Cellulolytic enzymes associated with the fruit rots of Citrus sinensis caused by Aspergillus aculeatus and Botryodiplodia theobromae

Botryodiplodia theobromae and Aspergillus aculeatus were inoculated in carboxymethylcellulose (CMC) medium and on filter papers. Hydrolysis of the CMC medium and degradation of the filter papers were observed, indicating the production of C1 and Cx cellulases by the two rot pathogens. The C1 and Cx enzymes were also detected in filtrates of rotted orange fruits obtained by infection with the two pathogens. The cellulases could not induce rot development on their own. However, when they were added to pectinases in an enzyme inoculum, the incubation period for inducing rot development was shorter, thus establishing a secondary role for the cellulases in the rot development. Optimum conditions for the action of the cellulases included a neutral pH and temperature ranging from 25 to 30 degrees C.     

A Comparison between the Cellulolytic Activity of White and Brown Rot Fungi. I. The Activity on Insoluble Cellulose

About 70 strains of white and brown rot fungi were cultivated on media, containing filter paper cellulose as the main carbon source. The cellulolytic activity of the culture filtrates was measured after different periods of growth by means of the turbidimetric method. The results obtained indicate a difference between the two types of wood decay fungi as to the capacity of attacking the cellulose used in the medium and in the cellulase test. No significant C₁activity was found in any of the brown rot cultures whereas all white rot fungi tested exerted a measurable activity on the test substrate. The effect of various carbohydrates and some proteins as inducers of cellulase activity was studied. Especially cellobiose and lactose were active on white rot fungi in this respect, particularly in the presence of yeast extract. Also some brown rot fungi exerted C₁-activity after incubation on glucose or cellobiose.     

Effects of an organophosphorus insecticide on the growth and cellulolytic activity of fungi

The organophosphorus insecticide Selecron [O-(4-bromo-2-chlorophenyl) O-ethyl S-n-propyl-phosphorotioate] at 10 and 50 ppm significantly decreased respiration, mycelial protein, extracellular protein and mycelial dry weight of Aspergillus fumigatus, A. terreus and Myceliophthora thermophila when grown at 45°C. C_x and C₁ cellulases of tested fungi were significantly decreased. However, C₁ cellulase of A. fumigatus was slightly increased.     

Factors affecting growth and cellulose hydrolysis by the thermotolerant aspergillus nidulans from composts

The ability of Aspergillus nidulans (EIDAM) WINT to grow and sporulate at various temperatures and to degrade soluble and insoluble forms of cellulose were studied. A. nidulans was found to grow and sporulate best at 37°C in continuous light and alternating light-darkness respectively. The fungus was able to cause losses in the dry weights of filter papers on incubation and made appreciable growth on CMC and hemicellulose. The culture filtrates contained cellulases which hydrolysed filter papers and CMC to reducing sugars, and were only able to produce these enzymes in the presence of cellulose or its derivatives in the growth medium. The CM-cellulases had peak activity at pH 5.2 and at 50°C while optimal FP-activity occurred at a pH of 5.5 and at 45°C. The participatory role of A. nidulans in composting is discussed.     

Enzymatic degradation of steam-pretreated Lespedeza stalk (Lespedeza crytobotrya) by cellulosic-substrate induced cellulases

The cellulase production by Trichoderma viride, cultivated on different substrates, namely steam-pretreated Lespedeza, filter paper, microcrystalline cellulose (MCC) or carboxymethyl cellulose (CMC), was studied. Different cellulase systems were secreted when cultivated on different substrates. The cellulolytic enzyme from steam-pretreated Lespedeza medium performed the highest filter paper activity, exoglucanase and endoglucanase activities, while the highest β-glucosidase activity was obtained from the enzyme produced on filter paper medium. The hydrolytic potential of the enzymes produced from different media was evaluated on steam-pretreated Lespedeza. The cellulase from steam-pretreated Lespedeza was found to have the most efficient hydrolysis capability to this specific substrate. The molecular weights of the cellulases produced on steam-pretreated Lespedeza, filter paper and MCC media were 33, 37 and 40 kDa, respectively, and the cellulase from CMC medium had molecular weights of 20 and 43 kDa. The degree of polymerization, crystallinity index and micro structure scanned by the scanning electron microscopy of degraded steam-pretreated Lespedeza residues were also studied.     

