黄孢原毛平革菌生产锰过氧化物酶的发酵条件研究

目的 :研究黄孢原毛平革菌产锰过氧化物酶的发酵条件。方法 :对培养条件进行优化 ,采用正交设计法对培养基组分进行优化。结果与结论 :优化培养条件为 :接种量 1 6× 10 6 个孢子 L ,pH 4 4～ 4 8,温度 36℃～ 4 0℃ ,转数 12 0r/min。优化培养基参数为 :葡萄糖 5g L ,酒石酸铵 1 3mmol L ,吐温 - 80 1 2g L ,Mn2 + 0 9mmol L。     

黄孢原毛平革菌木素降解酶系的研究进展

黄孢原毛平革菌木素降解酶系主要由木素过氧化物酶、锰过氧化物酶和乙二醛氧化酶组成。由于该酶系特殊的降解机制,除了木质素,它能降解许多种类的有机污染物,因此在环保方面有巨大的应用前景。本文主要综述了国内外对该酶系的研究进展。     

Polymerisation of lignins by ligninases from Phanerochaete chrysosporium

Abstract Four major hemoproteins were purified by isoelectric focusing from an extracellular crude enzyme preparation, produced by the white rot fungus Phanerochaete chrysosporium under carbon-limited conditions. Both the crude enzyme and the purified proteins oxidised milled wood lignin, HCl-dioxane-extracted straw lignin and alkali straw lignin in the presence of hydrogen peroxide. The oxidation resulted mainly in further polymerisation of the lignins and was enhanced by addition of veratryl alcohol to the reaction mixture. Alkali straw lignin was also polymerised by horseradish peroxidase, although veratryl alcohol had no influence on this reaction.     

Fungal pretreatment of lignocellulose by Phanerochaete chrysosporium to produce ethanol from rice straw

Phanerochaete chrysosporium is a wood‐rot fungus that is capable of degrading lignin via its lignolytic system. In this study, an environmentally friendly fungal pretreatment process that produces less inhibitory substances than conventional methods was developed using P. chrysosporium and then evaluated by various analytical methods. To maximize the production of manganese peroxidase, which is the primary lignin‐degrading enzyme, culture medium was optimized using response surface methodologies including the Plackett–Burman design and the Box–Behnken design. Fermentation of 100 g of rice straw feedstock containing 35.7 g of glucan (mainly in the form of cellulose) by cultivation with P. chrysosporium for 15 days in the media optimized by response surface methodology was resulted in a yield of 29.0 g of glucan that had an enzymatic digestibility of 64.9% of the theoretical maximum glucose yield. In addition, scanning electronic microscopy, confocal laser scanning microscopy, and X‐ray diffractometry revealed significant microstructural changes, fungal growth, and a reduction of the crystallinity index in the pretreated rice straw, respectively. When the fungal‐pretreated rice straw was used as a substrate for ethanol production in simultaneous saccharification and fermentation (SSF) for 24 h, the ethanol concentration, production yield and the productivity were 9.49 g/L, 58.2% of the theoretical maximum, and 0.40 g/L/h, respectively. Based on these experimental data, if 100 g of rice straw are subjected to fungal pretreatment and SSF, 9.9 g of ethanol can be produced after 96 h, which is 62.7% of the theoretical maximum ethanol yield. Biotechnol. Bioeng. 2009; 104: 471–482 © 2009 Wiley Periodicals, Inc.     

Manganese peroxidase from the white-rot fungus Phanerochaete chrysosporium is enzymatically active and accumulates to high levels in transgenic maize seed

Manganese peroxidase (MnP) has been implicated in lignin degradation and thus has potential applications in pulp and paper bleaching, enzymatic remediation and the textile industry. Transgenic plants are an emerging protein expression platform that offer many advantages over traditional systems, in particular their potential for large-scale industrial enzyme production. Several plant expression vectors were created to evaluate the accumulation of MnP from the wood-rot fungus Phanerochaete chrysosporium in maize seed. We showed that cell wall targeting yielded full-length MnP, whereas cytoplasmic localization resulted in multiple truncated peroxidase polypeptides as detected by immunoblot analysis. In addition, the use of a seed-preferred promoter dramatically increased the expression levels and reduced the negative effects on plant health. Multiple independent transgenic lines were backcrossed with elite inbred corn lines for several generations with the maintenance of high-level expression, indicating genetic stability of the transgene.     

