Differences In Enzyme Production Research Articles

Elucidating Thermothielavioides terrestris secretome changes for improved saccharification of mild steam-pretreated spruce

BackgroundThe efficient use of softwood in biorefineries is hampered by its recalcitrance to enzymatic saccharification. In the present study, the fungus Thermothielavioides terrestris LPH172 was cultivated on three steam-pretreated spruce materials (STEX180°C/auto, STEX210°C/auto, and STEX210°C/H2SO4), characterized by different hemicellulose content and structure, as well as on untreated biomass. The aim of the study was to map substrate-induced changes in the secretome of T. terrestris grown on differently treated spruce materials and to evaluate the hydrolytic efficiency of the secretome as supplement for a commercial enzyme mixture.ResultsThe cultivation of T. terrestris was monitored by endo-cellulase, endo-xylanase, endo-mannanase, laccase, and peroxidase activity measurements. Proteomic analysis was performed on the secretomes induced by the spruce materials to map the differences in enzyme production. Growth of T. terrestris on STEX180°C/auto and STEX210°C/auto induced higher expression level of mannanases and mannosidases of the GH5_7 CAZy family compared to cultivation on the other materials. Cultivation on untreated biomass led to overexpression of GH47, GH76, and several hemicellulose debranching enzymes compared to the cultivation on the pretreated materials. T. terrestris grown on untreated, STEX180°C/auto and STEX210°C/auto induced three arabinofuranosidases of the GH43 and GH62 families; while growth on STEX210°C/H2SO4 induced a GH51 arabinofuranosidase and a GH115 glucuronidase. All secretomes contained five lytic polysaccharide monooxygenases of the AA9 family. Supplementation of Celluclast® + Novozym188 with the secretome obtained by growing the fungus grown on STEX180°C/auto achieved a twofold higher release of mannose from spruce steam-pretreated with acetic acid as catalyst, compared to the commercial enzyme cocktail alone.ConclusionsMinor changes in the structure and composition of spruce affect the composition of fungal secretomes, with differences in some classes explaining an increased hydrolytic efficiency. As demonstrated here, saccharification of spruce biomass with commercial enzyme cocktails can be further enhanced by supplementation with tailor-made secretomes.

Evaluation of the operating conditions for the solid-state production of pectinases by Aspergillus niger in a bench-scale, intermittently agitated rotating drum bioreactor

Pectinase production by Aspergillus niger LB-02-SF was studied in a bench-scale rotating drum bioreactor. With 30% of the bioreactor volume occupied, agitation promoted increasing fungal growth and decreasing enzyme production, whereas with 50% occupation, an opposite trend occurred. The interval between agitation events was evaluated for an occupation volume of 70%, and no difference in enzyme production was observed for the 90 and 120 min intervals; for the 60 min intervals, however, the activity was reduced. Cultivations performed without temperature control attained 45 °C and showed maximum enzyme activity almost four times superior to cultivations controlled at 30–37 °C. Air flow rates between 0.18 and 0.90 L of air per kilogram of medium per minute were evaluated for 30 and 50% of the occupation volume. Fungal growth and pectinase production were favoured by the smallest and largest occupation volumes, respectively, with an intermediate air flow. The results of this work indicate that the best conditions for biomass growth are not the best for the production of pectinases.

English

Halophilic bacteria are a group of salt tolerant microorganisms; the production of enzymes by halophilic bacteria, due to their activities in harsh environments, is considered as a major field of investigation. Hence, the present study was conducted for the isolation of halothermophilic bacteria from Howz Soltan salt lake. For this purpose 40 samples were collected from five regions of the Howz Soltan lake. The samples were diluted and cultivated on Molten haloid agar (MH) with different salt concentrations (5-35%). Then the plates were incubated at 35-70°C in aerobic conditions for 7-10 days. The strains were screened for amylase production based on differences in enzyme production. In total 205 strains were grown on media including 5-15% salt concentrations. Of all, 18 strains were hyperhalophilic and could grow on media including 20-35% salt concentrations at 40-70°Ði. Three (3) isolates were identified as best amylase producers. In addition, the results indicated that maximum enzyme activity was observed at 48 h (18.145 U/ml). The optimal pH, temperature and NaCl concentration for amylase production were 7.0-7.5, 40°C and 15% (2.5 M), respectively. Taxonomic and phylogenetic analysis using 16SrRNA gene sequencing revealed that they belonged to Bacillus licheniformis and Gracilibacillus while the third isolate was closer to β proteobacterium. Key words: Halothermophilic bacteria, amylase, Howz Soltan Lake.

