毛栓菌(Tramets trogii)液体培养产漆酶的研究

实验研究了碳源、氮源和综合因素对毛栓菌(Tramets trogii)液体培养产漆酶和生长情况的影响。碳源为玉米面、氮源为豆饼粉时,菌株的产漆酶酶活最高;碳源为可溶性淀粉,氮源为麦麸时,对菌株的生长有利;在液体条件下,菌株产漆酶的最佳培养基组成为玉米面3.5%,豆饼粉4.0%,KH2PO40.3%,MgSO40.15%,VB10.04%。     

Ligninolytic Enzymes of the White Rot Basidiomycete Trametes trogii

The white rot fungus Trametes trogii strain BAFC 463 produced laccase, manganese peroxidase, lignin peroxidase and cellobiose dehydrogenase, as well as two hydrogen peroxide‐producing activities: glucose oxidizing activity and glyoxal oxidase. In high‐N (40 mM N) cultures, the titres of laccase, MnP and GLOX were 27 (6.55 U/ml), 45 (403.00 mU/ml)and 8 (32,14 mU/ml) fold higher, respectively, than those measured in an N‐limited medium. This is consistent with the fact that the ligninolytic system of T. trogii is expressed constitutively. Lower activities of all the enzymes tested were recorded upon decreasing the initial pH of the medium from 6.5 to 4.5. Adding veratryl alcohol improved GLOX production, while laccase activity was stimulated by tryptophan. Supplying Tween 80 strongly reduced the activity of both MnP and GLOX, but increased laccase production. The titre of MnP was affected by the concentration of Mn in the culture medium, the highest levels were obtained with 90 μM Mn (II). LiP activity, as CDH activity, were detected only in the mediumsupplemented with sawdust. In this medium, laccase production reached a maximum of 4.75 U/ml, MnP 747.60 mU/ml and GLOX 117.11 mU/ml. LiP, MnP and GLOX activities were co‐induced, attaining their highest levels at the beginning of secondary metabolism, but while MnP, laccase, GLOX and CDH activities were also present in the primary growth phase, LiP activity appears to beidiophasic. The simultaneous presence of high ligninolytic and hydrogen peroxide producing activities in this fungus makes it an attractive microorganism for future biotechnological applications.     

Copper induction of lignin-modifying enzymes in the white-rot fungus Trametes trogii

Trametes trogii, a white rot basidiomycete involved in wood decay worldwide, produces several ligninolytic enzymes, laccase being the dominant one, with higher titers than those reported for most other white rot fungi studied up to date. The effect of copper on in vitro production of extracellular ligninolytic activities was studied. CuSO(4)·5H(2)O concentrations from 1.6 μM to 1.5 mM were tested in a synthetic medium with glucose 20 g/L and asparagine 3 g/L. The addition of copper (up to 1 mM) did not affect growth but strongly stimulated ligninolytic enzyme production; faster decolorization of the polymeric dye Poly R-478 was observed as well. Maximal production of manganese peroxidase, laccase, and glyoxal oxidase [1.28 U/mL, 93.8 U/mL (with a specific activity of 720 U/mg protein), and 0.46 U/mL respectively] was attained with 1 mM CuSO(4)·5H(2)O. However, higher copper concentrations inhibited growth and notably decreased manganese peroxidase production, although they did not affect laccase secretion. Laccase activity in the culture filtrate was maximal at 50 C and pH 3.4, and the enzyme was completely stable at pH 4.4 and above, and at 30 C for up to 5 d. Denaturing polyacrylamide gel electrophoresis of extracellular culture fluids showed two laccase activity bands (mol wt 38 and 60 kDa respectively). The pattern of isoenzyme production was not affected by medium composition but differed with culture age.     

培养条件对毛栓菌漆酶分泌的影响

研究了碳源、氮源、愈创木酚、香兰素及培养条件对漆酶分泌的影响;结果表明,麦草粉作碳源、(NH_4)_2SO_4作氮源有利于漆酶的分泌,适宜浓度的愈创木酚和香兰素等对漆酶的产生有一定的作用;pH在3.0～8.0的范围内对漆酶的分泌影响差别不大,培养温度、接种量、通气量对漆酶的分泌有较大影响。     

