Enzymatic Characterization of Pectinex XXL,a Pectinase Produced by Aspergillus niger,and Its Application in Fruit Juice Production

Pectinex XXL,a commercially prepared pectinase,was investigated for its potential application in the fruit juice industry.Polygalacturonic acid was used as the substrate for determining the enzymatic properties of Pectinex XXL using the DNS method.According to the results,the optimal pH for Pectinex XXL activity was 4.5,and the enzyme was stable in the pH range of 3.0~4.5.The optimal pH and pH stability range are consistent with those of some tropical and subtropical fruits.The optimal temperature for Pectinex XXL activity was 60℃,and the enzyme remained stable after one hour in a water bath set at 40℃.Additionally,the enzymatic activity was not inhibited in the presence of 1 mmol/L of Na^(+),Mg^(2+),Ba^(2+),Co^(2+),Zn^(2+),and Fe^(2+),whereas it was slightly inhibited in the presence of 2 mmol/L of K^(+)and Fe^(2+)and partially inhibited in the presence of 1 and 2 mmol/L of Ca^(2+)and Mn^(2+),demonstrating its good stability in acids and excellent thermal catalytic performance.Based on the above experimental results,depectinization experiments were performed on plantain and cherry tomato juices using different amounts of Pectinex XXL.After one hour reaction with 16 U/mL of the enzyme,the yields of the plantain and cherry tomato juices were substantially increased by 119.03%and 15.97%,respectively,while their light transmittance was remarkably enhanced by 37.65%and 12.35%,respectively.Furthermore,the enzyme reduced the viscosity of the plantain and cherry tomato juices by 88.29%and 29.50%,respectively.The juice production experiments confirmed that this enzyme can significantly improve the yield and light transmittance of plantain juice,while effectively reducing its viscosity.These findings indicate the potential of Pectinex XXL in the industrial production of plantain juice.

Evaluation of Water Transfer Capacity of Poplar with Pectinase Treated under the Solar Interface Evaporation

Poplar wood,which was used as the absorption material for the solar-driven interfacial evaporation,was treated for 3 days,6 days and 9 days with the pectinase,and then was simulated for photothermal evaporation test at one standard solar radiation intensity(1 kW⋅m^(−2)).The effects of pectinase treatment on cell passage and water migration capacity of poplars were analyzed by the mercury intrusion porosimetry,the scanning electron microscope and fractal theory.It was found that the pit membrane and the ray parenchyma cells of poplar wood were degraded and destroyed after pectinase treatment.Compared with the untreated poplar wood,the evaporation rate of three sections of the specimen was changed.Especially the evaporation rate of radial and tangential direction was significantly increased.At the same time,based on the experimental data and fractal dimension deduction,fractal characteristics could be found in that the structure of poplars treated with pectinase.The porosity decreased with the increase of the fractal dimension in a certain range.It was shown that it is feasible to evaluate solar-driven water migration capacity by using fractal theory.

Adenosine cyclic phosphate with ultrasonic-assisted pectinase extraction alleviated allergic reactions in RBL-2H3 through inhibiting the influx of intracellular Ca^(2+)

Jujube contains abundant cyclic adenosine monophosphate(cAMP)and the ultrasonic-assisted pectinase extraction(UAPE)conditions for obtaining the maximum cAMP yield from jujube were optimized.Orthogonal array design was applied to evaluate the effects of 4 variables by UAPE on cAMP yield.The results showed that the optimal cAMP yield(783.0μg/g)was derived at ratio of liquid to solid 5 mL/g,ratio of pectinase to raw material 1.5%,time 60 min and temperature 40℃.Moreover,the effect of cAMP on the anti-allergic function of action induced by immunoglobulin E(IgE)and its meschanism was investigated through establishing the sensitized cell model in rat basophilic leukemia(RBL-2 H3)cells using dinitrophenylated(DNP)-bovine serum albumin(BSA)-IgE.The results showed that cAMP interfered with sensitized cells,effectively inhibited the occurrence of basophil degranulation in dose dependence,and significantly reduced the activity ofβ-hexosamindase(β-hex),at the optimal concentration of 50μg/mL.The level of anti-inflammatory factor interleukin-10(IL-10)was promoted and the content of pro-inflammatory factor tumor necrosis factor-α(TNF-α)was suppressed by cAMP.In addition,influx of intracellular Ca^(2+) was repressed effectively.Our results demonstrate that jujube cAMP regulated the cytokine balance in the allergy pathway through blocking the influx of extracellular Ca^(2+),with the prevention of allergy symptoms.

