced MMP-1 secretion while attenuating the collagen levels. Pectinase and beta-glucosidase treated CE (ECE) showed increased anti-photoaging effects against UVA-induced changes in MMP-1 and collagen production. Camellenodiol (CMD), a known triterpenoid from C. japonica, isolated as the active ingredient of ECE and its anti-photoaging effect was screened. Results showed that CMD ameliorated the UVA-induced deterioration in collagen levels by suppressing MMP-1 production in transcriptional level. CMD treatment downregulated the phosphorylation of p38, ERK, and JNK MAPKs along their downstream effectors, c-Fos, and c-Jun. In conclusion, enzyme-assisted extraction of C. japonica flowers was suggested to enhance the anti-photoaging properties suggestively through high bioactive content such as CMD.

New insights into the specificity and processivity of two novel pectinases from Verticillium dahliae.

Pectin, the major non-cellulosic component of primary cell wall can be degraded by polygalacturonases (PGs) and pectin methylesterases (PMEs) during pathogen attack on plants. We characterized two novel enzymes, VdPG2 and VdPME1, from the fungal plant pathogen Verticillium dahliae. VdPME1 was most active on citrus methylesterified pectin (55-70%) at pH 6 and a temperature of 40 °C, while VdPG2 was most active on polygalacturonic acid at pH 5 and a temperature of 50 °C. Using LC-MS/MS oligoprofiling, and various pectins, the mode of action of VdPME1 and VdPG2 were determined. VdPME1 was shown to be processive, in accordance with the electrostatic potential of the enzyme. VdPG2 was identified as endo-PG releasing both methylesterified and non-methylesterified oligogalacturonides (OGs). Additionally, when flax roots were used as substrate, acetylated OGs were detected. The comparisons of OGs released from Verticillium-susceptible and partially resistant flax cultivars identified new possible elicitor of plant defence responses.

From Secretion in Pichia pastoris to Application in Apple Juice Processing: Exo-Polygalacturonase from Sporothrix schenckii 1099-18.

BACKGROUND: Polygalacturonases are a group of enzymes under pectinolytic enzymes related to enzymes that hydrolyse pectic substances. Polygalacturonases have been used in various industrial applications such as fruit juice clarification, retting of plant fibers, wastewater treatment drinks fermentation, and oil extraction. OBJECTIVES: The study was evaluated at the heterologous expression, purification, biochemical characterization, computational modeling, and performance in apple juice clarification of a new exo-polygalacturonase from Sporothrix schenckii 1099-18 (SsExo-PG) in Pichia pastoris. METHODS: Recombinant DNA technology was used in this study. Two different pPIC9K plasmids were constructed with native signal sequence-ssexo-pg and alpha signal sequence-ssexo-pg separately. Protein expression and purification performed after plasmids transformed into the Pichia pastoris. Biochemical and structural analyses were performed by using pure SsExo-PG. RESULTS: The purification of SsExo-PG was achieved using a Ni-NTA chromatography system. The enzyme was found to have a molecular mass of approximately 52 kDa. SsExo-PG presented as stable at a wide range of temperature and pH values, and to be more storage stable than other commercial pectinolytic enzyme mixtures. Structural analysis revealed that the catalytic residues of SsExo- PG are somewhat similar to other Exo-PGs. The KM and kcat values for the degradation of polygalacturonic acid (PGA) by the purified enzyme were found to be 0.5868 µM and 179 s-1, respectively. Cu2+ was found to enhance SsExo-PG activity while Ag2+ and Fe2+ almost completely inhibited enzyme activity. The enzyme reduced turbidity up to 80% thus enhanced the clarification of apple juice. SsExo-PG showed promising performance when compared with other commercial pectinolytic enzyme mixtures. CONCLUSION: The clarification potential of SsExo-PG was revealed by comparing it with commercial pectinolytic enzymes. The following parameters of the process of apple juice clarification processes showed that SsExo-PG is highly stable and has a novel performance.

Allergen Homologues, Pathogenesis-Related 1, Polygalacturonase, and Pectin Methyl Esterase from a Japanese Hop.