Cellulase production by a thermophilic clostridium species

Strain M7, a thermophilic, anaerobic, terminally sporing bacterium (0.6 by 4.0 μm) was isolated from manure. It degraded filter paper in 1 to 2 days at 60 C in a minimal cellulose medium but was stimulated by yeast extract. It fermented a wide variety of sugars but produced cellulase only in cellulose or carboxymethyl-cellulose media. Cellulase synthesis not only was probably repressed by 0.4% glucose and 0.3% cellobiose, but also cellulase activity appeared to be inhibited by these sugars at these concentrations. Both C₁ cellulase (degrades native cellulose) and C_x cellulase (β-1,4-glucanase) activities in strain M7 cultures were assayed by measuring the liberation of reducing sugars with dinitrosalicylic acid. Both activities had optima at pH 6.5 and 67 C. One milliliter of a 48-h culture of strain M7 hydrolyzed 0.044-meq of glucose per min from cotton fibers. The cellulase(s) from strain M7 was extracellular, produced during exponential growth, but was not free in the growth medium until approximately 30% of the cellulose was hydrolyzed. Glucose and cellobiose were the major soluble products liberated from cellulose by the cellulase. ZnCl₂ precipitation appeared initially to be a good method for the concentration of cellulase activity, but subsequent purification was not successful. Isoelectric focusing indicated the presence of four C_x cellulases (pI 4.5, 6.3, 6.8, and 8.7). The rapid production and high activity of cellulases from this organism strongly support the basic premise that increased hydrolysis of native cellulose is possible at elevated temperature.     

Characterization of the cellulase complex from Cellulomonas grown on bagasse pith

Summary The effect of physico-chemical parameters on the cellulolytic activity of Cellulomonas sp. IIbc grown on sugarcane bagasse pith was investigated, and the optimum ranges for enzyme activity were established. The cellulases were more stable when incubated at the optimum growth temperature (32°C) than under optimum activity conditions (45°C for -glucosidases and 50°C for CMC- and FP-cellulases). The -glucosidases were the thermostability-limiting enzymes of the complex. Two types of endoglucanases could be recognized according to their adsorption properties on bagasse: one weakly-bound and one tightly-bound type, the latter constituting approximately 73% of the extracellular endoglucanases at exponential growth phase. Four forms active on filter paper and three active on CMC were obtained by HPLC separation of the extracellular fraction of the culture at stationary phase.Abbreviations CMC carboxymethylcellulose - FP filter paper     

Protein recovery by continuous flotation

Summary Bovine serum albumin (BSA) was recovered from aqueous solutions by foam flotation. The protein concentrations in foam liquid C _S, in feed C _Pand in residue C _Rwere determined. The protein enrichment C _S/C_Pand the separation C _S/C_Ras well as the protein fraction in the foam liquid % BSA and foam liquid volume flow were determined as functions of the medium properties, operational conditions, and equipment parameters as well as concentrations of solid particles. At low protein concentrations in feed (e.g., C _P=40 mg · l^-1), and at 40° C, high performance was attained (C _X=2,000 mg · l^-1, C _R=4.4 mg · l^-1, C _S/C_P=50, C _S/C_R=450, 90% BSA. Continuous foam flotation is an efficient procedure for the recovery of low concentrations of proteins from liquid cultures.Abbreviations BSA bovine serum albumine - C _P protein concentration in feed (mg · l^-1) - C _R protein concentration in residue (mg · l^-1) - C _S protein concentration in foam liquid (mg · l^-1) - C _S/C_R protein separation (-) - C _S/C_P protein enrichment (-) - V _P feed rate (ml · min^-1) - V _R residue flow rate (ml · min^-1) - V _S foam liquid volume flow (ml · min^-1) - N number of theoretical stages in an ideal cascade (-) - temperature (° C) - mean residence time (min)     

