Autohydrolysis extraction process as a pretreatment of lignocelluloses for their enzymatic hydrolysis

Autohydrolysis was studied as a pretreatment to enhance sugar yields from enzymatic hydrolysis of wheat and rape straw, beech, birch and poplar sawdust. Reaction temperatures were 185°C to 212°C and the reaction time 20 min. The pretreated slurries were hydrolyzed with “Novo” cellulase and Fusarium sp. 27 cellulase at 45°C and pH 4.8 for 24 h with addition of Fusarium sp. 27 cellbound cellobiase. From 85% to 90% sugar content of substrates were converted to reducing sugars after 24 h enzymatic hydrolysis, with exception of poplar wood. 10.8 g biomass was obtained after cultivation of Fusarium sp. 27 with water solution hemicellulose fraction from 100 g beech sawdust autohydrolyzed at 200°C during 20 min.     

Enzyme catalysed production of vegetable oils containing omega-3 polyunsaturated fatty acid

Summary The application of enzymatic interesterification for production of vegetable oils containing omega-3 polyunsaturated fatty acids was investigated. Six veteable oils were used as substrates, together with omega-3 polyunsaturated fatty acid, and reactions were catalysed by immobilized Mucor miehei lipase in organic solvent. The degree of incorporation of eicosapentaenoic acid and docosahexaenoic acid into corn oil, sunflower oil, peanut oil, olive oil and soybean oil were 17.71, 17.59, 16.79, 14.89, 13.91 and 10.48%, respectively, after a 12 h incubation period.     

Comparative Study on Caustic and Enzymatic Cleanings of Stainless Steel Surface Fouled with β-Lactoglobulin

Desorption behavior of β-lactoglobulin (β-Lg), one of the main constituents of fouling deposits in milk processing, from stainless steel surfaces during caustic and enzymatic cleanings was studied by using a glass column packed with stainless steel particles fouled with β-Lg. Both in caustic and enzymatic cleanings, the amount of β-Lg remaining on the stainless steel particles decreased according to first-order kinetics at the initial stage, and gradually reached a constant value. The desorption rate constant at the initial stage and the residual amount of β-Lg after 2 h of cleaning were evaluated as the measures of cleaning efficiency under various conditions. In caustic cleaning, these two values were strongly affected by the concentration of NaOH. The initial desorption rate increased with increasing flow rate of the caustic solution, suggesting a mass transfer effect. In enzymatic cleaning, the maximum desorption rate constant was obtained at around the optimum pH for the enzyme reaction. The temperature dependence of the initial desorption rate constant was stronger in caustic cleaning than in enzymatic cleaning.     

Degradation of diclofenac,trimethoprim, carbamazepine,and sulfamethoxazole by laccase from Trametes versicolor: Transformation products and toxicity of treated effluent

Abstract

The degradation of diclofenac (DCF), trimethoprim (TMP), carbamazepine (CBZ), and sulfamethoxazole (SMX) by laccase from Trametes versicolor was investigated. Experiments were conducted using the pharmaceuticals individually, or as a mixture at different initial concentrations (1.25 and 5?mg/L each). The initial enzymatic activity of all the treated samples was around 430–460?U_(DMP)/L. The removal of the four selected pharmaceuticals tested individually was more effective than when tested in mixtures under the same conditions. For example, 5?mg DCF/L was completely removed to below its detection limit (1?µg/L) within 8?h in the individual experiment vs. after 24?h when dosed as a mixture with the other pharmaceuticals. A similar trend was visible with other three pharmaceuticals, with 95 vs. 39%, 82 vs. 34% and 56 vs. 49% removal after 48?h with 5?mg/L of TMP, CBZ, and SMX tested individually or as mixtures, respectively. In addition, at the lower initial concentration (1.25?mg/L each), the removal efficiency of TMP, CBZ, and SMX in mixtures was lower than that obtained at the higher initial concentrations (5?mg/L each) during both the individual and combined treatments. Four enzymatic transformation products (TPs) were identified during the individual treatments of DCF and CBZ by T. versicolor. For TMP and SMX, no major TPs were observed under the experimental conditions used. The toxicity of the solution before and after enzymatic treatment of each pharmaceutical was also assessed and all treated effluent samples were verified to be non-toxic.     