BACKGROUND: Japanese hop is an important cause of weed pollinosis in East Asia. Its pollen is abundant in autumn. This pollen is known to be the cause of many allergic diseases. However, molecular characteristics of its allergens have not been elucidated. OBJECTIVE: In this study, we produced recombinant proteins of allergen homologues from Japanese hop by the analysis of expressed sequence tags (EST), and evaluated its allergenicity. METHODS: cDNA library was constructed using as little as 50 ng of total RNA from Japanese hop pollen. Allergen homologues were identified by the initial screening of 963 EST clones. Recombinant proteins were overexpressed in the E. coli expression system and purified using Ni-nitrilotriacetic acid-agarose. Purified proteins were analyzed by ELISA. RESULTS AND DISCUSSION: Japanese hop pathogenesis-related 1 protein (PR-1) shares 37.0 to 44.4% of amino acid sequence identity with Art v 2, Cuc m 3, and Cyn d 24. Pectin methyl esterase (PME) shows 23.2 to 50.2% of identities to Act d 7, Ole e 11, and Sal k 1. Polygalacturonase (PGs) shows 16.7 to 19.3% of identities to Phl p 13, Cry j 2, Cha o 2, Jun a 2, Pla a 2, and Pla or 2. IgE antibodies from Japanese hop allergy patients' sera recognized PR-1 (3.4%), PME (13.8%), PGs (3.7%), and profilin (13.8%), respectively. CONCLUSION: Novel allergenic components were identified, even though low IgE reactivity was displayed reflecting the low degree of cross-reactivity with other pollen allergens. We believe that these molecules have worth further studies.

Environmental pollution reducing strategy for scouring of undegummed sisal fibers using xylanase and pectinase enzymes.

This study was undertaken to investigate the potential of bioscouring in the processing of undegummed sisal fibers, using xylano-pectinolytic enzymes. Optimum bioscouring was obtained at pH 8.5 and 50 mM buffer molarity, using xylanase (10 IU) and pectinase (8 IU), with a material to liquor proportion of 1:25 (g:ml), EDTA (2 mM) and Tween 80 (0.5%), at 50 °C temperature with agitation rate of 55 rpm and treatment period of 60 min. Enzymatic treatment of sisal fibers enhanced the brightness and whiteness by 11.52 and 6.83%, respectively, and reduced the yellowness by 7.14% in comparison to control. The use of xylanase and pectinase enzymes completely replaced the chemical scouring method for removing non-cellulosic impurities. Thus, enzymatic scouring is energy saving and ecofriendly, since it completely eliminated the use of toxic chemicals used in alkaline scouring. An increase of 23.75% and 11.58% in brightness and whiteness of enzymatically scoured cum bleached fibers, as compared to chemically scoured cum bleached fibers was finally obtained, along with reduction in yellowness by 27.99%. This is the first report demonstrating environmentally sustainable enzymatic approach for scouring of undegummed sisal fibers, using enzymes, simultaneously produced from a bacterial isolate.

Effect of high hydrostatic pressure-assisted pectinase modification on the Pb2+ adsorption capacity of pectin isolated from sweet potato residue.

Novel pectin derived from sweet potato residue was modified by high hydrostatic pressure (HHP)-assisted pectinase and then used for Pb2+ removal from aqueous solutions. The removal characteristics and mechanisms were also investigated. Results showed that modified sweet potato pectin exhibited greater adsorption performances for Pb2+ than that of natural ones, and showed excellent eco-friendly properties and good potential for adsorption of some other heavy metals (such as Cu2+). The adsorption curves were much more conformed to Langmuir model, and the highest capacity for Pb2+ adsorption was 263.15 mg/g with 1.00% pectin at pH 7. Chemical adsorption process of pectin for Pb2+ absorption involved O-containing functional groups (O-H, COO-), cation exchange, and along with electrostatic interactions. Overall, the results in this study indicated that sweet potato pectin modified with HHP-assisted pectinase had the potential to become an environmentally friendly coagulant-flocculant agent for the heavy metal adsorption, especially for Pb2+.

Dietary enzymatically-treated Artemisia annua L. supplementation could alleviate oxidative injury and improve reproductive performance of sows reared under high ambient temperature.