Synergy between pretreatment lignocellulose modifications and saccharification efficiency in two brown rot fungal systems

Brown rot wood-degrading fungi distinctly modify lignocellulose and completely hydrolyze polysaccharides (saccharification), typically without secreting an exo-acting glucanase and without removing lignin. Although each step of this two-step approach evolved within the same organism, it is unknown if the early lignocellulose modifications are made to specifically facilitate their own abbreviated enzyme system or if enhancements are more general. Because commercial pretreatments are typically approached as an isolated step, answering this question has immense implication on bioprocessing. We pretreated spruce and pine blocks with one of two brown rot fungi, Gloeophyllum trabeum or Fomitopsis pinicola. Wood harvested at weeks 1, 2, 4, and 8 showed a progression of weight loss from time zero due to selective carbohydrate removal. Hemicellulose losses progressed faster than cellulose loss. This “pretreated” material was then saccharified with commercially relevant Trichoderma reesei cellulases or with cellulases from the brown rot fungi responsible for degrading the wood to test for synergy. With increased decay, a significant increase in saccharification efficiency was apparent but not limited to same-species enzyme sources. We also calculated total sugar yields, and calculations that compensate for sugars consumed by fungi suggest a shorter residence time for fungal colonization than calculations based solely on saccharification yields.     

Purification and Properties of Cellulases from an Alkalophilic Streptomyces Strain

An alkalophilic Streptomyces strain, KSM-9, producing extracellular cellulases was isolated from soil. Three kinds of cellulases that preferentially hydrolyzed carboxymethylcellulose (CMC) were purified from the strain and designated as CMCase I, II and III. The optimum pH of CMCase I (M_r, 32,000) is 8.5 while those of CMCase II (M_r, 32,500) and III (M_r, 92,000) are at around pH 6.0. CMCase I hydrolyzed CMC in a more random fashion than the other two enzymes.     

Efficient production of Al(OH)3‐immobilized laccase with a Heterobasidion annosum strain selected by microplate screening

Aims: Wild‐type white rot fungi are the most important production organisms for laccase, a promising oxidative biocatalyst with numerous applications. This study aimed at identifying novel highly productive strains, finding optimal cultivation conditions for laccase production and establishing a simple immobilization procedure. Methods and Results: By using a newly developed 96‐well microplate cultivation method, 23 species of white rot fungi, represented by 29 strains, were directly compared with regard to the amount of secreted laccase. Both, with glucose and spruce saw dust as growth substrate a Heterobasidion annosum strain and a Physisporinus vitreus strain were the most productive (730–2200 U l^?1 of secreted laccase). Cultivation conditions for laccase production with H. annosum were optimized in larger‐scale liquid cultures. Aeration with a sparger lead to a 3·8‐fold increase in laccase activity when compared to nonaerated flask cultures. More than 3000 U l^?1 laccase was produced in glucose medium supplemented with yeast extract and the inducer veratryl alcohol. Culture supernatant was incubated with short‐range ordered Al(OH)₃ particles to directly immobilize and concentrate laccase by adsorption. Active laccase was recovered in 40% yield and the Al(OH)₃‐adsorbed laccase was suitable for repeated decolourization of indigo carmine. Conclusions: Microplate cultivation allowed a large‐scale comparison of the capacity of different fungal species for laccase production. Laccase secretion of a highly productive H. annosum strain was found to vary strongly with different cultivation conditions. Adsorption to Al(OH)₃ proved to be suitable as direct immobilization technique. Significance and Impact of the Study: The microplate screening method simplifies strain and medium development for laccase production. Two novel fungal strains suitable for laccase production were identified. Procedures for simple and efficient production of immobilized H. annosum laccase were established.     