Addendum to Issue 1 - ENZITEC 2012Simultaneous biosynthesis of biomass-degrading enzymes using co-cultivation of Aspergillus niger and Trichoderma reesei

Abstract

The biotransformation of lignocellulosic materials into biofuels and chemicals requires the simultaneous action of multiple enzymes. Since the cost of producing an efficient enzyme system maybe high, mixed cultures of microorganisms maybe an alternative to increase enzymatic production and consequently reduce costs. This study investigated the effects of different inoculum ratios and inoculation delays on the biosynthesis of cellulases and xylanases during co-cultivation of Aspergillus niger and Trichoderma reesei under solid-state fermentation (SSF). While the monoculture of T. reesei was more efficient for CMCase production than the co-cultivation of A. niger and T. reesei, a significant increase in β-glucosidase and xylanase production was achieved by co-cultivation of both species. The maximum CMCase activity of 153.91 IU/g was obtained with T. reesei after 48 h of cultivation, while the highest β-glucosidase activity of 119.71 IU/g (after 120 h) was obtained by co-cultivation of A. niger and T. reesei with a 3:1 inoculum ratio (A. niger: T. reesei). The greatest xylanase activity observed was 589.39 IU/g after 72 h of mixed culturing of A. niger and T. Reesei with a 1:1 inoculum ratio. This is the first study where the effects of inoculum ratio and inoculation delay in mixed culture of T. reesei and A. niger under SSF have been systematically assessed, and it indicates co-cultivation as a feasible alternative to increase enzymatic production.     

Selection of an extracellular esterase-producing microorganism

Summary The conventional esterase plate-screening technique has been modified in order to obtain a better differentiation of high producing strains. A short exposure to Lugol's iodine solution after colony growth enhanced the contrast between the precipitation halo and the background. Batch cultures of a selected strain characterized as belonging to theBacillus subtilis group showed a high level of extracellular activity at pH 6.6 to 8.0 and 35°C. In crude extracts the optimum enzymatic activity was obtained at 35°C and pH 7.0.     

Penduliflorain I: A Cysteine Protease Isolated from <Emphasis Type="Italic">Hohenbergia penduliflora</Emphasis> (A.Rich.) Mez (<Emphasis Type="Italic">Bromeliaceae</Emphasis>)

Penduliflorain I, a new plant endopeptidase, was isolated and characterized from Hohenbergia penduliflora. Crude extract was obtained from stems. A partially purified enzyme preparation was obtained by ethanol precipitation. This preparation showed maximum activity between pH 7.5 and 8.5, was stable at ionic strength (20% decrease in proteolytic activity could be detected after 2 h in 0.4 M sodium chloride solution), and exhibited high thermal stability (inactivation required heating for 20 min at 75 °C). Inhibition and activation assays indicated the cysteine nature of the enzymatic preparation. Penduliflorain I was purified by anion exchange chromatography (Q-Sepharose HP) by FPLC system. Homogeneity was confirmed by mass spectroscopy. Molecular mass of the enzyme was 23 412.847 Da (MALDI-TOF–MS). Kinetic parameters were determined for PFLNA (K _m = 0.3227 mM and k _cat = 4.27 s⁻¹). The N-terminal sequence (AVPQSIDWRDYGAVTTDKNQ) of isolated protease showed considerable similarity to other cysteine proteases obtained from stems or fruits of different Bromeliaceae species.     