Effect of agitation speed on the morphology of Aspergillus niger HFD5A-1 hyphae and its pectinase production in submerged fermentation

AIM: To investigate the impact of agitation speed on pectinase production and morphological changing of Aspergillus niger(A. niger) HFD5A-1 in submerged fermentation. METHODS: A. niger HFM5A-1 was isolated from a rotted pomelo. The inoculum preparation was performed by adding 5.0 m L of sterile distilled water containing 0.1% Tween 80 to a sporulated culture. Cultivation was carried out with inoculated 1 × 107 spores/m L suspension and incubated at 30 ℃ with different agitation speed for 6 d. The samples were withdrawn after 6 d cultivation time and were assayed for pectinase activity and fungal growth determination. The culture broth was filtered through filter paper(Whatman No. 1, London) to separate the fungal mycelium. The cell-free culture filtrate containing the crude enzyme was then assayed for pectinase activity. The biomass was dried at 80 ℃ until constant weight. The fungal cell dry weight was then expressed as g/L. The 6 d old fungal mycelia were harvested from various agitation speed, 0, 50, 100, 150, 200 and 250 rpm. The morphological changing of samples was then viewed under the light microscope and scanning electron microscope.RESULTS: In the present study, agitation speed was found to influence pectinase production in a batch cultivation system. However, higher agitation speeds than the optimal speed(150 rpm) reduced pectinase production which due to shear forces and also collision among the suspended fungal cells in the cultivation medium. Enzyme activity increased with the increasing of agitation speed up to 150 rpm, where it achieved its maximal pectinase activity of 1.559 U/m L. There were significant different(Duncan, P < 0.05) of the pectinase production with the agitation speed at static, 50, 100, 200 and 250 rpm. At the static condition, a well growth mycelial mat was observed on the surface of the cultivation medium and sporulation occurred all over the fungal mycelial mat. However with the increased in agitation speed, the mycelial mat turned slowly to become a single circular pellet. Thus, it was found that agitation speed affected the morphological characteristics of the fungal hyphae/mycelia of A. niger HFD5A-1 by altering their external as well as internal cell structures.CONCLUSION: Exposure to higher shear stress with an increasing agitation speed could result in lower biomass yields as well as pectinase production by A. niger HFD5A-1.

Research Progress of Pectinase

Pectinase is a group of enzymes that can decompose complex pectin into small molecules such as galacturonic acid. It is mainly produced by microorganisms by liquid and solid fermentation and immobilized cell method. Its representative strain is Aspergillus niger,and yeast and actinomycete are also high-yield strains for screening pectinase. Pectinase can be used in many fields of life and production,such as clarification of fruit juice and wine,pulping of paper making industry,degumming of textile,extracting of traditional Chinese medicine components,animal feed and other industries,which is economic,green,and efficient and pollution-free. Moreover,pectinase is one of the four major enzymes in the world,and its practical application has very important significance,and the prospect of pectinase application is also important.

Effects of Neodymium on Growth, Pectinase Activity and Mycelium Permeability of Fusarium oxysporum

The diameter of the colony of Fusarium oxysporum in solid medium, and the mycelium growth, pectinase activity, and mycelium permeability of Fusarium oxysporum in liquid medium under varying concentrations of Nd^3＋ （0, 2, 4, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 300, and 400 mg·L^-1） were measured. The results indicated that the growth of Fusarium oxysporum was stimulated in solid medium when the concentration of Nd^3＋ ranges from 2 to 180 mg·L^-1, whereas it was inhibited when Nd^3 ＋ concentration was greater than 200 mg· L^-1. The colonies were fewer and smaller when Nd^3 ＋ was used in the solid medium. The growth of Fusarium oxysporum was inhibited in liquid medium when Nd^3＋ was used. The inhibition rate showed by the dry weight of mycelium ranged from 4.83% to 52.18% and inereased with Nd^3 ＋ concentration. The pectinase activity decreased compared with that of controls. When the concentration of Nd^3 ＋ was 10 and 400 mg· L^- 1, the pectinase activity decreased by 95 % at both concentrations. Mycelium cell membrane permeability increased when Nd^3 ＋ concentrations ranged from 10 to 400 mg· L^-1 but decreased when Nd^3＋ concentration was 2 mg· L^-1.