The medicinal plant Artemisia annua L. is well known for its antimalarial compound artemisinin and the antioxidant capacity of its active ingredients. However, low bioavailability of Artemisia annua L. limits its therapeutic potential, fermentation of Artemisia annua L. can improve its bioavailability. This study was aimed to investigate the effects of dietary supplementation of enzymatically-treated Artemisia annua L. (EA) on reproductive performance, antioxidant status, milk composition of heat-stressed sows and intestinal barrier integrity of their preweaning offspring. 135 multiparous sows of average parity 4.65 (Landrace × large white) at day 85 of pregnancy were randomly distributed into 3 treatments. Sows in the control group were housed at control rooms (temperature: 27.12 ± 0.18 °C, temperature-humidity index (THI): 70.90 ± 0.80) and fed the basal diet. Sows in the HS, HS + EA groups were fed the basal diet supplemented with 0 or 1.0 g/kg EA respectively, and reared at heat stress rooms (temperature: 30.11 ± 0.16 °C, THI: 72.70 ± 0.60). Heat stress increased the malondialdehyde (MDA) content, reduced the activities of total antioxidant capacity (T-AOC) and total superoxide dismutase (T-SOD) of sows and piglets, and seriously compromised the antioxidant capacity of the sows and the intestinal integrity of their offspring. However, dietary supplementation of 1.0 g/kg EA reduced the MDA content, increased the activities of T-SOD and T-AOC in serum, colostrum, and milk of heat-stressed sows, and increased colostrum yield and 14-d milk fat content. EA supplementation also increased piglet weaning weight and the activities of T-SOD and T-AOC in serum. In addition, the abundances of intestinal tight junction proteins claudin-1 and occludin were up-regulated in piglets in EA-supplemented group. In conclusion, dietary EA supplementation at 1.0 g/kg can alleviate the oxidative stress in heat-stressed sows, improve the antioxidant capacity in both sows and their offspring, and promote the intestinal barrier integrity in their offspring. EA may be a potent dietary supplement that ameliorates oxidative stress in livestock production by improving the antioxidant capacity.

Ultrasound-assisted enzymatic extraction of anthocyanins from grape skins: optimization, identification, and antitumor activity.

Grape skins produced during the grape juice production and processing contain abundant anthocyanins and other active compounds. Consequently, this study optimized the extraction conditions for ultrasound-assisted enzymatic extraction (UAEE) of anthocyanins from grape skins via response surface methodology coupled with genetic algorithm. The optimum extraction parameters to achieve the highest anthocyanins yield (3.01 ± 0.04) mg/g from grape skins by UAEE were obtained under an extraction temperature of 50 °C, ultrasonic power of 400 W, pectinase dosage of 0.16%, and extraction time of 28 min. The AB-8 macroporous resin combined Sephadex LH-20 techniques were further employed to purify the anthocyanins extracts obtained under optimum extraction conditions (AEOEC), and the main anthocyanins were identified using high-performance liquid chromatography tandem mass spectrometry. The purified anthocyanins contained two anthocyanins in terms of delphinidin-3,5-O-diglucoside and cyanidin-3-O-rutinoside with purity of 91.35% and 92.64%, respectively. Ultimately, we further evaluated the antitumor activity of AEOEC and two purified anthocyanins on breast cancer. The results indicated that the antitumor effect of AEOEC on breast cancer MCF-7 cells was better than that of two purified anthocyanins. In addition, AEOEC could memorably increase intracellular reactive oxygen species levels and apoptosis of MCF-7 cells, and arrest MCF-7 cells in the G2/M phases. The findings provide an effective and feasible method for anthocyanins extraction and reduce the environmental burden of this waste.

Thermostable and alkalistable exopolygalacturonase of Bacillus pumilus dcsr1: Characteristics and applicability.

The bioactive form of thermostable and alkali stable pectinase of Bacillus pumilus dcsr1 is a homodimer of the molecular mass of 60 kDa with a pI of 4.6. The enzyme is optimally active at 50 °C and pH 10.5, and its Michaelis constant (Km), maximum rate of reaction (Vmax), activation energy (Ea), and temperature quotient (Q10) values (for citrus pectin) are 0.29 mg mL-1, 116 µmole mg-1 min-1, 74.73 KJmol-1 and 1.57, respectively. The enzyme has a shelf life of one and a half years at room temperature as well as 4 °C. The activity of the enzyme is stimulated by Mn2+ and Ca2+ and inhibited by Hg+, Cd2+, Co2+, Zn2+, Fe2+, Pb2+, EDTA and urea to a varied extent. The conformational studies of the enzyme revealed a high ß-sheet content in the bioactive dimer, and high α-helix in the inactive monomer. The Circular Dichroism (CD) spectra of the dimer in the presence of inhibitors suggested a marked decrease in ß-sheet, and a significant increase in α-helix, suggesting a key role of ß-sheets in the enzyme catalysis. Based on the end product analysis, the enzyme is an exopolygalacturonase with a unique ability of transglycosylation. When ramie fibers were treated with the enzyme, removal of gummy material (pectin) was visible, confirming its applicability in the degumming process.