Extracellular enzymatic activity of Malassezia spp. isolates

Extracellular enzymatic activity of different species of Malassezia spp was evaluated. Thirty-three isolates of animal origin (dogs and cats) and stock culture samples were studied. Twenty isolates of M. pachydermatis, 8 of M. furfur, 2 of M. sympodialis and M. globosa and one of M. restricta, M. obtusa and M. slooffiae were examined. The enzymatic activity was investigatedusing Api Zym system. The enzymatic patterns showed light differences. Esterase lipase, Phosphatase acid and Naphtol-AS-BI-phosphohydrolase were produced in significant amounts from most isolates excepted for M. restricta, confirming the limited enzymatic activity of this species. Data obtained from the other new species described after the revision of the genus, appear to be quite homogeneous. Dixon’s broth appeared to be a valid medium for the growth of all Malassezia spp. This revised version was published online in June 2006 with corrections to the Cover Date.     

Enzymatic synthesis of nucleoside analogues using immobilized 2′-deoxyribosyltransferase from <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis>

Covalent attachment of recombinant Lactobacillus reuteri 2′-deoxyribosyltransferase to Sepabeads EC-EP303 leads to the immobilized biocatalyst SLrNDT4, which displayed an enzymatic activity of 65.4 IU/g of wet biocatalyst in 2′-deoxyadenosine synthesis from 2′-deoxyuridine and adenine at 40°C and pH 6.5. Response surface methodology was employed for the optimization of SLrNDT4 activity. Optimal conditions for SLrNDT4 highest activity were observed at 40°C and pH 6.5. Immobilized biocatalyst retained 50% of its maximal activity after 17.9 h at 60°C, whereas 96% activity was observed after storage at 40°C for 110 h. This novel immobilized biocatalyst has been successfully employed in the enzymatic synthesis of different natural and therapeutic nucleosides effective against cancer and viral diseases. Among these last products, enzymatic synthesis of therapeutic nucleosides such as 5-ethyl-2′-deoxyuridine and 5-trifluorothymidine has been carried out for the first time. Importantly for its potential application, SLrNDT4 could be recycled for 26 consecutive batch reactions in the synthesis of 2,6-diaminopurine-2′-deoxyriboside with negligible loss of catalytic activity.     

Chitosanases in the autolysis of Mucor rouxii

The mycelium of Mucor rouxii reached a 50% degree of lysis after 50 days incubation, and was then stable with the incubation time. The pH of the medium was 4.3 when autolysis began, rising to pH 7.6 after 6 days of autolysis and remaining there for the duration of the experiment. Maximum degradation of mycelium occurs during the first days of autolysis. Glucosamine is present in the culture liquid during all the autolytic process. Enzymes implicated in the degradation of chitosan and chitin were studied in the culture fluid during autolysis. An exochitosanase activity was detected after a day of autolysis, and its activity increased during 20 days of autolysis and afterwards remained constant until the end of the process. An endochitosanase activity was detected in the culture fluid from the beginning of the autolysis, having its maximum activity after 34 days of incubation. Both activities show an optimum pH of 5.5, but the pH range of activity for endochitosanase was broader than for exochitosanase. Both activities were not inhibited by 0.5 mM glucosamine. Activities of the enzymes B-N-acetylglucosaminidase and chitinase were not found. The chitosan content in the cell walls decreased with the incubation time. In these cell walls the chitin content experienced an increase at the beginning of the autolysis, decreasing afterwards. The enzymatic complex obtained from autolyzed cultures of M. rouxii hydrolyzed 2-day-old cell walls of this fungus. The hydrolysis was 21% after 24 h of incubation, liberating glucose and glucosamine. As a consequence protoplasts from M. rouxii germinated spores were obtained with its own lytic enzymes in adequate osmotic conditions. The involvement of chitosanases in the autolysis of this fungus have been studied.     