毛栓菌漆酶的分离纯化及酶学性质研究

对毛栓菌产漆酶的分离、纯化及酶学性质进行研究。粗酶液经硫酸铵盐析、透析、DEAE-Sepharose柱层析,得到2种漆酶同工酶LacA和LacB。LacA和LacB回收率分别为17.1%和2.74%。SDS-PAGE电泳测得2漆酶的分子量分别为54.6 ku和7.7 ku;LacA和LacB最适作用温度分别为50℃和60℃;最适反应pH值分别为4.5和4.0;Cu2＋、Mg2＋对LacA有激活作用,对LacB影响不大;Ag＋对LacA和LacB表现为完全抑制;Fe3＋对LacA和LacB有一定的抑制作用;Ca2＋、Mn2＋、K＋、Na＋、Zn2＋对LacA和LacB影响不大。DTT、EDTA、DMSO、SDS对酶均有不同程度的抑制作用,且随其浓度的升高抑制作用增强。     

Effect of light on peroxidase and lignin synthesis in mungbean hypocotyls

Two days of light irradiance reduced the growth of mungbean hypocotyls as well as the levels of endogenous indole-3-acetic acid (IAA). In hypocotyls, both peroxidase and laccase activities were enhanced by light. The lignin content in mungbean hypocotyls was enhanced twofold by light. The inhibition of mungbean hypocotyl growth caused by light might be due to the decline of endogenous IAA, which could be degraded by a cationic peroxidase. The higher levels of lignin were correlated with the increased anionic peroxidase activity in light-treated tissues.     

AUXIN DESTRUCTION BY MARASMIUS PERNICIOSUS

Attempts were made to determine whether Marasmius perniciosus Stahel., causal agent of the witches' broom disease of cacao, produces substances that could elicit on the host some of the symptoms which are characteristic of the disease. In vitro the fungos did not produce significant amounts of cytokinins or auxins. However, culture filtrates destroyed indole-3-acetic acid (IAA) rapidly because of two different IAA-oxidizing enzymes: (1) a peroxidase (IAA oxidase) characterized by (a) dependence on Mn⁺⁺ and 2,4-dichlorophenol (DCP), (b) pH. optimum of 3.0, (c) inactivation at 85 C for 5 min, (d) stimulation of oxidative activity by addition of 0.3 μmole/ml H₂O₂; and (2) a laccase characterized by (a) no dependence on Mn⁺⁺ and DCP, (b) pH optimum of 6.0, (c) inactivation at 95 C for 5 min, (d) ability to oxidize IAA and phenolic substrates in the absence of H₂O₂. The peroxidase was completely inhibited by scopoletin (0.0002 m ) ; the laccase was inhibited by thiourea (0.1 m ). The laccase could be separated from the peroxidase by elution through a column of Sephadex G-100. It is suggested that the main pathogenic effect of the fungus may be associated with these auxin-inactivating systems.     

Biodegradation of olive oil mill wastewaterby Coriolus versicolor and Funalia trogii:effects of agitation, initial COD concentration, inoculum size and immobilization

The biodegradation of olive oil mill wastewater (OOMW) by Coriolus versicolor and Funalia trogii was investigated. Initial COD concentration, agitation and inoculum size were all found to be significant for biodegradation. Adding glucose, sulphate or nitrogen had no effect on biodegradation. During growth in optimum conditions, C.versicolor removed approximately 63% COD, 90% phenol and 65% colour within 6 days and F. trogii removed approximately 70% COD, 93% phenol and 81% colour of the OOMW used. The fungi also excreted large amounts of extracellular laccase into the medium. High biodegradation yields were also obtained by fungi immobilized in calcium alginate gels.     

Biodegradation of olive oil mill wastewaterby Coriolus versicolor and Funalia trogii:effects of agitation,initial COD concentration,inoculum size and immobilization

The biodegradation of olive oil mill wastewater (OOMW) by Coriolus versicolor and Funalia trogii was investigated. Initial COD concentration, agitation and inoculum size were all found to be significant for biodegradation. Adding glucose, sulphate or nitrogen had no effect on biodegradation. During growth in optimum conditions, C.versicolor removed approximately 63% COD, 90% phenol and 65% colour within 6 days and F. trogii removed approximately 70% COD, 93% phenol and 81% colour of the OOMW used. The fungi also excreted large amounts of extracellular laccase into the medium. High biodegradation yields were also obtained by fungi immobilized in calcium alginate gels.     

Chrysene bioconversion by the white rot fungus <Emphasis Type="Italic">Pleurotus ostreatus</Emphasis> D1

The effect of cultivation conditions on chrysene bioconversion by the fungus Pleurotus ostreatus D1 was shown. Under the laccase production conditions, transformation of this polycyclic aromatic hydrocarbon occurs with accumulation of the quinone metabolite. Under both the laccase and versatile peroxidase production conditions, chrysene degradation occurs, with the stages leading to phthalic acid formation and its further utilization. The formation of phthalic acid as a metabolite of chrysene degradation by white rot fungi was revealed for the first time. The data obtained suggest that the laccase revealed on the mycelial surface and the extracellular laccase are probably involved at the initial stages of chrysene metabolism, whereas versatile peroxidase seems to be required for oxidizing the metabolites formed.     

