Production of Ligninases and Degradation of Lignin in Agitated Submerged Cultures of Phanerochaete chrysosporium

Research on the extracellular hemeprotein ligninases of Phanerochaete chrysosporium has been hampered by the necessity to produce them in stationary culture. This investigation examined the effects of detergents on development of ligninase activity in agitated submerged cultures. Results show that addition of Tween 80, Tween 20, or 3-[(3-colamidopropyl)dimethylammonio]1-propanesulfonate to the cultures permits development of ligninase activity comparable to that routinely obtained in stationary cultures. The detergent-amended cultures express the entire ligninolytic system, assayed as the complete oxidation of [¹⁴C]lignin to ¹⁴CO₂. The detergent effect is evidently not merely in facilitating release of extant enzyme. Development of ligninolytic activity in the agitated cultures, as in stationary cultures, is idiophasic. Ion-exchange fast protein-liquid chromatography indicated that the heme protein profiles in agitated and stationary cultures are very similar. These findings should make it possible to scale up production of ligninolytic enzymes in stirred tank fermentors.     

Influence of Veratryl Alcohol and Hydrogen Peroxide on Ligninase Activity and Ligninase Production by Phanerochaete chrysosporium

Veratryl alcohol, added as a supplement to cultures of Phanerochaete chrysosporium, enhanced ligninase activity through protection of the ligninase against inactivation by hydrogen peroxide produced by this fungus in cultures. In the presence of veratryl alcohol, the loss of ligninase activity observed in non-protein-synthesizing cultures (cycloheximide-treated) equaled the extracellular protein turnover. When cultures were not supplemented with veratryl alcohol, inactivation of ligninase by hydrogen peroxide added to protein turnover, resulting in a more rapid loss of ligninase activity. Although all ligninase isoenzymes are sensitive to inactivation by hydrogen peroxide, only the isoenzyme of the highest specific activity (80.6 nkat · mg of protein⁻¹; M_r, 41,800; pI, 3.96) was found to be protected by veratryl alcohol. The concentration of veratryl alcohol necessary for full protection of ligninase activity varied according to the concentration of hydrogen peroxide present in the medium, which depended on the nature of the carbon source (glucose or glycerol). It is proposed that the nature of the carbon source influences the overall ligninase activity not only directly, by affecting the rate and the type of synthesized ligninase, but also by affecting the rate of hydrogen peroxide production, bringing about different rates of inactivation.     

Ligninase production in agitated conditions by Phanerochaete chrysosporium

Abstract Extracellular H₂O₂-dependent ligninase activity of Phanerochaete chrysosporium was produced in agitated culture conditions when veratryl alcohol or veratraldehyde were added to the cultures. The enzyme production was suppressed by cycloheximide indicating that true protein synthesis occurred. The activated cultures were also able to degrade synthetic lignin. Reduction of veratraldehyde to corresponding alcohol during secondary metabolism was a good indicator of the effect of agitation on cell metabolism. Too high agitation speed led to complete inhibition of both the reduction reaction and the ligninolytic activity.     

Determination of ligninase activity in P. chrysosporium pellets with diffuse reflectance spectrophotometry

Diffuse reflectance spectrophotometry was applied for measuring ligninase activity in pellets of Phanerochaete chrysosporium. Enhanced ligninase activity in pellets and in growth medium were detected in cultures supplemented with oleic acid emulsified with Tween 80, Tween 80, hydrophilic (alcoholic) residue of Tween 80 hydrolysate, Tween 20, and (15OE)C_18:1. In cultures with low extracellular ligninase activity, low activity in pellets was also observed. Our results indicate that the stimulatory effect of the tested surfactants cannot be contributed solely to promotion of ligninases through the cell membrane. Correspondence to: D. Letan     

Role of Veratryl Alcohol in Regulating Ligninase Activity in Phanerochaete chrysosporium

Ligninase activity in Phanerochaete chrysosporium is stimulated by incubating cultures with various substrates for the enzyme, including veratryl (3,4-dimethoxybenzyl) alcohol, which is a secondary metabolite of this fungus. This study was designed to provide insight into the mechanism involved in this stimulation. Ligninase activity increased 2 to 4 h after the addition of exogenous veratryl alcohol to ligninolytic cultures. This increase was prevented by inhibitors of protein synthesis. Analysis of the extracellular proteins by high-performance anion-exchange liquid chromatography revealed increases in the amounts of some, but not all, ligninase species. The normal rapid decrease in ligninase activity in aging cultures was not prevented or retarded by veratryl alcohol, indicating that veratryl alcohol does not increase ligninase activity by protecting extant enzyme. We conclude that veratryl alcohol probably functions via an induction type of mechanism, affecting only certain ligninase species. Results with an isolated lignin indicate that lignin (or its biodegradation products) functions in the same way that veratryl alcohol does.     