Cellulase activity of some phytopathogenic fungi isolated from diseased leaves of broad bean

Forty-five fungal species and two varieties belonging to the 23 genera were collected from 50 samples of diseased leaves of broad bean collected from Qena Governorate in Egypt on glucose-Czapek’s, dichloran-chloramphenicol-malt extract and dichloran-chloramphenicol-peptone agar at 28 °C. The most common genera were Alternaria, Aspergillus, Cladosporium, Fusarium, Mycosphaerella and Penicillium. The most prevalent species were Alternaria alternata, Aspergillus flavus, A. fumigatus, A. niger, Cladosporium cladosporioides, Fusarium merismoides, Mycosphaerella tassiana and Penicillium chrysogenum. Among the eight dematiaceous Hyphomycetes phytopathogenic fungi screened for their abilities to produce both exo- and endo-β-1,4-glucanase enzymes (C₁ and C_x), five species had high C₁ enzyme production and two species had high C_x enzyme production. However, two and three species were moderately active in producing C₁ and C_x enzymes, respectively. The remaining species had low production of both C₁ and C_x enzymes. The highest cellulase activity was recorded by Alternaria citri and Cochliobolus spicifer for C₁ and by A alternata and A. citri for C_x enzyme. Maximum production of C₁ enzyme by A. citri and C. spicifer was obtained after six days of incubation at 30 °C with an initial pH of 6 in a culture medium containing lactose and calcium nitrate as the carbon and nitrogen sources, respectively. The highest C_x enzyme production was by A. alternata and A. citri,which was recorded after eight days of incubation at 30 °C with an initial pH of 6 in a culture medium containing carboxymethyl cellulose and sodium or calcium nitrate as the carbon and nitrogen sources, respectively.     

Comparative studies on simple lipids ofSclerotium cepivorum,the causal agent of white rot of onion,and other fungi of the class Deuteromycetes

Summary The simple lipids ofSclerotium cepivorum, the causal agent of white rot of onion and nine other fungal species of the same class were investigated. The fatty acid composition of the simple lipids of these fungi were determined by GLC. The main fatty acids common to these fungal species were C₁₆ (saturated) and C₁₈ (unsaturated) acids. The sterol fraction was isolated by column chromatography and its components were detected by GLC and mass spectrometry. Ergosterol and γ-Ergostenol were found mostly in all fungal species under investigation. However, two fungal species namelyAlternaria alternata andScolecobasidium constrictum showed no Ergosterol.     

Cellulase production from agricultural residues by recombinant fusant strain of a fungal endophyte of the marine sponge Latrunculia corticata for production of ethanol

Several fungal endophytes of the Egyptian marine sponge Latrunculia corticata were isolated, including strains Trichoderma sp. Merv6, Penicillium sp. Merv2 and Aspergillus sp. Merv70. These fungi exhibited high cellulase activity using different lignocellulosic substrates in solid state fermentations (SSF). By applying mutagenesis and intergeneric protoplast fusion, we have obtained a recombinant strain (Tahrir-25) that overproduced cellulases (exo-β-1,4-glucanase, endo-β-1,4-glucanase and β-1,4-glucosidase) that facilitated complete cellulolysis of agricultural residues. The process parameters for cellulase production by strain Tahrir-25 were optimized in SSF. The highest cellulase recovery from fermentation slurries was achieved with 0.2% Tween 80 as leaching agent. Enzyme production was optimized under the following conditions: initial moisture content of 60% (v/w), inoculum size of 10⁶ spores ml⁻¹, average substrate particle size of 1.0 mm, mixture of sugarcane bagasse and corncob (2:1) as the carbon source supplemented with carboxymethyl cellulose (CMC) and corn steep solids, fermentation time of 7 days, medium pH of 5.5 at 30°C. These optimized conditions yielded 450, 191, and 225 units/gram dry substrate (U gds⁻¹) of carboxylmethyl cellulase, filter-paperase (FPase), and β-glucosidase, respectively. Subsequent fermentation by the yeast, Saccharomyces cerevisiae NRC2, using lignocellulose hydrolysates obtained from the optimized cellulase process produced the highest amount of ethanol (58 g l⁻¹). This study has revealed the potential of exploiting marine fungi for cost-effective production of cellulases for second generation bioethanol processes.     