Comprehensive analysis of polygalacturonase gene family highlights candidate genes related to pollen development and male fertility in wheat (Triticum aestivum L.).

MAIN CONCLUSION: Four polygalacturonase gene family members were highlighted that contribute to elucidate the roles of polygalacturonase during the fertility conversion process in male-sterile wheat. Polygalacturonase (PG) belongs to a large family of hydrolases with important functions in cell separation during plant growth and development via the degradation of pectin. Specific expressed PGs in anthers may be significant for male sterility research and hybrid wheat breeding, but they have not been characterized in wheat (Triticum aestivum L.). In this study, we systematically studied the PG gene family using the latest published wheat reference genomic information. In total, 113 wheat PG genes were identified, which could be classified into six categories A-F according to their structure characteristics and phylogenetic comparisons with Arabidopsis and rice. Polyploidy and segmental duplications in wheat were proved to be mainly responsible for the expansion of the wheat PG gene family. RNA-seq showed that TaPGs have specific temporal and spatial expression characteristics, in which 12 TaPGs with spike-specific expression patterns were detected by qRT-PCR in different fertility anthers of KTM3315A, a thermo-sensitive cytoplasmic male-sterile wheat. Four of them specific upregulated (TaPG09, TaPG95, and TaPG93) or downregulated (TaPG87) at trinucleate stage of fertile anthers, and further aligning with the homologous in Arabidopsis revealed that they may undertake functions such as anther dehiscence, separation of pollen, pollen development, and pollen tube elongation, thereby inducing male fertility conversion in KTM3315A. These findings facilitate function investigations of the wheat PG gene family and provide new insights into the fertility conversion mechanism in male-sterile wheat.

Structural, physicochemical and emulsifying properties of sweet potato pectin treated by high hydrostatic pressure and/or pectinase: a comparative study.

BACKGROUND: Sweet potato (Ipomoea batatas L.) is the sixth most important food crop in the world, and China is the largest producer. Large amounts of sweet potato residues are generated during starch extraction, leading to environmental pollution and resource waste. However, these residues can be used as a viable source for pectin extraction. As a natural biopolymer with high molecular weight and complex structure, the usefulness of pectin has been limited, and it needs to be modified in order to improve its physicochemical properties, thus expanding its applications in the food industry. Therefore, the reported study was conducted to modify sweet potato pectin (SPP) using high hydrostatic pressure (HHP) and/or pectinase treatment, and to determine the effects of such treatment on structural, physicochemical and emulsifying properties. RESULTS: The results demonstrated that the molecular weight of SPP decreased following HHP and pectinase treatment, which was evidenced using scanning electron microscopy and atomic force microscopy. The degree of esterification was also decreased, confirmed by decreased intensity of the peak at 1739 cm-1 in the Fourier transform infrared spectrum and decreased peaks at 3.6 and 3.8 ppm in the 1 H NMR spectrum. Moreover, the content of monosaccharides and uronic acids increased and emulsifying properties improved after HHP and pectinase treatment. CONCLUSIONS: HHP-assisted pectinase treatment could be used as novel technique for the modification of pectin to give better emulsifying properties with great potential for application in the food industry. © 2020 Society of Chemical Industry.

Optimization of pectinase-assisted extraction of Annona muricata L. juice and the effect of liquefaction on its pectin structure.

BACKGROUND: Soursop (Annona muricata L.) is an underutilized tropical and subtropical fruit with high nutritional and therapeutic benefits. This fruit is faced with enormous post-harvest losses due to its high perishability. This work was aimed to optimize the pectinase-assisted extraction conditions of soursop juice using Doehlert design and to study the effect of pectinase on its pectin structure. RESULTS: The predicted models were validated for all the responses studied and the regression coefficients ranged from 0.905 to 0.987 (P ≤ 0.05). An incubation time of 172 min, enzyme concentration of 0.04% (w/w) and incubation temperature at 42.9 °C were found to be the optimal conditions for soursop juice extraction, which resulted in 75.20%, 3.74, 7.35 °Brix, 87.06%T, and 0.44% MAE for soursop juice yield (%), pH, total soluble solids (TSS) (°Brix), clarity (%T) and titratable acidity (% malic acid equivalent, MAE), respectively. Morphologically, untreated soursop pulp presented a non-uniform spherical surface; enzyme hydrolyzed soursop exhibited ruptured and wrinkled surface; meanwhile for the different pectin obtained, untreated soursop pectin depicted porous surface and enzyme hydrolyzed soursop pectin showed whirling rough surface. Fourier-transform infrared (FTIR) confirmed the presence of similar chemical group stretching and vibrations in commercial pectin and soursop pectin. CONCLUSION: Under the optimum conditions, the numerical predictions were similar to the experimental data obtained, thus confirming the validity of the models. Application of enzyme treatment caused the breakdown of pectin structure as illustrated by scanning electron microscopy (SEM) and FTIR analyses.

