Effect of aeration on lignin peroxidase production by Streptomyces viridosporus T7A

The effect of aeration on lignin peroxidase production by Streptomyces viridosporus T7A was studied in a bench-scale bioreactor using a previously optimized growth medium (0.65% yeast extract and 0.1% corn oil, pH7.0) at 37°C and natural pH. Airflow rates of 0.3, 1.0, and 1.5 vvm and a fixed agitation of 200 rpm were initially studied followed by 1.0 vvm and 200, 300, 400, and 500 rpm. The use of 1.0 vvm and 400 rpm increased enzyme concentration 1.8-fold (100–180 U/L) and process productivity 4.8-fold (1.4–6.7 U/[L·h]) in comparison with the use of 200 rpm and 0.3 vvm. The inexpensive corn oil, used as carbon source, besides its antifoam properties, proved to be nonrepressive for enzyme production.     

Lignin Peroxidase from Streptomyces viridosporus T7A: Enzyme Concentration Using Ultrafiltration

It is well known that lignin degradation is a key step in the natural process of biomass decay whereby oxidative enzymes such as laccases and high redox potential ligninolytic peroxidases and oxidases play a central role. More recently, the importance of these enzymes has increased because of their prospective industrial use for the degradation of the biomass lignin to increase the accessibility of the cellulose and hemicellulose moieties to be used as renewable material for the production of fuels and chemicals. These biocatalysts also present potential application on environmental biocatalysis for the degradation of xenobiotics and recalcitrant pollutants. However, the cost for these enzymes production, separation, and concentration must be low to permit its industrial use. This work studied the concentration of lignin peroxidase (LiP), produced by Streptomyces viridosporus T7A, by ultrafiltration, in a laboratory-stirred cell, loaded with polysulfone (PS) or cellulose acetate (CA) membranes with molecular weight cutoffs (MWCO) of 10, 20, and 50 KDa. Experiments were carried out at 25 °C and pH 7.0 in accordance to the enzyme stability profile. The best process conditions and enzyme yield were obtained using a PS membrane with 10 KDa MWCO, whereby it was observed a tenfold LiP activity increase, reaching 1,000 U/L and 90% enzyme activity upholding.     

Lignin peroxidase and protease production by Streptomyces viridosporus T7A in the presence of calcium carbonate

Streptomyces are good producers of enzymes of industrial interest, such as lignin peroxidase (LiP) and proteases. To optimize production of these enzymes by Streptomyces viridosporus T7A, two parameters were evaluated: carbon sources and calcium carbonate. Shake-flask fermentations were performed using culture media, with and without CaCO₃, contained yeast extract, mineral salts and either glucose, lactose, galactose, or corn oil. In the absence of calcium carbonate, the maximum values for LiP and protease activities occurred during the idiophase with LiP activity being favored by glucose, corn oil, and galactose, and protease activity being favored only by corn oil. Calcium carbonate affected the cell morphology by reducing the size of the pellets. Moreover, in the presence of the salt, LiP production was growth-associated in all media but the glucose medium. Higher enzyme levels were observed when galactose and glucose were used as carbon sources. Protease activity was repressed by both glucose and galactose, whereas corn oil was the best carbon source for the enzyme production. Calcium carbonate increased LiP production by up to 2.6-fold. Such improvement was not observed for protease production, suggesting a selective effect of CaCO₃ on LiP activity.     

Lignin biotransformations by an aromatic aldehyde oxidase produced byStreptomyces viridosporus T7A

Streptomyces viridosporus produces an intracellular aromatic aldehyde oxidase that oxidizes aromatic and α, β-unsaturated aromatic aldehydes to their corresponding acids. It also produces extracellular oxidase as shown by zones of clearing when grown on agar containing insoluble dehydrodivanillin (DHDV). This extracellular form may be responsible for oxidizing aldehyde groups in lignin. The extracellular oxidase was expressed maximally after 3 d growth in medium containing only yeast extract. However, higher levels were produced when lignocellulose was in the medium. The enzyme was partially purified and its molecular weight was approximated to be about 80,000 daltons. Mutant cultures that had lost the ability to produce zones of clearing on DHDV-containing agar solubilized smaller quantities of lignin as compared to the wild type, except for one strain. A partially purified oxidase preparation was shown to oxidize a natural lignocellulose substrate.     