Factors Involved in the Regulation of a Ligninase Activity in Phanerochaete chrysosporium

The regulation of an H₂O₂-dependent ligninolytic activity was examined in the wood decay fungus Phanerochaete chrysosporium. The ligninase appears in cultures upon limitation for nitrogen or carbohydrate and is suppressed by excess nutrients, by cycloheximide, or by culture agitation. Activity is increased by idiophasic exposure of cultures to 100% O₂. Elevated levels of ligninase and, in some cases, of extracellular H₂O₂ production are detected after brief incubation of cultures with lignins or lignin substructure models, with the secondary metabolite veratryl alcohol, or with other related compounds. It is concluded that lignin degradation (lignin → CO₂) by this organism is regulated in part at the level of the ligninase, which is apparently inducible by its substrates or their degradation products.     

Phospholipid and fatty acid enrichment of Phanerochaete chrysosporium INA-12 in relation to ligninase production

Summary Ligninase activity of Phanerochaete chrysosporium INA-12 was increased when vegetable oils emulsified with sorbitan polyoxyethylene monooleate (Tween 80) were added to growth medium. Maximal enzyme yield was 22.0 nkat·ml^-1 in olive oil cultures after 4 days incubation. P. chrysosporium INA-12 was also able to utilize tall oil fatty acids for ligninase synthesis. An extracellular lipase activity was detected during the primary phase of growth in culture containing vegetable oils. On the other hand, ligninase production was 1.5-fold enhanced when olive oil cultures were supplemented with soybean asolectin as a phospholipid source. In cultures supplied with olive oil plus asolectin, P. chrysosporium INA-12 mycelium exhibited a preferential enrichment of oleic acid (C18:1), phosphatidylcholine (PC) and lysophosphatidylcholine (LPC) as compared to lipid-free medium. PC and LPC enrichment was associated with an increased ratio of saturated versus unsaturated fatty acids of phospholipids.     

Influence of some surfactants and related compoundson ligninolytic activity of Phanerochaete chrysosporium

Abstract Several surfactants were tested as possible stimulators of ligninase production in agitated culture of Phanerochaete chrysosporium . In addition to Tween 80 and Tween 20, surfactants already known to have a stimulatory effect, polyoxyethylene oleate (15EO)C_18:1 and the hydrophilic fraction of Tween 80, were also found to enhance ligninase activity over 200-fold, indicating that surfactants as such may not be the true active substances. Cultures with increased ligninase activities exhibited preferential enrichment of total and polar lipids in palmitoleic acid and an increased unsaturation index.     

Oxidation of Thianthrene by the Ligninase of Phanerochaete chrysosporium

The oxidation of heterocyclic sulfur compounds reported to be part of the macrostructure of coal and petroleum was investigated. The oxidation of thianthrene solubilized in 10% dimethylformamide to thianthrene monosulfoxide in the presence of hydrogen peroxide was catalyzed by the ligninase from Phanerochaete chrysosporium.     

Effect of Environmental Conditions on Extracellular Protease Activity in Lignolytic Cultures of Phanerochaete chrysosporium

Two different types of extracellular protease activity were identified in the culture fluid of Phanerochaete chrysosporium wild-type BKM-F grown in submerged batch culture on N-limited media. The first activity, which appears to be inherent to the active growth phase, displayed a maximum on day 2 and decreased to a very low level on day 4. The second activity, which appeared at day 8 following the peak of ligninase activity, seems to be characteristic of late secondary metabolism and is stimulated by carbon starvation. Cultures started with half the amount of glucose of other cultures showed a remarkably earlier development of secondary activity. In contrast, the fed-batch addition of glucose started when ligninase activity was at a maximum (day 6) completely repressed secondary protease activity and enhanced ligninase production. The addition of exogenous veratryl alcohol increased the level of secondary protease activity, whereas the oxygen supply pattern significantly affected both the time course and the level of overall proteolytic activity. The addition of phenylmethylsulfonyl fluoride to growing cultures (0, 1, or 6 days) diminished overall protease activity, while it significantly enhanced ligninase activity. In all cases, the time courses of protease and ligninase activities were negatively correlated, indicating that protease activity promotes the decline of ligninase activity in batch culture.     

