Activity of cell wall-associated enzymes in ripening olive fruit

Enzymatically active cell wall isolaled from olive (Olea europaea) fruit was employed Hi investigate some hydrolytic enzymes bound to the cell wall and the changes in these during ripening. Seven glycosidases. β-glucosidase (EC 3.2.1.21) α-galactosidase (EC 3.2.1.22). β-galactosidase (EC 3.2.1.23). α-arabinosidase (EC 3.2.1.55), α-mannosidase (EC 3.2.1,24). β-xylosidase (EC 3.2.1.37) and β-N-acetylglucosamidase (EC 3.2.1.30). as well as Cx-cellulase (EC 3.2.1.4) and endo-polygalacturonase (EC 3.2.1.15). were identified in the cell wall preparation, at four stages of ripeness (mature green. changing colour, black and black-ripe). Activities of all these cell wall-associated enzymes fionicallv and covalently linked) were determined either by cell wall incubation with artificial substrate or after extraction from the cell wall with buffers of high salt concentration (Cx-cellulase). and were compared to those of forms solubilized from acetone powders with 500 nM citrate buffer (cytoplasmic and/or apoplastic plus ionically hound to cell wall) In general, the activities of low ionic strength buffer-soluble enzymes were found to be much higher than those of the bound enzymes. The bound enzymes are present in the fruit at the green colour stage, whereas the activities of the soluble enzymes only increased from the changing colour stage onwards. The tenacity of binding of enzymes to the wall was investigated by treating the walls with high salt and measuring residual activity. The nature of the ionic and covalent binding and the changes during ripening were also established for wall-hound glycosidase During ripening there was a marked change in the percentages of covalently- and tonically linked activities of β-glucosidase and β-galaclosidase: al the changing colour stages about 75–80% of the bound active in was present in high ionic strength buffer while al the black-ripe stage it was only 15–20. A possible role for these cell wall degradative enzymes in olive softening is discussed.     

Characterization and <Emphasis Type="Italic">in vitro</Emphasis> expression patterns of extracellular degradative enzymes from non-pathogenic binucleate <Emphasis Type="Italic">Rhizoctonia</Emphasis> AG-G

Many filamentous fungi produce an array of extracellular enzymes that acting in cell walls release elicitors of the plant defense response These enzymes may therefore be important in biocontrol applications. The aim of this study was to characterize extracellular degradative enzymes produced by a non-pathogenic binucleate isolate of Rhizoctonia AG-G. The fungus was grown in liquid culture supplemented with pectin, polygalacturonic acid or glucose as a carbon sources and filtrates of the culture media were analyzed for the detection of pectinolytic and glucan hydrolytic enzymes. Using only pectin as a carbon source, secretion of polygalacturonases and methylesterases was found. When the liquid medium was supplemented with polygalacturonic acid, only polygalacturonase activity was detected. However, when glucose was used as carbon source -1,3 and -1,6 glucanases activities were detected, using laminarin and pustulan as substrates, but none of the pectinolytic activities were found. These enzymes were partially purified and characterized. The -(1,3)(1,6) glucanase and polygalacturonase enzymes showed to be active against cell wall polysaccharides from potato sprouts. These enzymes may have an important role in fungus-plant cell wall interaction. This is the first study about the production of extracellular enzymes by non-pathogenic binucleate Rhizoctonia AG-G.     

Cell Wall-Bound Phenolic Acid and Lignin Contents in Date Palm as Related to its Resistance to Fusarium Oxysporum

The root cell walls of the resistant cultivars of the date palm were more resistant to the action of the cell wall-degrading enzymes (CWDE) of Fusarium oxysporum f. sp. albedinis than those of the susceptible cultivars. Date palm roots contain four cell wall-bound phenolics identified as p-hydroxybenzoic acid, p-coumaric acid, ferulic acid and sinapic acid. The contents of p-coumaric acid and ferulic acid in the resistant cultivars (IKL, SLY, BSTN) were about 2 times higher than those in the susceptible cultivars (BFG, JHL, BSK). The contents of p-hydroxybenzoic acid and sinapic acid in the resistant cultivars were 8.4 and 4.5 times, respectively, higher than those in the susceptible cultivars. The lignin contents in roots of the resistant cultivars were 1.8 times higher than those of the susceptible cultivars. The cell wall-bound phenols accumulated particularly in resistant cultivars reduced strongly the mycelial growth and the CWDE production in vitro.     

Enzymatic cleaning of biofouled thin-film composite reverse osmosis (RO) membrane operated in a biofilm membrane reactor

Application of environmentally friendly enzymes to remove thin-film composite (TFC) reverse osmosis (RO) membrane biofoulants without changing the physico-chemical properties of the RO surface is a challenging and new concept. Eight enzymes from Novozyme A/S were tested using a commercially available biofouling-resistant TFC polyamide RO membrane (BW30, FilmTech Corporation, Dow Chemical Co.) without filtration in a rotating disk reactor system operated for 58 days. At the end of the operation, the accumulated biofoulants on the TFC RO surfaces were treated with the three best enzymes, Subtilisin protease and lipase; dextranase; and polygalacturonase (PG) based enzymes, at neutral pH (~7) and doses of 50, 100, and 150 ppm. Contact times were 18 and 36 h. Live/dead staining, epifluorescence microscopy measurements, and 5 μm thick cryo-sections of enzyme and physically treated biofouled membranes revealed that Subtilisin protease- and lipase-based enzymes at 100 ppm and 18 h contact time were optimal for removing most of the cells and proteins from the RO surface. Culturable cells inside the biofilm declined by more than five logs even at the lower dose (50 ppm) and shorter incubation period (18 h). Subtilisin protease- and lipase-based enzyme cleaning at 100 ppm and for 18 h contact time restored the hydrophobicity of the TFC RO surface to its virgin condition while physical cleaning alone resulted in a 50° increase in hydrophobicity. Moreover, at this optimum working condition, the Subtilisin protease- and lipase-based enzyme treatment of biofouled RO surface also restored the surface roughness measured with atomic force microscopy and the mass percentage of the chemical compositions on the TFC surface estimated with X-ray photoelectron spectroscopy to its virgin condition. This novel study will encourage the further development and application of enzymes to remove biofoulants on the RO surface without changing its surface properties.     