Production of S-adenosyl- L-methionine by a mutant strain of Kluyveromyces lactis

S-Adenosyl-l-methionine (AdoMet) was produced by a mutant strain Kluyveromyces lactis AM-65 grown on whey. A full factorial design method of three factors – (NH₄)₂SO₄ (factor x ₁), corn steep liquor (factor x ₂) and l-methionine (factor x ₃) on three levels – was used to determine the optimal medium conditions for the production of AdoMet. A time course shake-flask experiment in optimal whey medium (x ₁=3.1 g l^–1, x ₂=12.7 g l^–1, x ₃=4.6 g l^–1) was also carried out and the results confirmed the results of the factorial design and subsequent quadratic modelling and optimization of AdoMet production which reached 90 mg g^–1 cell dry wt.     

Levan production using mutant strains of Zymomonas mobilis in different culture conditions

Several levan hyperproducing mutants of Zymomonas mobilis strains were selected by mutagenesis with N-methyl-N-nitro-nitrosoguanidine and caffeine. Highest levan production (41 g l^–1) was obtained with a mutant strain HL 29 in a culture medium containing 200 g sucrose l^–1 and 0.5 g (NH₄)₂SO₄ l^–1 stored at 7 °C for 29 days. This is the first report describing the levan synthesis by Z. mobilis at 7 °C.     

Assessment of saccharification efficacy in the cellulase system of the brown rot fungus Gloeophyllum trabeum

Brown rot fungi uniquely degrade wood by creating modifications thought to aid in the selective removal of polysaccharides by an incomplete cellulase suite. This naturally successful mechanism offers potential for current bioprocessing applications. To test the efficacy of brown rot cellulases, southern yellow pine wood blocks were first degraded by the brown rot fungus Gloeophyllum trabeum for 0, 2, 4, and 6 weeks. Characterization of the pine constituents revealed brown rot decay patterns, with selective polysaccharide removal as lignin compositions increased. G. trabeum liquid and solid state cellulase extracts, as well as a commercial Trichoderma reesei extract (Celluclast 1.5 L), were used to saccharify this pretreated material, using β-glucosidase amendment to remove limitation of cellobiose-to-glucose conversion. Conditions varied according to source and concentration of cellulase extract and to pH (3.0 vs. 4.8). Hydrolysis yields were maximized using solid state G. trabeum extracts at a pH of 4.8. However, the extent of glucose release was low and was not significantly altered when cellulase loading levels were increased threefold. Furthermore, Celluclast 1.5 L continually outperformed G. trabeum cellulase extracts, although extent of glucose release never exceeded 22.0%. Results suggest methodological advances for utilizing crude G. trabeum cellulases and imply that the suboptimal hydrolysis levels obtained with G. trabeum and Celluclast 1.5 L cellulases, even at high loading levels, may be due to brown rot modifications insufficiently distributed throughout the pretreated material.     

Some extracellular enzymes associated with two tomato fruit spoilage molds

The production of amylolytic, cellulolytic and pectinolytic enzymes by Aspergillus flavus and A. fumigatus was investigated. The two fungi were cultured on wheat offal and liquid crystalline carboxymethylcellulose media. A. flavus produced amylases on basal and starch containing media while A. fumigatus could only produce amylases on starch medium. The cellulolytic activities of filtrates from culture or infected fruits showed that A. flavus produced lesser quantities of cellulolytic enzymes than A. fumigatus. At 25 °C and at a pH range of 6–8, A. flavus best produces amylases and cellulases, while A. fumigatus showed highest activities of the two enzymes at 35–40 °C and at pH 7.0. Two pectinolytic enzymes — polymethylgalacturonase and pectinmethyltrans-eliminase — were identified in vivo with the two molds. An endopolygalacturonase in addition to these two pectinolytic enzymes was well associated with A. fumigatus.     