Effect of glucose oxidase on decolorization efficiency of crystal violet by Phanerochaete chrysosporium cultures

An enzymatic reaction using glucose oxidase (GOx) was applied for continues production of hydrogen peroxide and organic acid in Phanerochaete chrysosporium cultures for use simultaneously in catalytic cycle of peroxidases. Decolorization efficiency of crystal violet (CV) as a model pollutant was investigated in 16 d old cultures which overproduced manganese peroxidase (MnP) in response to daily GOx addition and control cultures (i.e. no GOx was added). However, the ability of overproduced cultures in decolorization of CV was not increased significantly, through addition of GOx (300?U/L)?+?glucose (10?Mm) to the culture medium at the start of decolorization, the time needed to obtain 87?±?0.5% removal of CV was reduced 10.7-fold in compared with the control culture. The best GOx concentration in culture medium for more efficient decolorization was obtained to be 300?U/L. These findings indicated that GOx in the presence of glucose could increase the degradation of CV not only by inducing ligninolytic activity in cultures but also as a subsidiary source for in situ H₂O₂ and organic acid production for catalytic activity of peroxidases in P. chrysosporium cultures.     

Suppressive effect of l-phenylalanine on manganese peroxidase in the white-rot fungus Phanerochaete chrysosporium

Abstract The effect of added l-amino acids and NH₄⁺ on manganese peroxidase activity in ligninolytic cultures of Phanerochaete chrysosporium were investigated. Among 11 amino acids (0.2 mM) tested, including phenylalanine, glutamate, glutamine, histidine, alanine, iso-leucine, ornithine, glycine, aspartate, proline, and arginine, phenylalanine was the most effective in suppression of manganese peroxidase synthesis. However, all the amino acids tested except proline completely suppressed the enzyme synthesis at 2 mM concentration.     

Fractionation of subcellular membranes of the secretory pathway from the peroxidase-producing white-rot fungus Phanerochaete chrysosporium

Abstract Mycelia from the basidiomycete Phanerochaete chrysosporium , producing lignin and manganese peroxidases, were homogenized and fractionated on a sucrose gradient. The main subcellular fungal membrane fractions were successfully separated. Lipid composition analyses of the isolated membranes as well as associated marker enzymes distribution gave evidence to similarities with membranes originating from plants. Lignin and manganese peroxidases were investigated by immunodetection in subcellular fractions. Our results show that lignin and manganese peroxidases are mainly associated with Golgi apparatus vesicles and, to a lesser extent, with endoplasmic reticulum and light density vesicles, but not with plasma membranes.     

Use of Phanerochaete chrysosporium Immobilized on Kissiris for Synthetic Dye Decolourization: Involvement of Manganese Peroxidase

The mineral Kissiris, which is formed from the thickened foam of volcanic lava, was tested to approximate its mineral composition using energy-dispersive X-ray (EDX) analysis. The solid mineral contains silicon dioxide at about 16 (w/w). The considerable surface roughness of Kissiris along with its extensive porosity made this natural solid cell support an attractive candidate for manganese peroxidase (MnP) production for synthetic dye decolourization, at low cost. The white rot fungus Phanerochaete chrysosporium immobilized on the mineral Kissiris was grown in both stationary and agitated cultures (rotary shaker, 100 rev/min) using either carbon- or nitrogen-limited growth medium to study the ability of the fungus to degrade the synthetic dye methylene blue (MB). The value of residual dye for MB used at 60 ppm was 6% within 8 days of the incubation of the nitrogen-limited culture under the shaken conditions. Production of (MnP) occurred simultaneously in nitrogen-limited culture medium with the added MnSO₄ at 100 ppm. The MnP activity was at relatively high level (170 U/l).     

Toward a control of lignin and manganese peroxidases hypersecretion by Phanerochaete chrysosporium in agitated vessels: Evidence of the superiority of pneumatic bioreactors on mechanically agitated bioreactors

Phanerochaete chrysosporium and cultivated both mechanically agitated and pneumatic bioreactors. In the pneumatic devices, the yields of lignin and manganese peroxidases as well as extracellular protein, were considerably increased as compared with mechanically agitated bioreactors. Lignin peroxidase and manganese peroxidase activities as high as 4500 U . L(-1) and 1812 U . L(-1) respectively, were produced in an airlift bioreactor. By using enzyme markers, the secretion pathway and the respiration were shown to be dramatically activated in pneumatic bioreactors. The general metabolism of the fungus, when cultivated in the conventional fermentors, is oriented toward the synthesis of biomass at the expense of the synthesis of peroxidases. The use of pneumatic devices for the production of extracellular peroxidases by P. chrysosporium, avoids shear effects due to turbine agitator in the conventional fermentors, and provides a good example for the production of shear-sensitive metabolites. (c) 1993 John Wiley & Sons, Inc.     