Development of a stirred tank reactor system for the production of lignin peroxidases (ligninases) byPhanerochaete chrysosporium BKM-F-1767

Summary Lignin peroxidases produced byPhanerochaete chrysosporium have several important potential industrial applications based on their ability to degrade lignin and lignin-like compounds. A stirred tank reactor system for the production of lignin peroxidases is described here. Included in this study is an examination of the mechanics of pellet biocatalyst formation and the optimization of an acetate buffered medium. Higher levels of lignin peroxidase were obtained with acetate buffer compared to the other buffer systems tested. Concentrations of 0.05% (w/v) Tween 80 and 0.4 mM veratryl alcohol gave optimal lignin peroxidase activity in acetate buffered medium. In shake flask cultures, mycelial fragments in the inoculum aggregated into pellets during the first eight hours of incubation and thereafter increased in size through the eighth day. The agitation rate in shake flask cultures affected pellet size, the number of pellets formed, and lignin peroxidase activity. Transfer of fungal pellets from shake flask culture to a continuously oxygenated baffled stirred tank reactor (STR) resulted in production of high lignin peroxidase titres comparable to those of shake flask cultures when the agitation rate, oxygen dispersion and foaming were closely controlled.     

Semi-continuous ligninase production using foam-immobilised Phanerochaete chrysosporium

Summary The immobilisation of Phanerochaete chrysosporium in cubes of polyurethane foam enables ligninase to be produced on a semi-continuous basis. At each successive harvest, cultures are purged with oxygen and ligninase activity induced with veratryl alcohol. Using 200 ml of a five-fold dilution of the batch culture medium in 1l flasks, harvests of ligninase with the same apparent protein profile by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) are obtained every 24 to 48 h.It is also possible to store cubes of foam containing pellets of P. chrysosporium so that further yields of ligninase comparable to fresh cultures can be produced within 48 h when desired.     

Production of Phanerochaete chrysosporium lignin peroxidase under various culture conditions

Lignin peroxidase production by the white-rot fungus Phanerochaete chrysosporium is markedly influenced by the buffer system employed. In immobilized P. chrysosporium cultures with carbon-limited glucose medium, the use of acetate buffer resulted in higher lignin peroxidase activities than tartrate. With acetate as the buffer in shake-flask cultures a 20% to over 100% improvement in lignin peroxidase production was obtained as compared to tartrate-buffered systems. Of trace elements, Cu²⁺, Mn²⁺ and Zn²⁺ seemed to have the greatest influence on lignin peroxidase production. Furthermore, an increase in the Cu²⁺ and Zn²⁺ concentrations resulted in considerably higher ligninase activities. Although it has been shown previously that high manganese levels repress ligninase production, for maximum ligninase production the presence of some Mn²⁺ appeared to be necessary. The concentration of phosphorus had surprisingly little effect on ligninase production. Highest lignin peroxidase activities were obtained with lower phosphorus concentrations, but reasonably high activities were obtained within the whole studied phosphorus range of 0.12–4.60 g l^–1. Diammonium tartrate alone was a better nitrogen source than a mixture of diammonium tartrate, proteose peptone and yeast extract. The addition of solid manganese (IV) oxide to 3-day-old immobilized biocatalyst cultures increased the maximum ligninase activity obtained by about one-third. Correspondence to: S. Linko     

彩绒革盖菌固体发酵生产木质素酶工艺优化研究*

采用均匀设计U15(5^8)和双温度培养法进行彩绒革盖菌固体发酵生产木质素酶,对漆酶、愈创木酚酶、多酚氧化酶的活力进行回归分析。结果表明：应用双温度培养法进行彩绒革盖菌固体发酵生产木质素酶时,在自然补给氧气,培养基pH自然(约6．5),并保持环境湿度约60％的条件下,20d是适宜的发酵周期;玉米浆、麸皮、(NH4)2SO4、水分适宜作为固体培养基的成分;少量的Tween-80有利于木质素酶的生产。     

Influence of some oils and surfactants on ligniolytic activity,growth and lipid fatty acids of Phanerochaete chrysosporium

Summary Ligninase production by Phanerochaete chrysosporium MZKIBK-B 186 was increased when the culture medium was supplemented with an emulsion of oleic acid. Addition of linseed oil enhanced fungal biomass synthesis. Under the growth conditions used in our tests, the fungus was capable of accumulating fatty acids from the culture medium into cell lipids. Addition of oleic acid, Tween 80, or 3-[(cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS), which are known to increase ligninase production by fungi, resulted in oleic acid enrichment of whole cell and polar lipids. Offprint requests to: D. Le相似文献   

Control of Lignin Peroxidase Production by Phanerochaete chrysosporium INA-12 by Temperature Shifting

There are two temperature optima connected with lignin peroxidase synthesis by Phanerochaete chrysosporium INA-12. One, at 37°C, is for the mycelium-growing phase; the other, at 30°C, is for the lignin peroxidase-producing phase. One of six extracellular proteins with ligninase activity increased when cultures were grown at 30°C for the entire fermentation period or when cultures were grown at 37°C for the first 2 days of incubation and then shifted to 30°C, compared with the activity of control cultures grown at 37°C for the entire fermentation period. The unsaturation of fatty acid (Δ/mole) of P. chrysosporium INA-12 mycelium decreased from 1.25 to 1.03 when the growth temperature was shifted from 20 to 40°C.     