A novel metabolic pathway for biodegradation of DDT by the white rot fungi, <Emphasis Type="Italic">Phlebia lindtneri</Emphasis> and <Emphasis Type="Italic">Phlebia brevispora</Emphasis>

1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) was used as the substrate for a degradation experiment with the white rot fungi Phlebia lindtneri GB-1027 and Phlebia brevispora TMIC34596, which are capable of degrading polychlorinated dibenzo-p-dioxin (PCDD) and polychlorinated biphenyls (PCBs). Pure culture of P. lindtneri and P. brevispora with DDT (25 μmol l⁻¹) showed that 70 and 30% of DDT, respectively, disappeared in a low-nitrogen medium after a 21-day incubation period. The metabolites were analyzed using gas chromatography/mass spectrometry (GC/MS). Both fungi metabolized DDT to 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD), 2,2-bis(4-chlorophenyl)acetic acid (DDA) and 4,4-dichlorobenzophenone (DBP). Additionally, DDD was converted to DDA and DBP. DDA was converted to DBP and 4,4-dichlorobenzhydrol (DBH). While DBP was treated as substrate, DBH and three hydroxylated metabolites, including one dihydroxylated DBP and two different isomers of monohydroxylated DBH, were produced from fungal cultures, and these hydroxylated metabolites were efficiently inhibited by the addition of a cytochrome P-450 inhibitor, piperonyl butoxide. These results indicate that the white rot fungi P. lindtneri and P. brevispora can degrade DBP/DBH through hydroxylation of the aromatic ring. Moreover, the single-ring aromatic metabolites, such as 4-chlorobenzaldehyde, 4-chlorobenzyl alcohol and 4-chlorobenzoic acid, were found as metabolic products of all substrate, demonstrating that the cleavage reaction of the aliphatic-aryl carbon bond occurs in the biodegradation process of DDT by white rot fungi.     

Bacterial iturins mediate biocontrol activity of Bacillus sp. against postharvest pear fruit‐rotting fungi

A potential antagonist, Bacillus sp. LYLB4 isolated from pear fruits, was tested for its antifungal activity against postharvest pear pathogens. LYLB4 had a remarkable antifungal effect on Botryosphaeria dothidea. Although it showed a weak inhibition effect on the growth of Rhizopus stolonifer on potato dextrose agar (PDA) plates, LYLB4 almost completely destroyed R. stolonifer during direct contact in potato dextrose broth (PDB). LYLB4 treatment was able to significantly reduce disease incidence (by 68.9% and 100%, respectively) and lesion diameter (by 68.7% and 100%, respectively) of ring rot caused by B. dothidea and soft rot caused by R. stolonifer in pears. LYLB4 also suppressed several other phytopathogens in vitro, suggesting a broad‐spectrum antagonistic activity against fungal pathogens. 16S rRNA and gyrA sequence analysis indicated that LYLB4 is closely related to B. velezensis. Genome mining indicated that LYLB4 had 11 secondary metabolites encoding clusters, but that the surfactin and fengycin gene clusters may not be functional because of a large deletion. Matrix‐assisted laser desorption ionization‐time of flight mass spectra (MALDI‐TOF‐MS) demonstrated that iturins were the major lipopeptides, and that C₁₆/C₁₇ Bacillomycin D synthesis was stimulated when LYLB4 was co‐cultured with B. dothidea compared to the control. Overall, these results demonstrate that the main biocontrol mechanism adopted by LYLB4 could be through the production of toxic metabolites and direct contact with pathogens.