Identification of low molecular weight aromatic compounds by gas chromatography–mass spectrometry (GC–MS) from kraft lignin degradation by three Bacillus sp.

Three bacterial strains identified as Paenibacillus sp., Aneurinibacillus aneurinilyticus and Bacillus sp. have been shown to decolourise kraft lignin in 6 days of incubation. The release of low molecular weight aromatic compounds by these bacterial strains during degradation of kraft lignin was analysed by GC–MS analysis. The total ion chromatograph (TIC) of ethyl acetate extract from kraft lignin sample inoculated by Paenibacillus sp. was similar to control except some minor changes in the chromatographic profile indicating incapability of this bacterium to modify kraft lignin. On the other hand the TIC of ethyl acetate extract from kraft lignin inoculated by A. aneurinilyticus and Bacillus sp. caused formation of several aromatic lignin-related compound that were not present in the extract of control. The compounds identified in extract of the sample degraded by A. aneurinilyticus were guaiacol, acetoguiacone, gallic acid and ferulic acid while t-cinnamic acid, 3,4,5 trimethoxy benzaldehyde, and ferulic acid by Bacillus sp. indicating oxidization of coniferylic (G units) and sinapylic (S units) alcohol of lignin polymer. Differences between the identified compounds from different bacterial treatment were strain-specific. Among the identified aromatic compounds, ferulic acid and 3,4,5-trimethoxy benzaldehyde could be useful to the industry of preservatives, aromas and perfumes.     

Production and characterization of ligninolytic enzymes ofBjerkandera adusta grown on wood meal/wheat bran culture and production of these enzymes using a rotary-solid fermenter

Manganese peroxidase (MnP) and lignin peroxidase (LiP) were produced by growing a white-rot fungusBjerkandera adusta statically, on a wood meal/wheat bran culture in flasks. MnP and LiP reached their maximum activity after 6 and 19 days of inoculation, respectively. Both MnP and LiP are thought to be important enzymes in lignin biodegradation byB. adusta. Ion exchange chromatography showed thatB. adusta produced a single LiP and a single MnP enzyme in wood meal/wheat bran culture. These enzymes were separated and characterized. The molecular weight of MnP was 46,500 with a pl of 3.9. The molecular weight of LiP was estimated to be 47,000 with a pl of 3.5. Spectral analysis demonstrated that both enzymes are heme proteins. Production of these enzymes was also achieved using a rotarysolid culture fermenter. MnP, LiP and veratryl alcohol oxidase were produced byB. adusta in the fermenter.     

Effect of culture conditions on the production of ligninolytic enzymes by white rot fungi Phanerochaete chrysosporium (ATCC 20696) and separation of its lignin peroxidase

The present work was carried out to determine the optimum culture conditions of Phanerochaete chrysosporium (ATCC 20696) for maximizing ligninolytic enzyme production. Additionally, separation of its lignin peroxidase was conducted. After experiments, an optimized culture medium/condition was constructed (per liter of Kirk’s medium): dextrose 10 g, ammonium tartrate 0.11 g, Tween-80 0.5 g, MnSO₄ 7 mg, and veratryl alcohol 0.3 g in 10 mM acetic acid buffer pH 4.5. Under the optimized experimental condition, both lignin peroxidase (LiP) and manganese peroxidase (MnP) were detected and reach the highest yield at 30°C on the 8th day culture. Salt precipitation methods was used in the extraction and purification processes. Results show that salt precipitation with 60% (NH₄)₂SO₄ yielded the best result, especially toward LiP. Enzyme separation was conducted and two fractions with LiP activity. LiP1 and LiP2 were produced using three columns sequentially: desalting column, Q FF ion exchange column and Sepharyl S-300 HR gel filtration. LiP1 and LiP2 had been purified by 9.6- and 7.6-fold with a yield of 22.9% and 18.6%, respectively. According to the data of sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE), the molecular weights of the enzymes are 38 kDa and 40 kDa, respectively.     

Interference of peptone and tyrosine with the lignin peroxidase assay.

The N-unregulated white rot fungus Bjerkandera sp. strain BOS55 was cultured in 1 liter of peptone-yeast extract medium to produce lignin peroxidase (LiP). During the LiP assay, the oxidation of veratryl alcohol to veratraldehyde was inhibited due to tyrosine present in the peptone and the yeast extract.     