Biotransformation of Glycosylated Saponins in Balloon Flower Root Extract into 3-O-ß-D-Glucopyranosyl Platycosides by Deglycosylation of Pectinase from Aspergillus aculeatus.

Platycodon grandiflorum root (Platycodi radix) saponins, platycosides, have been used as health supplements and food items for the treatment of respiratory disorders and pulmonary diseases. Deglycosylated saponins have been known to exert stronger biological effects than their glycosylated forms. In the present study, glycosylated platycosides in Platycodi radix extract were biotransformed into deglycosylated 3-O-ß-D-glucopyranosyl platycosides, including 3-O-ß-Dglucopyranosyl platycodigenin, 3-O-ß-D-glucopyranosyl polygalacic acid, and 3-O-ß-Dglucopyranosyl platyconic acid, by pectinase from Aspergillus aculeatus. This is the first report on the quantitative enzymatic production of 3-O-ß-D-glucopyranosyl platycosides. The chemical structures of 3-O-ß-D-glucopyranosyl platycosides were identified with LC/MS. Moreover, the biotransformation pathways of the three types of platycosides in Platycodi radix into 3-O-ß-Dglucopyranosyl platycosides were established.

Phenolic compounds extraction in enzymatic macerations of grape skins identified as low-level extractable total anthocyanin content.

Anthocyanins in wine principally depend on grape skin extractable anthocyanin content, that is, the amount of anthocyanins present in grape skin that are released to wine during the maceration stage. This amount of extractable anthocyanins is closely linked to the cell wall degradation of skin cells. Indeed, among other methodologies, the maceration in presence of different enzymes can be used to increase cell wall degradation, and therefore, the amount of anthocyanins extracted from grape skins to wine. Vitis vinifera L. cv. Tempranillo and Syrah red grapes have been identified as samples with low anthocyanin extraction potential by near infrared hyperspectral imaging. Grape skins have been macerated in the presence of cellulase, glucosidase, and pectinase. Then, color of the supernatants and phenolic compounds extracted from grape skins (total phenols, total flavanols, and total and individual anthocyanins) has been determined. Cellulase and glucosidase have shown a positive effect in the extraction of phenolic compounds from these grapes. Macerations carried out in the presence of cellulase have produced supernatants with a more intense color (lower lightness and higher chroma values), and a higher extraction of flavanols and anthocyanins than the respective control essays. However, pectinase treatments have produced the opposite effect, which could be partially explained by an eventual interaction between the cell wall polysaccharides liberated by pectinase and the phenolic compounds extracted. Synergy effects do not appear between cellulase and glucosidase. Moreover, the negative effect of the addition of pectinase might be due to the interactions between the cell wall material liberated by pectinase and the phenolic compounds extracted. PRACTICAL APPLICATION: In the present study, grape samples with a low anthocyanin extraction potential have been identified, and these samples have been macerated in the presence of different enzymes. The applied enzymes were three of the most common enzymes that are applied in the wine industry. Individual enzymes and mixtures have been applied to Syrah and Tempranillo grape skin samples and the results have been compared to control macerations. Knowledge in this topic will help the production of quality wines.

Inhibitory Effects of Pectinase-Treated Prunus Mume Fruit Concentrate on Colorectal Cancer Proliferation and Angiogenesis of Endothelial Cells.