Production of intracellular β-xylosidase from the submerged fermentation of citrus wastes by Penicillium janthinellum MTCC 10889.

Production of intracellular β-xylosidase was studied in cultures of Penicillium janthinellum grown on citrus fruit waste supplemented cultivation media. Both dried orange peel and sweet lime peel could induce the production of this enzyme. The working strain showed a pronounced optimum pH and temperature for β-xylosidase production at 6.0 and 27 °C, respectively. The enzyme production was found to remain stable for a long period of 120 h. Orange peel and sweet lime peel showed different responses in the presence of various nitrogen sources, probably due to their differences in hemicellulosic contents. This could be further confirmed by the difference in enzyme production after pretreatment with acid and alkali.

Enzyme activities of fungi associated with Picea abies needles

Abstract Decomposition of Picea abies needles and production of extracellular enzymes involved in decomposition of lignin, cellulose, hemicelluloses and other organic compounds were studied in fungal strains of interior needle colonizers isolated from needles in different stages of decomposition (attached to trees, and early and late decomposition stages in the litter horizon). In total, 12 strains of ascomycetes (members of Helotiales, Hypocreales, Dothideales, Diaporthales and Eurotiales) and four basidiomycetes (Polyporales, Agaricales and Russulales) were tested. Significant decomposition of needles was recorded for all fungal isolates. All isolates produced cellobiohydrolase, β-glucosidase, β-xylosidase, N-acetylglucosaminidase, α-glucosidase, phosphatase and arylsulfatase and most fungi also produced endocellulase, endoxylanase and laccase in needle litter. In addition, other hemicellulases were produced by all strains. Mn-peroxidase was only produced by two basidiomycetes. Although enzyme activities varied, fungi associated with needles on fallen trees exhibited enzyme production comparable with later litter colonizers, and there was no significant difference in enzyme production between ascomycete and basidiomycete strains.

Ecology of coarse wood decomposition by the saprotrophic fungus Fomes fomentarius

Saprotrophic wood-inhabiting basidiomycetes are the most important decomposers of lignin and cellulose in dead wood and as such they attracted considerable attention. The aims of this work were to quantify the activity and spatial distribution of extracellular enzymes in coarse wood colonised by the white-rot basidiomycete Fomes fomentarius and in adjacent fruitbodies of the fungus and to analyse the diversity of the fungal and bacterial community in a fungus-colonised wood and its potential effect on enzyme production by F. fomentarius. Fungus-colonised wood and fruitbodies were collected in low management intensity forests in the Czech Republic. There were significant differences in enzyme production by F. fomentarius between Betula pendula and Fagus sylvatica wood, the activity of cellulose and xylan-degrading enzymes was significantly higher in beech wood than in birch wood. Spatial analysis of a sample B. pendula log segment proved that F. fomentarius was the single fungal representative found in the log. There was a high level of spatial variability in the amount of fungal biomass detected, but no effects on enzyme activities were observed. Samples from the fruiting body showed high β-glucosidase and chitinase activities compared to wood samples. Significantly higher levels of xylanase and cellobiohydrolase were found in samples located near the fruitbody (proximal), and higher laccase and Mn-peroxidase activities were found in the distal ones. The microbial community in wood was dominated by the fungus (fungal to bacterial DNA ratio of 62-111). Bacterial abundance composition was lower in proximal than distal parts of wood by a factor of 24. These results show a significant level of spatial heterogeneity in coarse wood. One of the explanations may be the successive colonization of wood by the fungus: due to differential enzyme production, the rates of biodegradation of coarse wood are also spatially inhomogeneous.