Industrial Dye Decolorization by Laccases from Ligninolytic Fungi

White-rot fungi were studied for the decolorization of 23 industrial dyes. Laccase, manganese peroxidase, lignin peroxidase, and aryl alcohol oxidase activities were determined in crude extracts from solid-state cultures of 16 different fungal strains grown on whole oats. All Pleurotus ostreatus strains exhibited high laccase and manganese peroxidase activity, but highest laccase volumetric activity was found in Trametes hispida. Solid-state culture on whole oats showed higher laccase and manganese peroxidase activities compared with growth in a complex liquid medium. Only laccase activity correlated with the decolorization activity of the crude extracts. Two laccase isoenzymes from Trametes hispida were purified, and their decolorization activity was characterized. Received: 26 May 1998 / Accepted: 7 August 1998     

Decolourisation of the textile dye Astrazon Red FBL by Funalia trogii pellets.

The effects of various conditions such as initial pH, dye concentrations, amount of pellet, temperature and agitation on decolourising activity of Funalia trogii were investigated. These, except initial pH, were all found to be important for dye decolourising activity of F. trogii. The decolourisation of the dye involved adsorption of the dye compound by fungal pellets at the initial stage, followed by the decolourisation through microbial metabolism. Heat-killed pellets were also tested for their ability to decolourise Astrazon Red dye. These pellets adsorbed the dye and 55% decolourisation was obtained in 24 h. But at the second cycle there was only 24% decolourisation. Our observation showed that Astrazon Red dye decolourisation by heat-killed pellets was mainly due to biosorption. The longevity of the decolourisation activity of F. trogii pellets was also investigated in repeated batch mode. Variations in the amount of pellet increased % decolourisation and stability of pellets.     

Changes in peroxidase and IAA oxidase activities during cell elongation in Phaseolus hypocotyls

Cytoplasmic and salt-extracted peroxidase and IAA oxidase activities were studied in Phaseolus vulgaris hypocotyls treated with gibberellic acid (GA, 200 μM), naphthyl acetic acid (NAA, 100 μM) and distilled water control (DW). Peroxidase activity was assayed with four hydrogen donors during the initial phase of hypocotyl elongation. Though peroxidase activity showed a decreasing trend with time in all the hydrogen donors studied; considerable variation with different hydrogen donors was observed. NAA had maximum peroxidase activity as compared to DW or GA treatment. The activity showed a clear inverse correlation with hypocotyl growth. IAA oxidase activity showed a similar trend with growth as peroxidase activity. A highly significant correlation was observed between peroxidase and IAA oxidase activities and high molecular weight xyloglucan content (P<0.001). Finally, the possible role of peroxidase and IAA oxidase activities in hypocotyl elongation growth is discussed.     

Increased enzymatic activities and levels of superoxide anion and phenolic compounds in cultures of basidiomycetes after temperature stress

Selected strains of basidiomycetes (Abortiporus biennis, Trametes versicolor and Cerrena unicolor) were shown to produce enhanced extracellular peroxidase (EP), superoxide dismutase (SOD) and laccase activities following the exposure of 10-day-old fungal cultures to separate high and low temperature stress. The stressful conditions also caused an increase in the concentrations of phenol compounds and superoxide anion radicals in these cultures. At first, peroxidase activity was observed at 12 hours from the moment of temperature stress application. Laccase activity appeared at 96 hours after the maximum levels of superoxide anion radicals (48 h) and SOD activity (36–72 h). The concentration of phenolic substances grew steadily during the period of cultivation. These relations between laccase, SOD and EP as well as superoxide radicals and phenol levels in the environment of ligninolytic fungi seems to be important in the course of the biosynthesis or biodegradation of lignin, as the consequence of adaptation of these basidiomycetes to environmental temperature conditions.     