Pectinase is a well-known enzyme used in the food processing industry to produce fruit juice and concentrate. This study evaluated the anticancer and antiangiogenesis activities of pectinase-treated Prunus mume fruit concentrate (PC) and its phenolic components. PC treatment (250 to 1,000 µg/mL) resulted in decreased proliferation of SW480 human colorectal cancer cells through S-phase cell cycle arrest; however, equivalent concentrations of PC did not show toxicity toward CRL-1539 colon normal cells. Furthermore, PC-induced caspase-dependent apoptosis in SW480 cells, which was characterized by accumulation of apoptotic cell population, cell shrinkage, formation of apoptotic bodies, upregulation of proapoptotic Bax, cleaved PARP, caspase-3, caspase-8, and caspase-9, and downregulation of antiapoptotic Bcl-2. Antiangiogenesis effects of PC were assessed using human umbilical vein endothelial cells (HUVECs). We found that PC did not inhibit HUVECs proliferation at concentrations of 500 to 1,500 µg/mL. In addition, treatment with PC at nontoxic concentrations (500 to 1,000 µg/mL) blocked vascular endothelial growth factor induced cell migration, invasion, capillary-like tube formation, and angiogenesis from rat aortic rings. HPLC-PDA analysis showed that there were at least four different phenolics including 5-HMF, neochlorogenic acid, protocatechuic acid, and syringic acid. Taken together, these results indicated that PC could be used as a good source of phenolic compounds with selective anticancer and antiangiogenesis activities. PRACTICAL APPLICATION: Pectinases are one of the well-known enzyme used in the part of food processing. Treatment of pectinase is a useful strategy to reduce viscosity, turbidity, and pulp particles in the production of fruit juice, extract, and concentrate. In the present study, we found that pectinase-treated P. mume fruit concentrate significantly suppresses colorectal cancer proliferation and angiogenesis of human umbilical vein endothelial cells. The significance of our findings is that pectinase-treated P. mume concentrate may be used as a commercial functional food material to inhibit colorectal cancer and angiogenesis.

Effect of Bacillus tequilensis SALBT crude extract with pectinase activity on demucilation of coffee beans and juice clarification.

Pectinases are a group of enzymes, which catalyze the breakdown of pectin with numerous applications in various industries. Microbes are the predominant pectinase producers. In the present study, bacterial species were isolated from the soil of a vegetable and fruit dump yard area in a market. The species screened and isolated were identified as Bacillus tequilensis SALBT, and the media and culture conditions were optimized for enhanced production of total pectinases. Maximum pectinolytic activity was observed with 1.5% (w/v) pectin concentration with a combination of yeast extract as nitrogen source and MgSO4 as a metal ion source. Carbon/nitrogen in 2:1 ratio (w/v) yielded the maximum pectinase production with pH and temperature of the medium of 7.5°C and 40°C, respectively. Pectinase activity was determined by the dinitrosalicylic acid method. The pectinase production was relatively stable in the presence of various surfactants like Tween (20, 40, 60, and 80) and sodium dodecyl sulfate (SDS), whereas Triton X-100 showed an inhibitory effect. Mass production of the enzyme in optimized media and partial purification was performed by ammonium sulfate precipitation followed by dialysis. The approximate molecular weight of the partially purified pectinase was found to be 35 kDa by SDS-polyacrylamide gel electrophoresis. Application studies such as demucilaging coffee beans and juice clarification were also performed. The findings revealed that B. tequilensis SALBT with pectinase activity has the ability to remove the mucilage layer of pulped coffee seeds, and the partially purified pectinases found to be effective in clarifying juice.

Biotechnological potential of an exo-polygalacturonase of the new strain Penicillium janthinellum VI2R3M: biochemical characterization and clarification of fruit juices.

AIMS: The aim of this work was to characterize and apply a polygalacturonase of Penicillium janthinellum new strain VI2R3M. METHODS AND RESULTS: The polygalacturonase obtained from P. janthinellum VI2R3M was incubated in cultures of passion fruit peel and was partially purified by ion-exchange chromatography and gel filtration. The enzyme showed a relative molecular mass of 102·0 kDa, maximum activity at pH 5·0, temperature of 50°C, 100% stablity at 50°C and 80% stablity at pH 3·0-5·0. The apparent Km , Vmax and Kcat values for hydrolyzing polygalacturonic acid were 2·56 mg ml-1 , 163·1 U mg-1 and 277 s-1 respectively. The polygalacturonase presented exo activity and was activated by Mg2+ . The juices treated with polygalacturonase presented increases in transmittance with reduction in colour. CONCLUSIONS: The results suggest that the new lineage P. janthinellum VI2R3M presents a high yield of an exo-polygalacturonase induced by agro-industrial residues, with excellent activity and stability in acidic pH and at 50°C. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of agro-industrial residue to obtain the polygalacturonase can contribute to a decrease enzyme production cost. The results of the activity, stability to acidic pH and excellent performance in the clarification of juices show that the enzyme is promising for industrial application.