Hydrolysis of vegetable oils and triglycerides by thermotolerant and zoopathogenic species ofAspergillus from Nigerian palm produce

The ability ofAspergillus fumigatus Fres. andAspergillus nidulans (Eidam) Wint obtained from Nigerian palm produce to degrade vegetable oils and triglycerides and the production and activity of their extracellular lipases were studied. Both species readily hydrolysed palm oil and palm kernel oil among others liberating free fatty acids in the process. Good growth with mycelia production of both fungi were also recorded on the triglycerides used as sources of carbon at 37 °C with the best results obtained on palmitic and oleic acids, the predominant fatty acids in palm oil. Extracellular lipases were detected in the culture filtrates of both fungi within 48 h of incubation on an oat-meal chaff medium at 37 °C. Peak enzyme production occurred within the 10-day incubation period. The upases of both fungal species were most active at a pH of 5.6 and a temperature of 45 °C. The best glyceride for assaying the lipase activities of these fungi was trihexanoin while palm oil was a better vegetable oil than the conventional groundnut oil used for the same purpose. Because of the zoopathogenic nature of these fungi, attention is drawn to the potential health risks which their presence on the palm products where they were obtained pose to the consumers.     

The effect of exogenous phosphate deficiency on the activity of acid phosphatase of the root of two maize genotypes

In two maize genotypes, the effect of exogenous phosphate deficiency on acid phosphatase activity of the apical part of primary root was followed in dry and imbided grains. Higher acid phosphatase activity was found in the genotype LG 5. The enzyme activity increased after 18 h grain imbibition in the two genotypes. After 48 h germination no differences were found between the genotypes. After 24 h cultivation of root segments in a solution of 0.25 mM CaSO₄.2H₂O,0.1 mM MgSO₄.7H₂O, and further after 3,6 and 24 h cultivation in solutions with and without phosphate genotypic differences were found in acid phosphatase activity as well as in dry mass production. An increase in enzymatic activity due to exogenous phosphate deficiency was registered in the two genotypes only after 24 h cultivation.     

Ethanol production from hydrolysed agricultural wastes using mixed culture of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> and <Emphasis Type="Italic">Candida tropicalis</Emphasis>

Acid, alkaline and enzymatic hydrolysis of agricultural crop wastes were compared for yields of total reducing sugars with the hydrolysates being evaluated for ethanol production using a mixed culture of Zymomonas mobilis and Candida tropicalis. Acid hydrolysis of fruit and vegetable residues gave 49–84 g reducing sugars l⁻¹ and 29–32 g ethanol l⁻¹ was then obtained. Alkaline hydrolysis did not give significant amount of reducing sugars. Enzymatic hydrolysis of fruit and vegetable residues yielded 36–123 g reducing sugars l⁻¹ and 11–54 g ethanol l⁻¹.     

The effect of crop rotation of six forest tree species on peat-bark substrate enzymatic activity

The paper focuses on the effect of a nine-year utilisation of the peat-bark substrate and crop rotation of six main forest tree species on changes in the substrate enzymatic activity during successive rotation cycles. The study was conducted in the forest nursery in the years 1989–1997. Seedlings of Scots pine Pinus sylvestris, Norway spruce Picea abies, European larch Larix decidua, pendiculate oak Quercus robur, common beech Fagus silvatica, and silver birch Betula overrucosa were grown on peat-bark substrate. The activity of soil enzymes: betaglucosidase, invertase, urease, asparginase, acid phosphatase and dehydrogenases was assessed. The succession of three 3-year crop rotation cycles with species following each other according to the rotation plan was subject to observations. The obtained results have confirmed recent suppositions that the tree species and their rotation modify soil enzymatic activity. The enzymatic activity of the peat-bark substrate changed after each three-year crop rotation cycle and decreased with time. After the second crop rotation cycle the activity of betaglucosidase, urease, asparginase was found to be lower, and the activity of invertase and dehydrogenases — higher. After three crop rotation cycles the positive effect of appropriate species rotation on the enzymatic activity of the substrate was noted.     

