Polysaccharide hydrolyzing enzyme activity of bacteria, native to Apis florea gut

Introduction and Aim: Apis florea commonly known as “dwarf honey bee” harbors enormous gut bacteria that can digest complex carbohydrates and other food components. In this regard, the present investigation was focused on analyzing the polysaccharide degrading ability of bacteria isolated from the gut of honeybee, for their possible application in nutraceutical and pharmaceutical industries. Materials and Methods: Nine bacterial isolates were screened for carbohydrate degrading enzymes viz., amylase, pectinase, cellulase, tannase and laccase, using respective substrate by plate assay method. Further activities of amylase and pectinase were measured quantitatively by dinitrosalicylic acid (DNS) method. Results: All the nine selected isolates exhibited amylase and pectinase activities. However, only two isolates exhibited lignolytic and cellulolytic activity. None of the isolates showed tannin degradation. Maximum amylase activity (4.95 U/mg) was observed in Bacillus halotolerans af-M9 followed by Klebsiella oxytoca af-G4 (4.62 U/mg). With respect to pectinase activity Klebsiella pneumoniae af-E17 displayed higher activity (0.24 U/mg) followed by Klebsiella oxytoca af-G4 (0.20 U/mg). Conclusion: Habitat-specific innovations are being explored for novel compounds for therapeutic applications. This study throws a light on selection of carbohydrate degrading bacteria from a new source i.e., GUT of honeybee.

Effects of the Fungal Bioherbicide, Alternaria cassia on Peroxidase, Pectinolytic and Proteolytic Activities in Sicklepod Seedlings

Certain plant pathogens have demonstrated potential for use as bioherbicides for weed control, and numerous studies have been published on this subject for several decades. One of the early examples of an important fungal bioherbicide is Alternaria cassiae, isolated from the weed sicklepod (Senna obtusifolia). To gain further insight into biochemical interactions of this fungus and its host weed, we examined the effects of this bioherbicide on various enzymes associated with plant defense. Young sicklepod seedlings were challenged with A. cassiae spore inoculum and enzyme activities associated with plant defense (peroxidase, proteolytic, and pectinolytic) were assayed periodically over a 96-h time course on plants grown in continuous darkness or continuous light. Peroxidase activity increased with time in untreated control seedlings in both light and dark, but the effect was greater in the light. In A. cassiae-treated plants, peroxidase was elevated above that in control tissue at all sample times resulting in a 1.5 -fold increase above control in light-grown tissue and a 2- to 3-fold increase in dark-grown tissue over 48–96 h. Differences in leucine aminopeptidase activity in control versus A. cassiae-treated tissues were not significant until 48–96 h, when activity was inhibited in fungus-treated tissues by about 32% in light-grown tissue and 27% in dark-grown tissue after 96 h. Proteolytic activity on benzoyl-arginine-p-nitroanilide was not significantly different in treated versus control tissue in either light or dark over the time course. Pectinase activity increased in treated tissues at all time points as early as 16 h after spore application in light- or dark-grown plants. The greatest increases were 1.5-fold above control levels in light-grown plants (40–64 h) and 2-fold in plants grown in darkness (72–96 h). Data suggests that peroxidase may be involved as defense mechanism of sicklepod when challenged by A. cassia and that this mechanism is operative in young seedlings under both light and dark growth conditions. Differential proteolytic activity responses on these two substrates suggests the presence of two different enzymes. Increased pectinase activity during pathogenesis suggests that A. cassiae-sicklepod interaction results in an infectivity mechanism to degrade pectic polymers important to sicklepod cell wall integrity. These studies provide important information on some biochemical interactions that may be useful for improvements to biological weed control programs utilizing plant pathogens. Such information may also be useful in genetic selection and manipulation of pathogens for weed control.

PRODUCTION OF ENZYMES IN PREDOMINANT THERMOPHILIC FUNGI AVAILABLE FROM ORGANIC SUBSTRATES

Present investigation describes that the study site comes under Aurangabad Division Maharashtra and it falls in Deccan Plateau Zone of India. It was collected different types of organic substrates viz. vermiompost, poultary manure, baggase, farm yard manure (FYM), soil, Ash etc. Isolated thermophilic predominant fungi thermophilic fungi viz.Aspergillus niger, Mucor mucedo,Humicola insolens,Trichoderma harzianum,T. viride,Penicillium duponti,Fusarium oxysporun and Chaetomium thermophilum were carried out for the production of enzymes. Isolated predominant thermophilic fungi were evaluated on different types of enzymes. Among tested thermophilic fungi, the highest ativity was observed in C. thermophilium (20mm) followed by T. harzianum (19.50mm) In lipase, M. mucedo (15.40mm) was found maximum followed by F. oxysporun. Cellulase activity was found highest in A. nige (25mm) followed by others. In case of xylanase, catalase, peroxidase and esterase activities were found maximum, minimum and medium even negative in some fungi. Maximum pectinase activity was detected from H. insolens (52.26 @ 0 min) and (74.25 @ 10 min) and in case of M. mucedo, F. oxysporun and C. thermophilium was found most extreme while least in A. niger (30.12) and P. duponti (33.47) @ 0 minute. Key words: Organic Substrates, Thermophilic Fungi, Enzymes