Optimization of culture medium and conditions for penicillin acylase production by streptomyces lavendulae ATCC 13664

The culture medium for Streptomyces lavendulae ATCC 13664 was optimized on a shake-flask scale by using a statistical factorial design for enhanced production of penicillin acylalse. This extracellularenzyme recently has been reported to bea penicillin Kacylase, presenting also high hydrolytic activity against penicillin V and other natural aliphatic penicillins such as penicillin K, penicillin F, and penicillin dihydroF,. The factorial design indicated that the main factors that positively affect penicillin acylase production by S. lavendulae were the concentration of yeast extract and the presence of oligoelements in the fermentation medium, whereas the presence of olive oil in the medium had no effect on enzyme production. An initial concentration of 2.5% (w/v) yeast extract and 3 μg/mL of CuSO₄·5H₂O was found to be best for acylase production. In such optimized culture medium, fermentation, of the microorganism yielded 289 IU/L of enzyme in 72 h when employing a volume medium/volume flask ratio of 0.4 and a 300-rpm shaking speed. The presence of copper, alone and in combination with other metals, stimulated biomass as well as penicillin acylase production. The time course of penicillin acylase production was also studied in the optimized medium and conditions. Enzyme production showed catabolite repression by different carbon sources such as glucose, lactose, citrate, glycerol, and glycine.     

In vitro degradation of insoluble lignin in aqueous media by lignin peroxidase and manganese peroxidase

The abilities of lignin peroxidase (LIP) and manganese peroxidase (MNP) fromPhanerochaete chrysosporium to degrade an insoluble hardwood lignin in vitro in aqueous media were tested. Neither LIP nor MNP appreciably changed the mass or lignin content, although both produced small amounts of unique solubilized lignin fragments. Treatment with both LIP and MNP, however, decreased the mass by 11%, decreased the lignin content by 5.1% (4.2% as total weight), and solubilized unique lignin-derived molecules. These results suggest that LIP and MNP synergistically degrade high molecular weight insoluble lignin, but singly, neither enzyme is sufficient to effect lignin degradation.     

Protease production by Streptomyces sp. isolated from brazilian cerrado soil

Streptomyces are important microorganisms because of their capacity to produce numerous bioactive molecules. In the present work protease production, by Streptomyces sp. 594 isolated from a Brazilian Cerrado soil, was maximized by optimizing a low-cost culture medium composition (casitone and sugarcane molasses) using statistical experimental design. The final protease activity (56 U/mL) was 2.8-fold and 58-fold higher than that obtained in the beginning of this study, and in a previous work, using an actinomycete selection medium, respectively. Protease production, not growth associated, appeared to be modulated by an inducer system, whereby the C/N ratio seemed to play a significant role.     

Importance of C-Terminal Region for Thermostability of GH11 Xylanase from Streptomyces lividans

The amino acid sequences of xylanase B (XlnB) and xylanase C (XlnC) from Streptomyces lividans show significant homology. However, the temperature optima and stabilities of the two enzymes are quite different. XlnB exhibits an optimum temperature of 40 °C and retains 50% of its maximum activity at 43 °C, whereas the corresponding values for XlnC are 60 and 70 °C. To analyze these properties further, as well as to study the effect of the exchange of homologous segments in the C-terminal region, four chimeras designated as BSC, BFC, CSB, and CFB were constructed by substituting segments from the C-terminal homologous region of XlnB gene with that of XlnC and in turn substituting XlnC gene with that of XlnB. The purified chimeric enzymes were characterized with respect to pH/temperature activity, stability, and kinetic parameters. Most of enzymatic properties of chimeras were admixtures of those of the two parents. The chimeric enzymes were optimally active at 45–55 °C and pH 7.0. Both K _m and k _cat values of chimeric enzymes for p-nitrophenyl-β-d-cellobioside were admixtures of both parental enzymes, except that the k _cat value of chimeric BFC (2.79 s⁻¹) was higher than that of parental XlnC (1.99 s⁻¹). Notably, thermal stability of chimeric BSC and BFC was increased by 25 and 13 °C separately, as compared to one of parental XlnB, whereas the thermal stability of chimeric CSB and CFB was decreased by 23 and 21 °C, respectively, as compared to another parental XlnC. These results suggest that homologous C-terminal region in S. lividans GH11 xylanase appears to play an important role in determining enzyme characteristics, and exchanging of different segments of gene in this region might significantly alter or improve the enzymatic properties such as thermal stability.     