Stimulation of ligninolytic enzyme production in Phanerochaete chrysosporium by polyoxyalkanes

AIMS: Poly(ethylene glycol) (PEG) and some substances similar to PEG in chemical structure were tested as stimulators of ligninolytic enzyme production in shaken culture of Phanerochaete chrysosporium. METHODS AND RESULTS: The substances that caused high enzymatic activity were linear polymers [poly(ethylene glycol), poly(propylene glycol), poly(butylene glycol) and poly(vinyl alcohol)] and cyclic polymers (crown ether). They can have terminal groups other than -OH [PEG (di)methyl ether, PEG sulphate, PEG derivative with the amino group and xanthate]. The maximum lignin peroxidase activities were compared with the surface pressure caused by the stimulator. Addition of polymers composed of charged monomer units did not increase the enzymatic activity and the fungi did not grow at all on addition of polymers having a fixed positive charge. CONCLUSIONS: Lignin peroxidase activity was increased after the addition of polymers with uncharged monomer units. It was higher and its maximum was reached in a shorter time on addition of polymers with higher molecular weights. SIGNIFICANCE AND IMPACT OF STUDY: Beside Tweens there are several polymers that stimulate ligninolytic enzyme production in shaken culture of P. chrysosporium. Their characteristics are: similarity to PEG in chemical structure, having uncharged monomer units and high molecular weight.     

黄孢原毛皮革菌降解苯胺的工艺研究

通过正交试验优化筛选了适合黄孢原毛皮革菌降解苯胺的适宜培养基和摇瓶培养降解条件。结果表明：其适宜降解的液体培养基组成为：蔗糖20g／L,可溶性淀粉20g／L,(NH4)2SO4l0g／L,Mn^2 lμmol／L,Tween-800．3％,蛋白胨30g／L。适宜降解的摇瓶培养条件为：接种量为20％、pH为7．0、温度为30℃、培养时间为12d．此条件下的苯胺最高降解率可达95．5％。     

Oligomers of 4-chloroaniline are intermediates formed during its biodegration by Phanerochaete chrysosporium

Abstract Lignin peroxidase H2 (LP-H2) from Phanerochaete chrysosporium oxidized 4-chloroaniline to form several oligomers. Included among the compounds identified were: 4,4'-dichloroazobenzene, 2-(4-chloroanilino)-5-hydroxybenzoquinone-di-4-chloroanil and 2-amino-5-(4-chloroanilino) benzoquinone-di-4-chloroanil. In contrast to results by other, we showed that oligomers of 4-chloroaniline were also formed by the fungus in vivo. It was also demonstrated that, although these potentially toxic intermediates are made, they are also degraded.     

Isolation and characterization of Mn(III) tartrate from Phanerochaete chrysosporium culture broth

High initial Mn(II) concentration results in accumulation of a Mn(III) tartrate complex in the growth medium of Phanerochaete chrysosporium. Since Mn(III) is the major oxidant in ligninolysis by manganese peroxidase, the role of accumulated complex should not be neglected when degradation experiments by a crude culture filtrate are performed. To study the Mn(III) complex oxidative potential it was isolated by absorption to polyamide followed by desorption with an alkaline methanol solution. High performance liquid chromatography analysis and atomic absorption spectroscopy confirmed that the isolate was Mn(III) tartrate. Oxidation of 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonate) was used for testing the temperature and pH stability of the isolate that also intensively oxidized 2,6-dimethoxyphenol. In comparison with the non-isolated complex in the culture filtrate, the isolate showed increased temperature and pH stability. The oxidative potential of the isolated Mn(III) tartrate was additionally tested by decolorization of the synthetic dye Indigo carmine.     