Compartmentalization of Pectinase within Cellulose Hydrogel: An Efficient Technique to Enhance the Catalytic Properties of Pectinase for Industrial Applications

Pectinase catalyze the breakdown of pectin polymer and widely has been used in different industrial preparations such as fruit juice preparation, liquification and scarification of plant biomass as well as coffee and tea fermentation. In this study, the pectinase from Bacillus licheniformis was compartmentalized within cellulose beads hydrogel using encapsulation technique to make it reusable with easy recovery from reaction mixture. The compartmentalization improved catalytic properties of pectinase and ensured its reusability for continuous uses. It was observed that 5.0% cellulose concentration was enough to form stable cellulose hydrogel beads with retention of high relative of activity of pectinase. The hydrogel compartmentalization didn’t change the optima pH and temperature for maximum relative activity and both the hydrogel compartmentalized pectinase and free pectinase maximum relative at same pH and temperature but the hydrogel compartmentalization increased the stability of pectinase against various temperatures and pH, and hydrogel compartmentalized pectinase showed higher relative activities against various temperature and pH as compared to free pectinase. The hydrogel compartmentalization slightly reduced the affinity of pectinase toward the substrate (pectin) and hydrogel compartmentalized pectinase showed little bit higher Km value as compared to soluble pectinase. Cellulose hydrogel compartmentalization retained the pectinase activity for reutilization in different reaction preparation and hydrogel compartmentalized pectinase showed more than 80% of its original activity after three times reusing.

Isolation and Screening of Pectinase Producing Bacteria from Soil Sample

The vast majority of the industrial use of enzymes is covered from microorganisms. Microorganisms are favoured in industry because of their several advantages for example rapid growth, short life expectancy and simplicity in doing genetic alterations. Microbial enzymes are thus amply provided, very much standardized and promoted by many companies. Among various enzymes, Pectinases hold an exceptional place because of its different uses in various sectors like food, textile and biofuel industries.A total of 25% of total enzyme market is being shared by Pectinase alone.The current study was carried out to evaluate the pectinase activity of the pectinolytic bacteria. 40 Bacterial strains were isolated from different soil samples and screened for Pectinase production. Primary and Secondary screening showed 3 potential isolates I38 , I39 and I40 showing pectin degradation on Vincent’s media. Further, extracellular pectinase was partially purified by ammonium sulphate precipitation and dialysis. Sequential ammonium sulphate saturations from 20-80% i.e. (20, 40, 60 and 80%) showed 60% ammonium sulphate was optimum for precipitation of intracellular enzyme whereas 80% was optimum for extracellular enzyme.

Ultrastructural observation of Botrytis cinerea and physical changes in resistant and susceptible grapevines

The necrotrophic fungus Botrytis cinereais a major threat to grapevine cultivation worldwide. Here, a highly-resistant Chinese wild grapevine Vitis amurensis Rupr ‘Shuangyou’ (SY) and the susceptible V. vinifera ‘Red Globe’ (RG) were selected for study, and their pathogenic infection and biochemical responses to B. cinerea were evaluated. The results revealed more trichomes on and a thicker cuticle for leaves of SY than RG under scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Both SEM and TEM also showed that conidial germination, appressorium formation, and hyphal development of B. cinerea were delayed on the leaves of resistant SY. Fewer infected hyphae were also observed in leaves of resistant SY when compared with susceptible RG. The infected leaves of resistant SY harbored higher levels of cellulase and pectinase activity during the early infection stages of B. cinerea at 4 hours post-inoculation (hpi), and higher glucanase and chitinase activity were maintained in the inoculated leaves of SY from 4 hpi through 18 hpi. Lignin was deposited in the infected leaves of susceptible RG but not in resistant SY. Taken together, these results provide insights into the ultrastructural characterizations and physical changes in resistant and susceptible grapevines.

Transcriptome analysis insight into ethylene metabolism and pectinase activity of apricot (Prunus armeniaca L.) development and ripening

Ethylene metabolism is very important for climacteric fruit, and apricots are typical climacteric fruit. The activity of pectinase is closely related to fruit firmness, which further affects fruit quality. To better understand ethylene metabolism, pectinase activity and their molecular regulation mechanisms during the development and ripening of apricot fruit, ethylene metabolism, pectinase activity and the “Luntaibaixing” apricot fruit transcriptome were analyzed at different developmental stages. Ethylene metabolic precursors, enzyme activities and ethylene release increased during fruit development and ripening, with significant differences between the ripening stage and other stages (P < 0.05). Fruit firmness decreased significantly from the S1 to S5 stages, and polygalacturonase, pectin methylesterase, and pectin lyase activities were significantly higher in the S5 stage than in other stages. RNA sequencing (RNA-seq) analysis of fruit resulted in the identification of 22,337 unigenes and 6629 differentially expressed genes (DEGs) during development and ripening, of which 20,989 unigenes are annotated in public protein databases. In functional enrichment analysis, DEGs among the three stages were found to be involved in plant hormone signal transduction. Four key genes affecting ethylene metabolism, six key ethylene signal transduction genes and seven genes related to pectinase in apricot fruit were identified by KEGG pathway analysis. By RNA-sequencing, we not only clarified the molecular mechanism of ethylene metabolism during the ripening of "Luntaibaixing" apricot fruit but also provided a theoretical basis for understanding pectin metabolism in apricot fruit.