Production of C-terminal amidated recombinant salmon calcitonin in Streptomyces lividans

Salmon calcitonin (sCT) is one of the many bioactive peptides that require C-terminal amidation for full biologic activity. To produce fully bioactive sCT in large scale, we constructed Streptomyces lividans [pMSA], an engineering Streptomyces strain. In the expression vector, glycine-extended sCT, the substrate for amidation, and rat α-amidating enzyme cDNA were cloned under the control of the strong constitutive promoter from the Streptomyces fradiae aph gene in pIJ680. Both were expressed in a secretory manner by the recombinant strain using the expression and secretion signals of melC1. Extracellularly expressed recombinant sCT was purified to near homogeneity and characterized by enzyme immunoassay, followed by direct amino-terminal sequencing. High-performance liquid chromatography, matrixassisted laser desorption ionization-time-of-flight mass spectrometry, and bioassay in vivo demonstrated purified product to be equivalent to synthetic standard. Thus, the engineered Streptomyces strain can produce bioactive, C-terminal amidated recombinant sCT in the culture supernatant directly. The ease of the recombinant process, as well as its potential for scale-up, makes it adaptable to production demands for sCT, and it may be applied to other bioactive peptides that need C-terminal amidation.     

Use of steam explosion liquor from sugar cane bagasse for lignin peroxidase production by Phanerochaete chrysosporium

The possibility of using two by-products of the sugar cane industry, molasses and bagasse steam explosion liquor (SEL), for lignin peroxidase (LiP) production by Phanerochaete chrysosporium was investigated. For comparison, the fungus was initially cultivated in synthetic media containing either glucose, sucrose, xylose, or xylan as sole carbon sources. The effect of veratryl alcohol (VA) was also investigated in relation to the enzyme activity levels. Results showed that sucrose was not metabolized by this fungus, which precluded the use of molasses as a carbon source. Glucose, xylose, and xylan promoted equivalent cell growth. Enzyme levels in the absence of VA were lower than 28 UI/L and in the presence of VA reached 109 IU/L with glucose and 85 IU/L with xylose or xylan. SEL was adequate for P. chrysosporium LiP production as LiP activity reached 90 IU/L. When VA was added to this medium, enzyme concentration increased to 155 IU/L.     

Purification and Partial Characterization of Lignin Peroxidase from <Emphasis Type="Italic">Acinetobacter calcoaceticus</Emphasis> NCIM 2890 and Its Application in Decolorization of Textile Dyes

Lignin peroxidase was purified (72-fold) from Acinetobacter calcoaceticus NCIM 2890. The purified lignin peroxidase (55–65 kDa) showed dimeric nature. The maximum enzyme activity was observed at pH 1.0, between a broad temperature range of 50 and 70°C, at H₂O₂ concentration (40 mM) and the substrate concentration (n-propanol, 100 mM). Purified lignin peroxidase was able to oxidize a variety of substrates including Mn²⁺, tryptophan, mimosine, l-Dopa, hydroquinone, xylidine, n-propanol, veratryl alcohol, and ten textile dyes of various groups indicating as a versatile peroxidase. Most of the dyes decolorized up to 90%. Tryptophan stabilizes the lignin peroxidase activity during decolorization of dyes.     