INDOLE ACETIC ACID OXIDASE AND PEROXIDASE ACTIVITIES IN NORMAL AND CROWN GALL TISSUE CULTURES OF PARTHENOCISSUS TRICUSPIDATA

Lipetz , Jacques , and Arthur W. Galston . (Yale U., New Haven.) Indole acetic acid oxidase and peroxidase activities in normal and crown gall tissue cultures of Parthenocissus tricuspidata. Amer. Jour. Bot. 46(3) : 193-196. Illus. 1959.—Normal and crown gall cells of P. tricuspidata grown in pure culture were examined for IAA oxidase and peroxidase activities. No IAA oxidase activity could be demonstrated in dialyzed or undialyzed homogenates of either tissue; however, crown gall tissue, but not normal tissue, was found to produce an extracellular IAA oxidase which required Mn⁺⁺ and DCP as co-factors. Normal tissue, but not crown gall tissue was found to contain high levels of substances which spared IAA from destruction by a pea IAA oxidase preparation. Peroxidase activity was found to be higher in normal than in crown gall homogenates, but crown gall tissue released considerably more peroxidase into the external medium. The differences in the auxin requirements and growth rate between normal and crown gall cells appear not to be easily explicable in terms of differential auxin destruction.     

HORMONAL CONTROL OF PEROXIDASE ACTIVITY IN CULTURED PELARGONIUM PITH

Freshly excised Pelargonium pith tissue lacks peroxidase activity toward guaiacol or benzidine, but it develops such activity within 24–36 hr in aseptic culture. All the activity is manifested as a single enzyme moving toward the cathode during electrophoresis on starch gel at pH 9.0. This development of peroxidase activity is at first (up to ca. 50 hr in culture) inhibited and later (ca. 100–150 hr in culture) promoted by IAA. This dual effect of IAA resembles that previously reported for specific isoperoxidases in tobacco pith cells. Kinetin alone also inhibits peroxidase formation, but in the presence of IAA those concentrations which enhance growth enhance peroxidase formation as well.     

Effect of salinity stress on growth,peroxidase and IAA oxidase activities in vigna seedlings

The present work was carried out with the aim of studying the effect of salinity stress on growth and IAA oxidizing system (i.e. peroxidase and IAA oxidase) in vigna (Vigna unguiculata L.) seedlings. The seedlings were treated with two concentrations of sodium chloride (NaCl) 0.1 M and 0.25 M. Length, fresh and dry weight were the parameters considered for growth. Salinity effect was distinct in fresh weight and dry weight of different organs. Peroxidase and IAA oxidase activities were measured at different time intervals for both cytoplasmic and wall bound fractions. Peroxidase activity was maximum at higher stress conditions bringing about the hypocotyl growth restriction. Thus there was a clear inverse correlation between elongation and peroxidase activity. IAA oxidase activity also showed a similar trend for both cytoplasmic and wall bound fractions. The role of IAA oxidizing system in defense mechanism in response to salinity stress is discussed.     

Phlebia radiata laccase forms induced by veratric acid and xylidine in relation to lignin peroxidase and manganese-dependent peroxidase

White-rot basidiomycete Phlebia radiata grown in the nitrogen-poor liquid medium was tested for the inducing effect of 2,5-xylidine and veratric acid on laccase production and properties of its preparations. Also lignin peroxidase and manganese-dependent peroxidase were assayed under the same conditions. The maximum of laccase activity for veratric acid as a stimulator was done much earlier than that of xylidine. It was very sharp and disappeared quickly. At that time only weak lignin peroxidase and manganese-dependent peroxidase activities were noticed. The maximum of laccase induced by xylidine was observed much later and kept longer. Both laccase preparations showed the same properties. For biotechnological reasons, the production of laccase induced by the nontoxic veratric acid is much more economic and better acceptable than that induced by xylidine.     

Changes in production of lignin degrading enzymes during interactions between mycelia of the tropical decomposer basidiomycetes Marasmiellus troyanus and Marasmius pallescens

During the interaction of two tropical agaric fungi, Marasmius pallescens and Marasmiellus troyanus, on agar media, initial deadlock between the two mycelia was ultimately followed by take-over by M. troyanus. When shaken liquid cultures of these two fungi were mixed, a rapid increase in laccase and manganese peroxidase activity, but no lignin peroxidase, was detected in the culture supernatant. Even more rapid and elevated induction of laccase occurred when filter-sterilized supernatant of Marasmius pallescens was added to Marasmiellus troyanus cultures, but the reciprocal experiment (addition of M. troyanus supernatant to M. pallescens cultures), did not lead to any increase in laccase activity. Addition of autoclaved supernatant of M. pallescens also induced laccase activity from M. troyanus cultures, but over a period of days rather than hours. Although both M. troyanus, and to a lesser extent M. pallescens, are able to produce laccases in shaken liquid culture following addition of the inducer 2,5-dimethylalanine, these experiments suggest that the presence of heat-stable and heat-labile laccase inducers secreted by M. pallescens mycelia lead to induction of laccases by M. troyanus.