Pectinolytic activity of aspergillus niger and Aspergillus flavus grown on grapefruit (citrus Parasidis) peel in solid state fermentation

The present study was aimed at studying pectinolytic activity of resident fungi isolated from decomposing grapefruit (Citrus parasidis) peels in solid state fermentation. Grape fruit peel was subjected to natural fermentation and the fermenting fungi were isolated, characterized and identified using standard microbiological methods. The isolated fungi were in turn used for fermentation to determine their pectinolytic activity through solid state fermentation technique. Culture parameters such as incubation period, temperature, moisture content and addition of salts supplements were optimized during the research for five days. The identified fungi were Aspergillus Niger and Aspergillus flavus. The peak of pectinolytic activity was at day three of fermentation when the highest pectinase activity of 13.32 μmol/mg/min was recorded for A. Niger and 11.32 μmol/mg/min for A. flavus. Optimum temperature and pH for pectinase activity by A. Niger and A. flavus was at 40 0C and pH 7.5 and 7.7 respectively. The use of salt supplemented substrate did not alter enzyme activity. In conclusion, the isolated fungi could be promising organisms for pectinolytic enzyme production on grape peel as substrate. Keywords: Grapefruit, Pectinolytic Activity, Fungi, Fermentation, Aspergillus

Pectin Degradation in Fruit Juices by Pectinase from Meyerozyma sp. VITPCT75 Isolated from Phyllanthus emblica

This study aimed to identify and characterize a pectinase-producing novel yeast from the fermented juice of Phyllanthus emblica and apply the enzyme for fruit juice clarification. Among the five pectinase-producing yeasts, isolate-1 exhibited the highest pectinase activity and was further used in this study. Based on morphological, physiological, and 18SrRNAanalyses, isolate-1 was recognized as a new strain sharing 99% sequence homology with other Meyerozyma strains and was thus designated as Meyerozyma sp. VITPCT75. The strain produced pectinase optimally at a temperature and pH of 25oC and 7, respectively. Maximum pectinase production was observed after 4-days incubation. The enzyme exhibited optimum activity at the temperature of 25 °C and pH 7.0. The enzyme was more stable at a temperature and pH of 20 °C and 7, respectively. Storage stability studies revealed that the enzyme was stable at -20 °C. The cell-free supernatant was partially purified using ammonium sulfate and solvent precipitation. Acetone at a concentration of 20% assured an adequate partial purification. The molecular weight of pectinase was determined as 6 kDa. The enzymatic metal ion preference-related studies revealed that Ca²z, Kz, Cu²z, Fe²z, and Ba²z ions enhanced, Ni²z ions moderately inhibited, and Mn²z ions intensely inhibited the enzymatic activity. Neither Na+ and Mg2+ ions nor EDTA affected the enzyme activity. When subjected to fruit juice clarification, the enzyme significantly reduced the viscosity of the juice.

Role of Exopolygalacturonase-Related Genes in Potato-Verticillium dahliae Interaction

Verticillium dahliae is a hemibiotrophic pathogen responsible for great losses in dicot crop production. An ExoPG gene (VDAG_03463,) identified using subtractive hybridization/cDNA-AFLP, showed higher expression levels in highly aggressive than in weakly aggressive V. dahliae isolates. We used a vector-free split-marker recombination method with PEG-mediated protoplast to delete the ExoPG gene in V. dahliae. This is the first instance of using this method for V. dahliae transformation. Only two PCR steps and one transformation step were required, markedly reducing the necessary time for gene deletion. Six mutants were identified. ExoPG expressed more in the highly aggressive than in the weakly aggressive isolate in response to potato leaf and stem extracts. Its expression increased in both isolates during infection, with higher levels in the highly aggressive isolate at early infection stages. The disruption of ExoPG did not influence virulence, nor did it affect total exopolygalacturonase activity in V. dahliae. Full genome analysis showed 8 more genes related to polygalacturonase/pectinase activity in V. dahliae. Transcripts of PGA increased in the △exopg mutant in response to potato leaf extracts, compared to the wild type. The expression pattern of those eight genes showed similar trends in the △exopg mutant and in the weakly aggressive isolate in response to potato extracts, but without the increase of PGA in the weakly aggressive isolate to leaf extracts. This indicated that the △exopg mutant of V. dahliae compensated by the suppression of ExoPG by activating other related gene.