Changes in enzyme activities and soil microbial community composition along carbon and nutrient gradients at the Franz Josef chronosequence, New Zealand

During primary succession, the abundance of carbon (C) and nitrogen (N) in soil increases, while phosphorus (P) declines. These changes in nutrient concentrations in organic matter are likely to play an important role in controlling enzyme-mediated nutrient mineralization. We examined how enzyme activity and efficiency changed with successional time in organic and mineral soils taken from the 120 000-year-old Franz Josef soil development sequence, New Zealand, and the relationship between enzyme activity and efficiency and soil nutrient concentrations. We found that the activity of enzymes involved in P mineralization increased with site age across the Franz Josef chronosequence, while the activity of enzymes regulating C and N mineralization declined in organic but not mineral soil. Sulfatase activity peaked at an intermediate-aged site, possibly indicating a transient period of S limitation. The activity of phosphatase enzymes was negatively correlated with the concentration of P in the soil, whereas activity of C-, N- and S-hydrolyzing enzymes was not strongly dependent on nutrient concentrations. When assessed as efficiency (activity per unit microbial biomass), there were strong patterns of increasing efficiency of P-, and decreasing efficiency of C- and N-hydrolyzing enzymes with site age. We suggest that activity patterns for C-, N- and S-hydrolyzing enzymes were obscured by simultaneous and opposing changes in enzyme efficiency and microbial biomass. In mineral soil, efficiency of enzymes was negatively correlated with soil nutrient availability. In contrast, in organic soil, efficiency of C-, N- and S-hydrolyzing enzymes was positively correlated with soil P, while efficiency of P-hydrolyzing enzymes was negatively correlated with soil P. The increase in efficiency of P-hydrolyzing enzymes, and decrease in efficiency of C-, N- and S-hydrolyzing enzymes with site age was accompanied by a shift in microbial community composition towards higher relative abundances of fungi. Changes in enzyme efficiency with site age are likely to be due to both constitutive differences in enzyme production, and down-regulation of enzyme expression.

Cell type-specific inhibition of keratinocyte collagenase-1 expression by basic fibroblast growth factor and keratinocyte growth factor. A common receptor pathway.

Collagenase-1 is invariantly expressed by migrating basal keratinocytes in all forms of human skin wounds, and its expression is induced by contact with native type I collagen. However, net differences in enzyme production between acute and chronic wounds may be modulated by soluble factors present within the tissue environment. Basic fibroblast growth factor (bFGF, FGF-2) and keratinocyte growth factor (KGF, FGF-9), which are produced during wound healing, inhibited collagenase-1 expression by keratinocytes in a dose-dependent manner. However, KGF was >100-fold more effective than bFGF at inhibiting collagenase-1 expression, suggesting that this differential signaling is transduced via an FGF receptor that binds these ligands with different affinities. Reverse transcriptase-polymerase chain reaction analysis of human keratinocyte mRNA for fibroblast growth factor receptors (FGFRs) revealed expression of only FGFR-2 IIIb, the KGF-specific receptor, which also binds bFGF with low affinity, and FGFR-3 IIIb, which does not bind bFGF or KGF. FGFRs that bind bFGF with high affinity were not detected. Our results suggest that bFGF and KGF inhibit collagenase-1 expression through the KGF cell-surface receptor (FGFR-2 IIIb). Because bFGF induces collagenase-1 in most cell types, cell-specific expression of FGFR family members may dictate the regulation of matrix metalloproteinases in a tissue-specific manner.