Indigestible dextrin stimulates glucoamylase production in submerged culture of <Emphasis Type="Italic">Aspergillus kawachii</Emphasis>

Submerged batch cultures of Aspergillus kawachii grown on indigestible dextrin were investigated for potential improvements in glucoamylase (GA) production. In flask culture, specific GA productivities per dry weight biomass using dextrin and indigestible dextrin were 11.0 and 56.1 mU/mg-DW, respectively. Indigestible dextrin was a poor substrate for enzymatic hydrolysis. Rates of glucose formation from dextrin and indigestible dextrin by enzymatic hydrolysis were 0.477 and 0.100 mg-glucose/ml/h, respectively. For this reason, residual glucose concentrations in batch cultures grown on indigestible dextrin remained below 1.32 mg/ml where glucose-limiting conditions were easily maintained. Batch culture using indigestible dextrin had the same residual glucose profile as dextrin fed-batch culture, and nearly the same GA activity was obtained after 42.5 h of growth. However, between 42.5 and 66 h, the GA production rate of the indigestible dextrin batch culture (11.5 mU/ml/h) was higher than that of the dextrin fed-batch culture (6.5 mU/ml/h). During this period, a high amount of residual maltooligosaccharide was detected in the culture supernatant grown on indigestible dextrin. The high GA productivity observed in the indigestible dextrin batch culture may have resulted from the absence of glucose and the simultaneous presence of maltooligosaccharides throughout growth.     

Antimicrobial activity of broccoli (Brassica oleracea var. italica) cultivar Avenger against pathogenic bacteria,phytopathogenic filamentous fungi and yeast

Aims

The objective of this study was to show whether the edible part of broccoli has antibacterial and antifungal activity against micro‐organism of importance in human health and vegetable spoilage, and to test if this effect was partially due to antimicrobial peptides (AMPs).

Methods and Results

Crude extracts were obtained from florets and stems of broccoli cultivar Avenger and the inhibitory effect was demonstrated against pathogenic bacteria (Bacillus cereus, Staphylococcus xylosus, Staphylococcus aureus, Shigella flexneri, Shigella sonnei, Proteus vulgaris), phytopathogenic fungi (Colletotrichum gloeosporioides, Asperigillus niger) and yeasts (Candida albicans and Rhodotorula sp.). It was shown that samples treated with proteolytic enzymes had a reduction of approximately 60% in antibacterial activity against Staph. xylosus, suggesting that proteinaceous compounds might play a role in the inhibitory effect. Antimicrobial components in crude extracts were thermoresistant and the highest activity was observed under acidic conditions. It was shown that antifungal activity of broccoli's crude extracts might not be attributed to chitinases.

Conclusions

Organic broccoli cultivar Avenger has antimicrobial activity against pathogenic bacteria, yeast and phytophatogenic fungi. Data suggest that this effect is partially due to AMPs.

Significance and Impact of the Study

Broccoli's crude extracts have activity not only against pathogenic bacteria but also against phytophatogenic fungi of importance in agriculture. We suggest for first time that the inhibitory effect is probably due to AMPs.     

Reaction Kinetics of Immobilized α-Chymotrypsin in Organic Media 1. Influence at Solvent Polarity

Esterification of N-acetyl phenylalanine with ethanol catalyzed by immobilized α-chymotrypsin (EC 3.4.21.1) was studied in various reaction media. The effect of reaction medium polarity on enzymatic activity as well as equilibrium yield was measured. The reaction rate increased with increasing amounts of added water, reaching an optimum corresponding to water saturation of the reaction medium. Further additions of water resulted in decreased activity. Bell-shaped activity profiles were obtained for all reaction media tested. Reaction media consisting of pure solvents and of mixtures of solvents were used. The enzymatic activity and the equilibrium yield increased with decreased polarity of the medium. Maximum activity was found in a reaction medium consisting of 80% diisopropyl ether and 20% heptane. The enzymatic activity obtained at optimal water additions in the different solvents and solvents mixtures could be correlated to the solubility of water and the log P of the medium. The equilibrium yield of the reaction was much more closely correlated to the solubility of water than the log P. Much lower enzymatic activity was obtained when solvent mixtures producing water-miscible media were created, than in mixtures producing water-immiscible media, such as mixtures of acetonitrile and diisopropyl ether. The equilibrium yield could be increased by decreasing the water content in the reaction medium, which reduced the water activity.     