Effect of inducers on the decolorization and biodegradation of textile azo dye Navy blue 2GL by Bacillus sp. VUS

Bacillus sp. VUS decolorized azo dye Navy blue 2GL in 48 h at static anoxic condition in yeast extract medium, whereas it took only 18 h for the decolorization in presence of CaCl₂. Different inducers played role in the decolorization of Navy blue 2GL. CaCl₂ found to be the most effective inducer among all inducers tested. The activity of enzymes like lignin peroxidase, laccase and reductases viz. NADH-DCIP, azo and riboflavin induced during decolorization represents their role in the biodegradation. Extracellular LiP and intracellular laccase activity induced with CaCl₂. Yeast extract was best medium for faster decolorization than other media. UV–vis spectrophotometer analysis and visual examinations showed decolorization of dye. High performance liquid chromatography, Fourier transforms infrared spectroscopy showed degradation of dye. Gas Chromatography-Mass Spectroscopy revealed formation of 4-Amino-3-(2-bromo-4, 6-dinitro-phenylazo)-phenol and acetic acid 2-(-acetoxy-ethylamino)-ethyl ester as final products. Bacillus sp. VUS also decolorized synthetic effluent. Phytotoxicity study showed detoxification of Navy blue 2GL.     

Decolorization of Azo, Triphenyl Methane, Heterocyclic, and Polymeric Dyes by Lignin Peroxidase Isoenzymes from Phanerochaete chrysosporium

The ligninolytic enzyme system of Phanerochaete chrysosporium decolorizes several recalcitrant dyes. Three isolated lignin peroxidase isoenzymes (LiP 4.65, LiP 4.15, and LiP 3.85) were compared as decolorizers with the crude enzyme system from the culture medium. LiP 4.65 (H2), LiP 4.15 (H7), and LiP 3.85 (H8) were purified by chromatofocusing, and their kinetic parameters were found to be similar. Ten different types of dyes, including azo, triphenyl methane, heterocyclic, and polymeric dyes, were treated by the crude enzyme preparation. Most of the dyes lost over 75% of their color; only Congo red, Poly R-478, and Poly T-128 were decolorized less than the others, 54, 46, and 48%, respectively. Five different dyes were tested for decolorization by the three purified isoenzymes. The ability of the isoenzymes to decolorize the dyes in the presence of veratryl alcohol was generally comparable to that of the crude enzyme preparation, suggesting that lignin peroxidase plays a major role in the decolorization and that manganese peroxidase is not required to start the degradation of these dyes. In the absence of veratryl alcohol, the decolorization activity of the isoenzymes was in most cases dramatically reduced. However, LiP 3.85 was still able to decolorize 20% of methylene blue and methyl orange and as much as 60% of toluidine blue O, suggesting that at least some dyes can function as substrates for isoenzyme LiP 3.85 but not to the same extent for LiP 4.15 or LiP 4.65. Thus, the isoenzymes have different specificities towards dyes as substrates.     

Screening of tetrachlorodibenzo-<Emphasis Type="Italic">p</Emphasis>-dioxin-degrading fungi capable of producing extracellular peroxidases under various conditions

Forty-six pulp-bleaching fungi were screened for production of key enzymes for conversion of polychlorinated dibenzo-p-dioxins—lignin peroxidase (LiP), manganese peroxidase (MnP), and manganese-independent peroxidase (MiP)—under various conditions that would allow their utilization in the environment. Of 38 MnP-producing strains with MiP activity, 22 produced LiP. Three of the new isolates, Bjerkandera sp. strains MS191, MS325, and MS1167, were the best producers of the three different peroxidases, and had reasonable growth rates. The most promising Bjerkandera sp. strain, MS325, exhibited significant levels of LiP and MnP activities under various conditions, e.g., nutrient nitrogen-sufficient or -limited conditions, conditions with or without Mn(II), and changes in temperature (15–37°C). Furthermore, the ability of this strain to degrade 1,3,6,8-tetrachlorodibenzo-p-dioxin was confirmed. The results presented here indicate that utilization of Bjerkandera sp. strain MS325 on a practical scale in the environment has several advantages over many white rot fungi, which produce extracellular peroxidases only under specific conditions such as nutrient limitation.     