Catalase, Superoxide Dismutase and Listeriolysin O Production by Listeria monocytogenes in Broth Containing Acetic and Hydrochloric Acids

Cells of Listeria monocytogenes 10403S were propagated at 37°C in media acidified with either acetic or hydrochloric acid to determine the effect on the production of catalase (CA), superoxide dismutase (SOD) and listeriolysin O (LLO). The CA and LLO activities decreased while SOD activity increased as the pH of the growth media was decreased. Comparison of the acids indicated that neither acid caused significant differences in enzyme production except for SOD activity at pH 5.7. These results suggest that growth of L. monocytogenes in acid environments may influence the production of these enzymes, while growth in strong acids versus weak acids may not be significantly different.

Proteolytic activity in black-pigmented bacteroides species

Black-pigmented Bacteroides species are frequently found in dentoalveolar abscesses. One general mechanism of bacterial virulence is the production of extracellular enzymes which degrade connective tissue or molecules associated with host defense. In this study the proteolytic activity of 18 bacterial strains from 9 black-pigmented Bacteroides species was examined. Bacteroides gingivalis degraded the greatest number of substrates studied and produced the highest levels of enzymatic activity. B. gingivalis was the only species that degraded collagen and produced high levels of enzymes that degraded N-benzoyl-DL-arginine (BANA) and N-CBz-glycyl-glycyl-arginine. Bacteroides intermedius degraded several substrates including PZ peptide. Bacteroides endodontalis produced enzymes that degraded beta-naphthylamide derivatives of glycylproline and glycylphenylalanine. There were considerable differences in enzyme production between strains of the same species. Such heterogeneity between strains in the production of proteolytic enzymes may be relevant to the in vivo infections produced in the host.

Heterogeneity of virulence among strains of Bacteroides gingivalis

The ability of fresh isolates of B. gingivalis to establish abscesses in the mouse model was studied by comparing them with established laboratory strains of B. gingivalis. Eight fresh isolates obtained from plaque associated with periodontal disease and grown under similar conditions as established strains were injected subcutaneously on the back of the mouse. All of these strains produced secondary lesions on the abdomen. Septicemia was associated with seven of the strains. Two commonly used laboratory strains, W50 and W83, also produced secondary lesions and septicemia. Five other laboratory strains produced only localized abscesses. On histologic examination, the strains that produced disseminated disease showed invasion of connective disease by individual bacteria that were not in clumps. The strains that produced localized abscesses were characterized by growing in colonies or clumps in the abscess cavity. Four synthetic enzyme substrates were examined to determine whether the differences between invasive and non-invasive strains were due to differences in proteolytic enzyme production. No differences in enzyme production could be demonstrated with the selected substrates.

Production of polysaccharide degrading enzymes by Cochliobolus Sativus and Fusarium Culmorum grown in liquid culture

Cochliobolus sativus and Fusarium culmorum produced pectin degrading enzymes, xylanase and carboxymethylcellulase when grown in a salts medium with an appropriate carbon source. There was little difference in enzyme production by the two organisms except for the greatly enhanced endo-polymethylgalacturonase activity when F. culmorum was grown with citrus pectin as the carbon source. When grown on powdered wheat straw, xylanase and carboxy-methylcellulase activities were not present until the late stages of incubation. It is suggested that degradation of pectin and xylan may be as important as cellulolysis in the saprophytic survival of these organisms.

Cystine requirement of continuous human lymphoid cell lines of normal and leukemic origin.

A number of continuous, human lymphoid cell lines from healthy and leukemic donors have been studied. Those from healthy individuals were found to be cystine prototrophs (cys+), while those from leukemic donors were observed to be cystine auxotrophs (cys-). The former can grow in either cystine or cystathionine-containing media. The latter grow only in cystine-containing medium. The former contain readily measurable levels of cystathionase, the last enzyme in the mammalian cysteine biosynthetic pathway, while the latter contain much less of this activity. The apparent molecular weight, overall charge, pH versus activity, Km, and [pyridoxal phosphate] versus activity properties of the enzyme from both cell types were indistinguishable. No soluble cystathionase inhibitor could be detected in cys- cell lines. Thus, it is reasonable to hypothesize that those leukemic cell lines studied synthesize less active cystathionase protein than their cys+ counterparts and that this difference in enzyme production underlies the differential growth properties in cystathionine-containing medium.