Growth and protopectinase production of Geotrichum klebahnii in batch and continuous cultures with synthetic media

Geotrichum klebahnii ATCC 42397 produces a protopectinase (PPase-SE) with polygalacturonase (PGase) activity. The microorganism was aerobically cultivated in synthetic media. Glucose, fructose and xylose yielded the highest enzyme levels (10–11 PGase units ml⁻¹). Galacturonic acid repressed enzyme production and no growth was obtained with disaccharides and pectin. Specific enzyme activity obtained in an O₂-limited culture was similar to that found in nonlimited ones. A growth yield (Y _x/s) of 0.49 g of cell dry weight per gram of glucose consumed was obtained in a typical batch bioreactor culture. Enzyme production was growth associated, and no major products other than biomass and CO₂ were detected. The volumetric enzyme activity reached a maximum around D=0.3–0.4 h⁻¹ in glucose-limited continuous cultures. However, it varied strongly (together with microorganism morphology) even after retention times ≥8 at any D tested (0.035–0.44 h⁻¹) though the rest of the culture variables remained fairly constant. No correlation between morphology and enzyme activity could be obtained. Enzyme production was poor in urea- and vitamin-limited continuous cultures. In all cases, biomass and CO₂ accounted for ≅100% of carbon recovery though Y _x/s values were different. Journal of Industrial Microbiology & Biotechnology (2000) 25, 260–265. Received 20 April 2000/ Accepted in revised form 15 September 2000     

Detection of polygalacturonases and pectin esterases in lactic acid bacteria

Of 80 strains of lactic acid bacteria tested, only Lactobacillus casei strains HNK10 and L1–8, Lactobacillus plantarum Lc5 and Lactococcus lactis NN01 produced polygalacturonases (EC 3.2.1.) and/or pectin-esterases (EC 3.1.1.). Crude extracellular extracts of strain L1–8 were able to clarify pectin.     

Three polygalacturonases constitutively synthesized by Aspergillus alliaceus

Of three molecular forms of polygalacturonases synthesized by Aspergillus alliaceus on glucose media, two were exopolygalacturonases (exoPG₁ and exoPG₂) and one was an endopolygalacturonase (endoPG). Low-methoxylated beet pectin was the preferred substrate for the endoPG and exoPG₂ whereas pectic acid was the optimal substrate for exoPG₁. The highest activities of endoPG, exoPG₁ and exoPG₂ were at pH 5.5, 3.5, and 6.0 and at 35, 45 to 50 and 35°C, respectively. Disks of potato-tuber tissue were macerated by endoPG, but not by exoPG₁ or exoPG₂.The authors are with the Institute of Microbiology, Belarus Academy of Sciences, Zhodinskaya 2, 220141, Minsk, Belarus;     

One-step Concentration and Partial Purification of Aspergillus kawachii Non-acidic Polygalacturonases by Adsorption to Glass Fiber Microfilters

The non-acidic polygalacturonases produced by Aspergillus kawachii in a glucose/tryptone medium were adsorbed to a glass fiber microfilter that was used to clarify the fermentation broth. Maximum adsorption occurred at pH 3 under low ionic strength conditions. The adsorbed activity could be readily released with a buffer solution at pH 5. Based upon these observations, a separation process was developed which enabled the broth to be clarified and, at the same time, the non-acidic polygalacturonases to be concentrated 20-fold and purified 100-fold in a unique filtration step. The practical advantage of recovering polygalacturonases by a filtration process lies in the simplicity and efficiency of the operation involved. Received 27 July 2005; Revisions requested 24 August 2005; Revisions received 16 November 2005; Accepted 16 November 2005     

Purification and molecular characterization of a soybean polygalacturonase-inhibiting protein

A polygalacturonase-inhibiting protein (PGIP) was detected in soybean (Glycine max (L.) Merr.) seedlings. The protein was purified from germinating seeds and appeared to consist of at least three components with very close molecular weights (between 37 and 40 kDa) but each showing a unique N-terminal sequence. Primers specific for N-terminal and C-terminal nucleotide sequences of field bean (Phaseolus vulgaris L.) PGIP were used in a polymerase chain reaction (PCR) on soybean DNA, and only one amplification band was obtained. The amplified product was cloned and one of the PCR clones was sequenced. The nucleotide sequence comprises 942 bp with a single open reading frame which encodes a polypeptide of 313 amino-acid residues with a predicted molecular weight of 33984 Daltons and an isoelectric point of 8.21. Analysis of genome organization showed a single gene copy of PGIP with few related sequences, and wounding of soybean hypocotyls showed a strong induction of expression of the PGIP gene. The PGIP showed different activities toward three purified fungal endo-polygalacturonases (endo-PGs) (two endoPGs from Sclerotinia sclerotiorum and one endo-PG from Aspergillus niger). A possible involvement of soybean PGIP in plant defence against fungal pathogens is discussed.