Effect of veratryl alcohol and manganese (IV) oxide on ligninolytic activity in semi solid cultures of Phanerochaete chrysosporium

An inert carrier (nylon sponge), a non-inert carrier (barley straw) and the addition of veratryl alcohol or manganese (IV) oxide to the cultures were used to study the production of ligninolytic enzymes by Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) during semi solid state fermentation conditions. By supplementing the medium with these compounds we could stimulate the ligninolytic system of this fungus. The different carriers employed and the effect of adding veratryl alcohol or manganese (IV) oxide to the cultures were compared in order to determine the best system to produce high activities of ligninolytic enzymes. Lignin peroxidase (LiP) activities higher than 500 U/L and manganese-dependent peroxidase (MnP) activities about 1100 U/L were achieved.     

Production of lignin peroxidase by Phanerochaete chrysosporium immobilized on porous poly(styrene-divinylbenzene) carrier and its application to the degrading of 2-chlorophenol

Porous poly(styrene-divinylbenzene) carriers, for the immobilization of white rot fungus Phanerochaete chrysosporium have been prepared by the concentrated emulsion polymerization method. The concentrated emulsion consists of a mixture of styrene and divinylbenzene containing a suitable surfactant and an initiator as the continuous phase, and water as the dispersed phase. The polymerization of the monomers of the continuous phase generated the polymer carrier with a porcus structure. The white rot fungus Phanerochaete chrysosporium has been immobilized on porous poly(styrene-divinylbenzene) carriers and used for the batch production and the repeated batch production of lignin peroxidase in shake cultures based on a carbon-limited medium containing veratryl alcohol. The best results were achieved when a spore inoculum was used for immobilization instead of 1-day-old mycelial pellets, for both the batch production and the repeated batch production. The porous poly(styrene-divinylbenzene) immobilized Phanerochaete chrysosporium and freely suspended mycelial pellets were used as biocatalysts for the degradation of 2-chilorophenol in a 2-L bioreactor. The porous poly(styrene-divinylbenzene) particle (diameter congruent with 0.2 cm) immobilized spores exhibited a much higher activity in the degradation of 2-chlorophenol than the freely suspended mycelial pellets. (c) 1994 John Wiley & Sons, Inc.     

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.     

Effects of Zn2+ and Cu2+ on growth,lignin degradation and ligninolytic enzymes in Phanerochaete chrysosporium

The influence of Zn²⁺ (6.0 × 10^–3 –18.0 × 10^–3 M) and Cu²⁺ (4 × 10^–4 –1.2 × 10^–4 M) in the basal medium on mycelial growth (dry weight), activities of lignin peroxidase (Lip), manganese peroxidase (Mnp), solubilization, and mineralization (¹⁴CO₂ evolution) of lignin during a period of 3 weeks was studied in Phanerochaete chrysosporium strain MTCC-787. Highest mycelial growth was obtained at 0.6 M Zn²⁺ and 0.4 M Cu²⁺ levels. Enzyme activities were found to increase up to the highest levels of both the trace elements. However, Zn²⁺ had a relatively more stimulatory effect on Lip production and the reverse was true in case of Cu²⁺. [¹⁴C]Lignin solubilization was also promoted by higher levels of both trace elements. Mineralization of [¹⁴C]lignin was optimal at 6.0 M Zn²⁺ and 1.2 M Cu²⁺. The stimulatory effect of Zn²⁺ on Lip production was correlated with higher rates of [¹⁴C]lignin mineralization.     

Establishment of the white rot fungus Phanerochaete chrysosporium on unsterile straw in solid substrate fermentation systems intended for degradation of pesticides

The effects of different inoculum-loading rates and pre-treatment of wheat straw with formic acid and hot water (50 °C) on the establishment of Phanerochaete chrysosporium on unsterile straw were studied in laboratory scale and in a 1.5-m³ bioreactor. The establishment of P. chrysosporium on unsterile straw was satisfactory. Phanerochaete chrysosporium and other fungi, which developed simultaneously, were able to produce the activity necessary to degrade two herbicides, bentazon and MCPA (4-chloro-2-methylphenoxyacetic acid) in 20 days (65 and 75%, respectively). The decrease of both herbicides coincided with the presence of the activity of the lignin-degrading enzymes lignin peroxidase and manganese peroxidase/laccase. Extensive growth of P. chrysosporium or other lignin-degrading fungi on unsterile straw would be excellent for inexpensive solid substrate systems intended for degradation of pesticides.