藜芦醇和吐温80对白腐菌产木质素降解酶的影响及在偶氮染料脱色中的作用

担子菌PM2在限氮液体培养下,分泌木质素过氧化物酶和锰过氧化物酶;藜芦醇、吐温 80的补充,提高了该菌锰过氧化物酶的产生,获得的最大锰过氧化物酶Mnp酶活为254.2u/L、190.2 u/L,分别是对照的3.4倍和2.5倍。选择三种偶氮染料,在染料体系下,进一步分析藜芦醇、吐温 80对担子菌PM2产过氧化物酶及染料脱色的影响。结果表明,担子菌PM2分泌的锰过氧化物酶Mnp与染料脱色有关,脱色程度受其分子结构特征影响;吐温80的补充,更有利于染料的脱色降解,48h后三种染料均可达到80%以上的脱色率。     

Continuous production of lignin peroxidase by immobilized Phanerochaete chrysosporium in a pilot scale bioreactor

Lignin peroxidase was continuously produced by nylon-web or polyurethane immobilized Phanerochaete chrysosporium ATCC 24725 in a modified Biostate E® bioreactor, agitated either with the air and oxygen flow alone or in combination with a mechanical stirrer. Lignin peroxidase production started rapidly, and activities as high as ∼600 U l⁻¹ were reached as early as in 3 days. At best a total activity yield of ∼10 000 U was obtained during one week's continuous production with the maximum activity of ∼750 U l⁻¹ with nylon-web immobilized fungus, veratryl alcohol as an activator, and 2,2-dimethylsuccinate as buffer, although the relatively inexpensive benzyl alcohol and sodium tartrate peformed satisfactorily as the activator and buffer, respectively.     

Selection and Improvement of Lignin-Degrading Microorganisms: Potential Strategy Based on Lignin Model-Amino Acid Adducts

The purpose of this investigation was to test a potential strategy for the ligninase-dependent selection of lignin-degrading microorganisms. The strategy involves covalently bonding amino acids to lignin model compounds in such a way that ligninase-catalyzed cleavage of the models releases the amino acids for growth nitrogen. Here we describe the synthesis of glycine-N-2-(3,4-dimethoxyphenyl)ethane-2-ol (I) and demonstrate that growth (as measured by mycelial nitrogen content) of the known lignin-degrading basidiomycete Phanerochaete chrysosporium Burds. with compound I as the nitrogen source depends on its production of ligninase. Ligninase is shown to catalyze the oxidative C—C cleavage of compound I, releasing glycine, formaldehyde, and veratraldehyde at a 1:1:1 stoichiometry. P. chrysosporium utilizes compound I as a nitrogen source, but only after the cultures enter secondary metabolism (day 3 of growth), at which time the ligninase and the other components of the ligninolytic system (lignin → CO₂) are expressed. Compound I and related adducts have potential not only in the isolation of lignin-degrading microbes but, perhaps of equal importance, in strain improvement.     

Effect of oxygenation conditions on submerged cultures of Phanerochaete chrysosporium

Summary The effect of the oxygen supply pattern on the onset and development of the lignolytic enzyme system of Phanerochaete chrysosporium was studied in submerged culture employing the serum bottle approach. Periodic or continuous flushing through the head phase, and continuous bubbling through the liquid phase with either oxygen (O₂) or air were applied. The nature of the O₂ supply had a crucial regulatory effect not only on the formation of lignin-degrading peroxidases but also on their decay and on the production of extracellular protease activity and polysaccharides. Continuous oxygenation or aeration increased the glucose consumption rate, extracellular protease activity and polysaccharides. Gassing with air, whether continuous or periodic, sustained Mn-peroxidase activity while ligninase was undetectable. Continuous O₂ supply speeded up ligninase decay, displaying a sharper maximum, while a broader maximum and slower decay of ligninase activity were observed when supplying periodic O₂. Cultures initially grown with free exposure to air displayed a higher but sharper ligninase activity maximum when shifted to continuous rather than periodic O₂ supply. In general, the higher levels of either polysaccharides or protease activity corresponded to the lower levels and faster decay of ligninase and Mn-peroxidase activities. Offprint requests to: H. E. Grethlein