The selective visualization of lignin peroxidase,manganese peroxidase and laccase,produced by white rot fungi on solid media

A visual method for the selective screening of lignin degrading enzymes, produced by white rot fungi (WRF), was investigated by the addition of coloring additives to solid media. Of the additives used in the enzyme production media, guaiacol and RBBR could be used for the detection of lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase. Syringaldazine and Acid Red 264 were able for the detection of both the MnP and laccase, and the LiP and laccase, respectively, and a combination of these two additives was able to detect each of the ligninases produced by the WRF on solid media.     

Production,partial purification and characterization of ligninolytic enzymes from selected basidiomycetes mushroom fungi

In recent years, many research on the quantity of lignocellulosic waste have been developed. The production, partial purification, and characterisation of ligninolytic enzymes from various fungi are described in this work. On the 21st day of incubation in Potato Dextrose (PD) broth, Hypsizygus ulmarius developed the most laccase (14.83 × 10⁻⁶ IU/ml) and manganese peroxidase (24.11 × 10⁻⁶ IU/ml), while Pleurotus florida produced the most lignin peroxidase (19.56 × ⁻⁶ IU/ml). Laccase (Lac), lignin peroxidase (LiP), and manganese peroxidase (MnP), all generated by selected basidiomycetes mushroom fungi, were largely isolated using ammonium sulphate precipitation followed by dialysis. Laccase, lignin peroxidase, and manganese peroxidase purification findings indicated 1.83, 2.13, and 1.77 fold purity enhancements, respectively. Specific activity of purified laccase enzyme preparations ranged from 305.80 to 376.85 IU/mg, purified lignin peroxidase from 258.51 to 336.95 IU/mg, and purified manganese peroxidase from 253.45 to 529.34 IU/mg. H. ulmarius laccase (376.85 IU/mg) with 1.83 fold purification had the highest specific activity of all the ligninolytic enzymes studied, followed by 2.13 fold purification in lignin peroxidase (350.57 IU/mg) and manganese peroxidase (529.34 IU/mg) with 1.77-fold purification. Three notable bands with molecular weights ranging from 43 to 68 kDa and a single prominent band with a molecular weight of 97.4 kDa were identified on a Native PAGE gel from mycelial proteins of selected mushroom fungus. The SDS PAGE profiles of the mycelial proteins from the selected mushroom fungus were similar to the native PAGE. All three partially purified ligninolytic isozymes display three bands in native gel electrophoresis, with only one prominent band in enzyme activity staining. The 43 kDa, 55 kDa, and 68 kDa protein bands correspond to laccase, lignin peroxidase, and manganese peroxidase, respectively.     

高效木质素降解菌的筛选及其对玉米秸秆的降解效果

本研究利用愈创木酚和苯胺蓝固体培养基对菌株进行初筛,利用形态学和分子生物学对筛选出的菌株进行鉴定,以黄孢原毛平革菌Phanerochaete chrysosporium CGMCC 5.0776为对照,利用其对玉米秸秆进行预处理并测定木质素和纤维素的降解率,测定筛选菌株在预处理玉米秸秆过程中漆酶、锰过氧化物酶（manganese peroxidase,MnP）和木质素过氧化物酶（lignin peroxidase,LiP）活性。结果表明:利用愈创木酚和苯胺蓝固体培养基,从16株白腐真菌菌株中筛选出2株具有较高漆酶或MnP活性的菌株,鉴定其为桦栓孔菌Trametes betulina (L.) Pilát（ZT-153）和亚黑管孔菌Bjerkandera fumosa (Pers.) P. Karst.（ZT-307）,测定T. betulina ZT-153和B. fumosa ZT-307对玉米秸秆酸不溶木质素降解效率分别为13.60%和21.87%,较对照P. chrysosporium CGMCC 5.0776高1.58%和9.85%,对纤维素的降解率较低,分别为4.10%和4.50%。2株菌株在预处理玉米秸秆过程中,T. betulina ZT-153表现出漆酶和MnP活性,B. fumosa ZT-307只表现出LiP活性。其中B. fumosa ZT-307对玉米秸秆酸不溶木质素的降解效率最高,在秸秆资源的综合利用方面具有较好的潜力和应用前景。     