Isolation of Pectinase Producing Bacteria from the Rhizosphere of <i>Andrographis paniculata</i>Nees and 16S rRNA Gene Sequence Comparison of Some Potential Strains

Pectinases, the enzymes which break down pectic substances, have a wide range of applications in food, agriculture and environmental sectors. In the present study, attempts were made to isolate highly efficient pectinase producer from the rhizosphere of a medicinal plant, Andrographis paniculata Nees, known as the “King of bitters”. The total heterotrophic bacterial count of the rhizosphere soil of A. paniculata Nees ranged from 1.53 × 109 to 2.52 × 109 cfu/g. A total of 65 bacterial colonies were randomly selected from the nutrient agar plates, purified and assessed for pectinase activity. Out of the 65 isolates, 62 (95.38%) showed varying degree of pectinase activity in plate assay using pectin as a sole source of carbon. Among the pectinase producing strains, JBST36 showed best pectinase activity which is followed by the JBST22 and JBST27. Morphological characterization, biochemical tests and 16S rRNA gene sequencing were performed to identify the three most potential strains. Based on the morphological, biochemical and molecular data, JBST22 was identified as Bacillus flexus and the other two were identified as Bacillus subtilis. Furthermore, nucleotide sequences of the 16S rRNA gene of these 3 strains were compared and a phylogenetic tree was constructed. The study reveals that there are at least 66 base differences in the 16S rRNA gene sequences of B. flexus JBST22 and the B. subtilis JBST36.

Pectinases yeast production using grape skin as carbon source

Pectinases are used in Enology for some different utilities. Enzymatic preparations from moulds are a mixed of different enzymes with strong and unspe-cific activities. Some Saccharomyces cerevisiae pro-duce pectinases which can be used instead of com-mercial preparations. The objectives of this work were to study the enzyme secretion by one Saccharo-myces cerevisiae (CECT 11783) for growing on grape skin (industry oenological by-product) as carbon source. Preliminary experiments showed that the strain produced pectinases for growing on grape skin without any other carbon source. Statistical treat-ment (factorial design 25) was applied to evaluate the influences of related factors (agitation, temperature, presence of peptone and detergent in the medium and time of growth) Variables with the most significant interactions for pectinase production were agitation and nitrogen source concentration. Response surface methodology showed that a first order model was not adequate for results. Nevertheless, the built of a sec-ond order model offered a polynomial equation which surface predicted a maximum of activity (52.68 enzymatic units) for specific values of the studied variables (147.8 rpm of agitation and 15.9 g of pep-tone/ L culture medium).

Lucrative pectinase production by novel strain Pseudozyma sp. SPJ with statistical optimization techniques using agro-industrial residues

Production of high titers of an alkaline, extracellular and thermo-tolerant pectinase by a newly isolated yeast Pseudozyma sp. SPJ was carried out under solid state fermentation. Citrus peel, the inexpensive agro-industrial residue used as substrate, was experienced to be unsurpassed. Response surface methodology was conducted to optimize the culture conditions for Pseudozyma sp. SPJ for hyper production of pectinase. Plackett Burman design was applied to identify the most effective culture variables. Out of nine variables studied, incubation time, moisture content and ammonium sulfate were detected as most important. A full factorial Central Composite Design was used to optimize the levels of these variables, which resulted in 17-fold increase （71.19 IU/g to 1215.66 IU/g dry substrate） in the enzyme yield. The results of analysis of variance and multiple regression analysis implies that the effect of incubation time （p 〈 0.000） and moisture content （p 〈 0.018） is more than ammonium sulfate. And also the interaction of moisture content with ammonium sulfate （p 〈 0.002） is more significant.