Medium Optimization Based on Statistical Methodologies for Pristinamycins Production by <Emphasis Type="Italic">Streptomyces pristinaespiralis</Emphasis>

The optimization of nutrient levels for the production of pristinamycins by Streptomyces pristinaespiralis CGMCC 0957 in submerged fermentation was carried out using the statistical methodologies based on the Plackett–Burman design, the steepest ascent method, and the central composite design (CCD). First, the Plackett–Burman design was applied to evaluate the influence of related nutrients in the medium. Soluble starch and MgSO₄·7H₂O were then identified as the most significant nutrients with a confidence level of 99%. Subsequently, the concentrations of the two nutrients were further optimized using response surface methodology of CCD, together with the steepest ascent method. Accordingly, a second-order polynomial regression model was finally fitted to the experimental data. By solving the regression equation from the model and analyzing the response surface, the optimal levels for soluble starch and MgSO₄·7H₂O were determined as 20.95 and 5.67g/L, respectively. Under the optimized medium, the yield of pristinamycins in the shake flask and 5-L bioreactor could reach 1.30 and 1.01g/L, respectively, which is the highest yield reported in literature to date.     

Optimization of the Production Medium for Biosynthesis of Antifungal Antibiotic Ak-111-81 by Phosphate-Deregulated Mutant of <Emphasis Type="Italic">Streptomyces hygroscopicus</Emphasis>

Experimental mathematical designs were applied for optimization of a nutrient medium for biosynthesis of the antifungal antibiotic AK-111-81 by phosphate-deregulated mutant of Streptomyces hygroscopicus 111-81. Antifungal antibiotic AK-111-81 possesses well-expressed activity against Fusarium graminearum and other phytopathogenic fungi. The level of the production of the antibiotic AK-111-81 on this medium is more than three times higher than on the initial medium. The optimized quantitative composition of the nutrient culture media is (g/l): glucose, 20; soy meal, 18; ammonium succinate, 3; CaCO₃, 1.     

Influence of media nutrients on synthesis of lignin peroxidase from Aspergillus sp

The effect of carbon and nitrogen sources, lignocellulosic substrates, and metal ions on lignin peroxidase (LiP) activity of Aspergillus sp., which was isolated from a mangrove area, was studied. Glucose (1%) was found to be the best carbon source. Among the various lignocellulosic substrates used, coir pith at 3% concentration increased LiP activity twofold on the second day of incubation. Peptone and KNO₃ completely inhibited the enzyme synthesis while (NH₄)₂SO₄ at 12.5 mM produced maximum activity. Since seawater contained all the requisite metal ions, any added ions had a negative effect on activity. Cu²⁺ had the most inhibiting effect while K⁺ the least. When all the optimized conditions were provided, in nitrogen- and carbon-sufficient medium, a maximum LiP activity of 345 U/mL was obtained on the second day of incubation.     

Medium optimization for polysaccharide production of Cordyceps sinensis

As a potential anticarcinogenic agent, polysaccharides from Cordyceps sinensis have been demonstrated to possess strong antioxidation activity. The aim of the present research was to study the optimal medium to produce polysaccharides of C. sinensis by using response surface methodology (RSM). The composition of optimized medium for polysaccharide production calculated from the regression model of RSM was 6.17% sucrose, 0.53% corn steep powder, 0.5% (NH₄)₂HPO₄, and 0.15% KH₂PO₄ at pH 4.44, with a predicted maximum polysaccharide production of 3.17 g/L. When applying this optimal medium, the maximum polysaccharide production was 3.05 and 3.21 g/L in a shake flask and a 5-L jar fermentor, respectively. When the pH was controlled at a higher level such as pH 5.0, both cell growth and polysaccharide production were inhibited. A low pH of 2.85 was required for maximum production of polysaccharides.     