Hyperactivation and thermostabilization of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis> lignin peroxidase by immobilization in xerogels

This is a continuation of our previous paper on production of lignin peroxidase (LiP) by Phanerochaete chrysosporium in solid substrate fermentation (SSF) medium of corncobs. The enzyme was purified by ammonium sulphate precipitation and ion-exchange fast protein liquid chromatography. Maximum yield of LiP was 13.7 U/gds (units per gram dry substrate) after 5 days of SSF with 70% moisture and 20% (v/w) inoculum. The approximate molecular mass of purified LiP, estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was 38 kDa. The pH and temperature optima for the LiP were 4 and 40°C, respectively. Immobilization of LiP in hydrophobic xerogels caused hyperactivation of LiP and enhanced its thermostability properties. The K _M and V _max values for immobilized LiP were 10.56 mg/ml and 16.67 μmol/min (120.49 U/mg of protein) as compared to 13 mg/ml and 11.76 μmol/min (85 U/mg of protein), respectively, for free LiP using veratryl alcohol as substrate.     

Purification and characterization of cutinase from Bacillus sp. KY0701 isolated from plastic wastes

A total of approximately 400 bacterial strains were isolated from 73 plastic wastes collected from 14 different regions. Nineteen isolates that form clear zones both on tributyrin and poly ε-caprolactone (PCL) agar, were identified based on 16S rRNA gene sequences. Among these, Bacillus sp. KY0701 that caused the highest weight loss of PCL films in minimal salt medium, was selected for cutinase production. The highest enzyme activity (15 U/mL) was obtained after 4 days of incubation at 50°C, pH 7.0 and 200?rpm in a liquid medium containing 1.5% (w/v) apple cutin and 0.1% (w/v) yeast extract. The purified enzyme was stable at high temperatures (50–70°C) and over a wide pH range (5.5–9.0). The relative activity of cutinase was at least 75% in the percent of various organic solvents. The apparent K_m and V_max values of the cutinase for p-nitrophenyl butyrate were 0.72?mM and 336.8?µmol p-nitrophenol/h/g, respectively. In addition, it showed high stability and compatibility with commercial detergents. These features of cutinase obtained from Bacillus sp. KY0701 make it a promising candidate for application in the detergent and chemical industries. In our best knowledge, this is the first report for cutinase production and characterization produced by a Bacillus strain.     

Purification and characterization of lignin peroxidases from <Emphasis Type="Italic">Penicillium decumbens</Emphasis> P6

Summary Peroxidases are essential enzymes in biodegradation of lignin and lignite which have been investigated intensively in the white-rot fungi. This is the first report of purification and characterization of lignin peroxidase from Penicillium sp. P6 as lignite degradation fungus. The results indicated that the lignin peroxidase of Penicillium decumbens P6 had physical and chemical properties and a N-terminal amino acid sequence different from the lignin peroxidases of white-rot fungi. The lignin peroxidase was isolated from a liquid culture of P. decumbens P6. This enzyme had a molecular weight of 46.3 KDa in SDS-PAGE and exhibited greater activity, temperature stability and wider pH range than those previously reported. The isolation procedure involved (NH₄)₂SO₄ precipitation, ion-exchange chromatography on DEAE-cellulose and CM-cellulose, gel filtration on Sephadex G-100, and non-denaturing, discontinuous polyacrylamide gel electrophoresis. The K _m and V _max values of this enzyme using veratryl alcohol as substrate were 0.565 mmol L ⁻¹ and 0.088 mmol (mg protein) ⁻¹ min ⁻¹ respectively. The optimum pH of P6 lignin peroxidase was 4.0, and 70.6 of the relative activity was remained at pH 9.0. The optimum temperature of the enzyme was 45 °C.     