Effect of yeast cell disruption on ADH activity for redox reactions with in situ cofactor regeneration in a continuous solid/gas bioreactor

It has recently been demonstrated that dried cells of Saccharomyces cerevisiae were able to produce alcohols and aldehydes in a solid/gas reactor with in situ cofactor regeneration. Since diffusion of gaseous substrates may be limited by the membrane and cell wall, cell disruption by sonication was used to improve oxidoreduction with ethanol and butyraldehyde as substrates. Results showed that partial cell disruption enhances the maximum conversion yield with the best results obtained after 2 min of sonication. Beyond this time, the ADH activity decreased. Better stability was observed in the pellet obtained after centrifugation indicating the importance of cell environment for enzyme stability. Tests on purified mitochondria showed that the ADH activity in cells was mainly cytoplasmic. The addition of oxidized cofactor did not change either the activity or the stability of the catalyst in a gaseous medium. The effect of water activity was studied on material obtained after 2 min of disruption and a reduction of critical water activity needed for revealing enzymatic activity was observed. With increasing a_w, the enzyme was active at a_w=0.3 while a water activity of 0.4 was required before disruption. Nevertheless, the best compromise between activity and stability was obtained in both cases for a water activity of 0.57.     

Fusion of yeast protoplasts and isolated nuclei of Fusarium moniliforme

Protoplasts of a xylose-fermenting yeast strain (a fusion product of Pachysolen tannophilus and Saccharomyces cerevisiae) were fused with isolated nuclei of the xylan degrading filamentous fungus Fusarium moniliforme. Polyethyleneglycol 4000 was used as the fusogenic agent. Fourteen stable hybrids showing xylanase activity were obtained. It can be assumed that this ability was acquired from the nuclear genome of the fungus, since the parental yeast strain did not show any xylanase activity. The enzymatic activity was determined quantitatively. The parental strain of the fungus reached its maximum xylanase activity of 796 nkat/ml at 96 h of growth. Four of the hybrids had a xylanase activity of between 211 and 297 nkat/l at 24 h of growth. Zymograms of these hybrids showed the presence of xylanases when grown on xylan as the sole carbon source. Using pulse field electrophoresis gels, no difference between the chromosome pattern of the fusion products and the parental yeast strain was observed.     

In vitro refolding process of urea-denatured microbial transglutaminase without pro-peptide sequence

Efficient refolding process of denatured mature microbial transglutaminase (MTG) without pro-peptide sequence was studied in the model system using urea-denatured pure MTG. Recombinant MTG, produced and purified to homogeneity according to the protocol previously reported, was denatured with 8M urea at neutral pH and rapidly diluted using various buffers. Rapid dilution with neutral pH buffers yielded low protein recovery. Reduction of protein concentration in the refolding solution did not improve protein recovery. Rapid dilution with alkaline buffers also yielded low protein recovery. However, dilution with mildly acidic buffers showed quantitative protein recovery with partial enzymatic activity, indicating that recovered protein was still arrested in the partially refolded state. Therefore, we further investigated the efficient refolding procedures of partially refolded MTG formed in the acidic buffers at low temperature (5 degrees C). Although enzymatic activity remained constant at pH 4, its hydrodynamic properties changed drastically during the 2h after the dilution. Titration of partially refolded MTG to pH 6 after 2h of incubation at pH 4.0 improved the enzymatic activity to a level comparable with that of the native enzyme. The same pH titration with incubation shorter than 2h yielded less enzymatic activity. Refolding trials performed at room temperature led to aggregation, with almost half of the activity yield obtained at 5 degrees C. We conclude that rapid dilution of urea denatured MTG under acidic pH at low temperature results in specific conformations that can then be converted to the native state by titration to physiological pH.