In vitro Evaluation of Feed Enzyme Compound Produced by Aspergillus sulphureus in Solid-state Fermentation

ABSTRACT： Three trials were conducted to analyze a multi-enzyme compound produced by Aspergillus sulphureus in solid-state fermentation （SSF） as a po- tential feed additive. The results of the first trial showed that there were at least 5 non-starch polysac- charide enzymes： xylanase, 13-ghicanase, pectinase, mannase and carboxy methyl cellulase （CMCase） contained in the compound. Xylanase and fl-glucanase showed good activities at pH 2.5-7.0, which were in the range of 649-1046 U/g and 444-648 U/g, respec- tively. Pectinase showed good activity in acidic solu- tion （pH 2.5-3.0）,which ranged from 195 to 917 U/g. Mannase showed high activity of 235-298 U/g at pH 3.5-4.5 and the activity of CMCase was relatively constant at pH 2.5-7.0, which was in the range of 38.2-78.6 U/g. The second trial was aimed to test the stability of the enzymes in gastric liquor （pH 2.6） of finishing pigs and Na2 HPO4-gastric liquor （ pH 5.5 ）.After 6 h incubation at 40℃ in gastric liquor,the re- tained activity of xylanase, 13-glucanase, pectinase, mannase and CMCase was 26.3% ,65.0% ,71.0%, 74.8% and 85.6%, respectively. While after 6 h in- cubation at 40℃ in Na2I-IPO4-gastric liquor, the re- tained activity of xylanase, [3-glucanase, pectinase, mannase and CMCase was 87.9% ,91.1% ,92.3%, 95.0%, and 97.5%, respectively. The third trial was carried out in a jejunum liquor （ pH 5.8,200 mL）, which contained 0.2 g of the multi-enzyme compound and 10 g of soybean hull or wheat bran, respectively. After 8 h incubation at 40℃, 18.7% of soybean hull and 20.1% of wheat bran could be degraded to solu- ble saccharide, respectively. Compared with the tradi- tional methods for feed enzyme testing which involve feeding animals for 1-3 months, enzyme assay in this way was relatively convenient.

Search for Molecular Markers of Wheat Resistance to Fungal Pathogens

Functional traits, potentially associated with resistance to infection, were investigated in cultivars of bread wheat. Plant responses to infection by hemibiotrophic fungus Stagonospora (Septoria) nodorum were studied under laboratory conditions. Infection-induced up-regulation of genes coding for class III peroxidase, oxalate oxidase and protease inhibitor was greater in leaves with reduced disease symptoms (percentage of chlorotic or necrotic leaf area). Similar association was detected between activity of pectinase inhibitors and disease severity. Resistant cultivar also differed from the susceptible one by increased content of Н2О2 in infected tissues and more intensive deposition of lignin. We discuss possibility of using these functional traits in plant breeding for increased stress tolerance.

Endophytic mycobiota of wild medicinal plants from New Valley Governorate,Egypt and quantitative assessment of their cell wall degrading enzymes

The present study isolated and identified 32 species of endophytic mycobiota belonging to 18 genera associated with 8 wild medicinal plants collected from El-Kharga Oasis,New Valley Governorate,Egypt.Fusarium was the most common genus followed by Alternaria and Aspergillus.Convolvulus arvensis was the plant with the highest number of endophytes over the other plant species,while Moringa oleifera reported the lowest number of endophytes.In addition,the entomopathogenic fungus Beauveria bassiana;was recorded for the first time from leaves of Portulaca oleracea.One hundred and twenty-three isolates representing 32 species were screened for their abilities to produce pectinase,carboxy methyl cellulase(CMCase)and avicellase enzymes on sucrose free-Cz supplemented,individually with 1%pectin or 1%CMC or 1%avicel as a sole carbon source,respectively.Ninety-four isolates produced pectinase while 66 isolates produced cellulases.The quantitative assays of the three enzymes for high-producers were performed in submerged fermentation using sucrose-free Cz broth.Aspergillus was the superior in the production of the three enzymes with the potent strains were A.terreus AUMC 14287 for CMCase(22.0 IU/ml/min)and avicellase(47.868 IU/ml/min)and A.terreus AUMC 14278 for pectinase(225.43 IU/ml/min).