Production of ligninolytic enzymes by Fusarium solani strains isolated from different substrata

A comparative study on the extracellular ligninolytic enzymatic activity of five strains of Fusarium solani in a carbon-limited medium under shaking, revealed a differential production of these enzymes. Aryl alcohol oxidase (AAO) activity was observed only in the supernatant of strain CLPS no. 568 with levels higher than 57 mU ml⁻¹. Free extracellular laccase activity was detected in strains CLPS nos. 493, 568 and 570, strain no. 568 being the one which showed the highest activity (over 8.6 mU ml⁻¹). Free extracellular lignin peroxidase (LiP) activity was not detected in any isolate tested, whereas low levels of manganese-dependent peroxidase (MnP) and manganese-independent peroxidase (MIP) activities were detected in certain isolates used. The AAO activity of F. solani on primary α-alcohols such as veratryl alcohol, is reported for the first time; this enzyme activity is hydrogen-peroxide independent. This is also the first report for extracellular MnP and MIP activities of F. solani. This revised version was published online in November 2006 with corrections to the Cover Date.     

>Decolourisation and depolymerisation of solubilised low-rank coal by the white-rot basidiomycete Phanerochaete chrysosporium

Peroxidases secreted by the white-rot basidiomycete Phanerochaete chrysosporium can oxidise a wide range of recalcitrant compounds including lignin and aromatic xenobiotics. Since low-rank coals such as brown coal and lignite retain structural features of the parent lignin, we investigated the possibility that P. chrysosporium is capable of acting on a brown coal, with the production of useful low-molecular-mass compounds. In nitrogen-limiting liquid medium containing 0.03% solubilised Morwell brown coal, P. chrysosporium was found to convert about 85% of the coal after 16 days incubation to a form not recoverable by alkali-washing and acid-precipitation. The modal molecular mass of the residual coal macromolecules was reduced from the initial 65kDa to 32 kDa. Extensive bleaching of the coal coincided with the presence of extracellular lignin peroxidase (LiP) and manganese-dependent peroxidase (MnP), although both LiP and MnP activity were lower in cultures containing coal. These reductions are accounted for by interference with the enzyme assays by solubilised coal and by binding of MnP to precipitated coal. LiP was about eight times more sensitive than MnP to inhibition by solubilised coal. In nitrogen-sufficient medium containing solubilised coal, neither coal modification nor LiP activity were observed, suggesting that LiP is an essential component of the bleaching process.     

Laccase and other ligninolytic enzyme activities of selected strains ofTrametes spp. from different localities and substrates

Eighty-three strains belonging to three species of the genus Trametes FR. (T. versicolor, T. hirsuta and T. ochracea) collected in different localities and on different substrates were screened for laccase production. The production of other lignin-modifying enzymes--manganese peroxidase (MnP) and lignin peroxidase (LiP)--and the decolorization ability were also determined in 21 of them. Production variability was relatively high and no significant correlation was found between the origin of the strains (locality, substrate) and the enzyme production. Dikaryons of all 3 species (but not of all their strains) exhibited LiP activity, which was not detected in the respective monokaryons.     

Isolation and Characterization of Mercury Resistant Bacillus sp. from Soils with an Extensive History as Substrates for Mercury Extraction in Mexico

Metal phytoextraction assisted by bacteria plays an important role in bioremediation systems. In this work, mercury-resistant bacterial strains were isolated from soils with high levels of mercury (San Joaquin, Queretaro State, Mexico) and identified as Bacillus sp. based on the 16S rDNA gene sequence analysis. The bacterial strains were found to exhibit different multiple mercury-resistance and carbon source utilization characteristics. The mercury reduction ability was tested through a volatilization assay. The bacterial isolates were also evaluated for their ability to promote growth and mercury uptake in tomato plants. In a roll towel assay, the maximum vigor index of tomato plants was obtained with the inoculation of Bacillus sp. A2, A12, B11, B15 and C1, while in a pot assay, the maximum vigor index was obtained with the inoculation of Bacillus sp. A6, A7 and B20, compared with un-inoculated controls in the presence of HgCl₂. Maximum Hg accumulation in the roots and shoots of tomato plants was obtained only with Bacillus sp. A7 in the roll towel assay, whereas in the pot assay, maximum accumulation was obtained with Bacillus sp. A12 compared with un-inoculated controls. Our results show that mercury accumulation in tissue is enhanced by these plant growth promoting bacterial strains, which recommends their possible use as microbe-assisted phytoremediation systems in mercury-polluted soils.     