Effect of chitosan on peroxidase activity and isoenzyme profile in hairy root cultures of Armoracia lapathifolia

Hairy root cultures of Armoracia lapathifolia established by infection with Agrobacterium rhizogenes LBA 9402 present a level and isoenzyme pattern of peroxidases (POD) comparable to nontransformed roots. Elicitation with chitosan (10, 50, and 100 mg/L) was used in order to improve POD production. Total POD activity increased about 170% after 48h of treatment with chitosan 100 mg/L. Elicitation effect on soluble and ionically cell-wall-bound POD fractions of A. lapathifolia hairy roots was analyzed. POD activity of the ionically cell-wall-bound protein fraction increased in the presence of chitosan in a dose-response manner. No effect on soluble POD fractions was observed, but the isoenzyme pattern analyzed by isoelectrofocusing showed an increase of an acidic isoenzyme (pI=3.4) after the elicitation treatment. The ionically cell-wall-bound protein fraction showed only basic isoenzymes, with an increase of an isoenzyme of pI=8.7, after the elicitation treatment.     

Bacitracin production by a new strain ofBacillus subtilis

A new strain ofBacillus subtilis C 126 was isolated from sugar cane fermentation and produced an antibiotic that inhibited the growth ofMicrococcus flavus. The production of the antibiotic in culture medium followed to extraction withn-butanol, thin layer chromatography, and microbiological tests indicated that a polypeptide antibiotic was produced. The fraction obtained by Sephadex G-25 column and analyzed by HPLC indicated that bacitracin complex was produced.     

Biosurfactants production by Pseudomonas aeruginosa FR using palm oil

Biosurfactants production by a strain of Pseudomonas aeruginosa using palm oil as a sole carbon source was investigated. The experiments were carried out in 500-mL conical flasks containing 100 mL of mineral media supplemented with palm oil as the sole carbon source. The P. aeruginosa FR strain was able to reduce surface tension of three tested inorganic media. Rotation velocities from 100 to 150 rpm provided free-cell fermented media with the lowest surface tension of approx 33 mN/m. Emulsification index results of even 100% were achieved when diesel was used as oil phase. Eight surface-active compounds produced by the bacterium were identified by mass spectrometry.     

Mn2+ alters peroxidase profiles and lignin degradation by the white-rot fungus Pleurotus ostreatus under different nutritional and growth conditions

The white-rot fungus Pleurotus ostreatus produces two types of extracellular peroxidases: manganese-dependent peroxidase (MnP) and versatile peroxidase (VP). The effect of Mn²⁺ on fungal growth, peroxidase activity profiles, and lignin degradation by P. ostreatus was studied in liquid culture and under solid-state fermentation conditions on perlite, the latter resembling the natural growth conditions of this fungus. The fungus was grown in either a defined asparagine-containing basidiomycete selective medium (BSM) or in a rich peptone medium (PM). Biomass production, as determined by respiration experiments in solid-state fermentation and liquid cultures and fungal growth on Petri dishes, was higher in the PM than in the BSM. Mn²⁺ affected biomass production only in the PM on Petri dishes. In the nonamended PM, high levels of MnP and VP activity were detected relative to the nonamended BSM. Nevertheless, a higher rate of ¹⁴C-lignin mineralization was measured in the Mn²⁺-amended BSM, as determined during the course of 47 d of fermentation. Mn²⁺ amendment of the PM increased mineralization rate to that obtained in the Mn²⁺-amended BSM. The enzyme activity profiles of MnP and VP were studied in the BSM using anion-exchange chromatography. In the nonamended BSM, only minute levels of MnP and VP were detected. On Mn²⁺ amendment, two MnP isoenzymes (B1 and B2) appeared. Isoenzyme B2 was purified and showed 100% identity with the MnP isoenzyme purified in our previous study from PM-solid-state fermentation (P6). P6 was found to be the dominant isoenzyme in terms of activity level and gene expression compared with the VP isoenzymes. Based on these results, we concluded that Mn²⁺ plays a key role in lignin degradation under different nutritional and growth conditions, since it is required for the production of MnP in P. ostreatus.