Degradation of the herbicide bentazon as related to enzyme production by Phanerochaete chrysosporium in two solid substrate fermentation systems

Enzyme production and degradation of the herbicide bentazon by Phanerochaete chrysosporium growing on straw (solid substrate fermentation, SSF) and the effect of nitrogen and the hydraulic retention time (HRT) were studied using a small bioreactor and batch cultures. The best degradation of bentazon was obtained in the low nitrogen treatments, indicating participation of the ligninolytic system of the fungus. The treatments that degraded bentazon also had manganese peroxidase (MnP) activity, which seemed to be necessary for degradation. Pure MnP (with Mn(II) and H₂O₂) did not oxidize bentazon. However, in the presence of MnP, Mn(II) and Tween 80, bentazon was slowly oxidized in a H₂O₂-independent reaction. Bentazon was a substrate of pure lignin peroxidase (LiP) and was oxidized significantly faster (22,000–29,000 times) as compared to the MnP-Tween 80 system. Although LiP was a better enzyme for bentazon oxidation in vitro, its role in the SSF systems remains unclear since it was detected only in treatments with high nitrogen and high HRT where no degradation of bentazon occurred. Inhibition of LiP activity may be due to phenols and extractives present in the straw.     

Lignin Peroxidases, Manganese Peroxidases, and Other Ligninolytic Enzymes Produced by Phlebia radiata during Solid-State Fermentation of Wheat Straw

The white rot fungus Phlebia radiata 79 (ATCC 64658) produces lignin peroxidase (LiP), manganese peroxidase (MnP), glyoxal oxidase (GLOX), and laccase in the commonly used glucose low-nitrogen liquid medium. However, the enzymes which this fungus utilizes for selective removal of lignin during degradation of different lignocellulosic substrates have not been studied before. Multiple forms of LiP, MnP, GLOX, and laccase were purified from P. radiata culture extracts obtained after solid-state fermentation of wheat straw. However, the patterns of extracellular lignin-modifying enzymes studied were different from those of the enzymes usually found in liquid cultures of P. radiata. Three LiP isoforms were purified. The major LiP isoform from solid-state cultivation was LiP2. LiP3, which has usually been described as the major isoenzyme in liquid cultures, was not expressed during straw fermentation. New MnP isoforms have been detected in addition to the previously reported MnPs. GLOX was secreted in rather high amounts simultaneously with LiP during the first 2 weeks of growth. GLOX purified from P. radiata showed multiple forms, with pIs ranging from 4.0 to 4.6 and with a molecular mass of ca. 68 kDa.     

Biodegradation of kraft lignin by a bacterial strain Comamonas sp. B-9 isolated from eroded bamboo slips

Aims: The aim was to obtain evidences for lignin degradation by unicellular bacterium Comamonas sp. B‐9. Methods and Results: Comamonas sp. B‐9 was inoculated into kraft lignin‐mineral salt medium (KL‐MSM) at pH 7·0 and 30°C for 7 days of incubation. The bacterial growth, chemical oxygen demand (COD) reduction, secretion of ligninolytic enzymes and productions of low‐molecular‐weight compounds revealed that Comamonas sp. B‐9 was able to degrade kraft lignin (KL). COD in KL‐MSM reduced by 32% after 7 days of incubation. The maximum activities of manganese peroxidase (MnP) of 2903·2 U l^?1 and laccase (Lac) of 1250 U l^?1 were observed at 4th and 6th day, respectively. The low‐molecular‐weight compounds such as ethanediol, 3, 5‐dimethyl‐benzaldehyde and phenethyl alcohol were formed in the degradation of KL by Comamonas sp. B‐9 based on GC‐MS analysis. Conclusions: This study confirmed that Comamonas sp. B‐9 could utilize KL as a sole carbon source and degrade KL to low‐molecular‐weight compounds. Significance and Impact of the Study: Comamonas sp. B‐9 may be useful in the utilization and bioconversion of lignin and lignin‐derived aromatic compounds in biotechnological applications. Meanwhile, using Comamonas sp. B‐9 in treatment of wastewater in pulp and paper industry is a meaningful work.