白腐菌Trametes hirsuta BYBF在三相流化床生物反应器产漆酶的研究

应用三相流化床生物反应器对Trametes hirsuta BYBF进行了产漆酶的研究,结果表明,菌丝球在流化床内稳定,能达到较好的流化状态,其最佳的条件为接种量10%,通气量0.3L/min,发酵5d后酶活即可达到3.36U/mL。并对白腐菌进行了重复分批培养,与摇床发酵相比,不仅提高了漆酶活力,而且也大大缩短了产酶周期,提高了生产效率。     

白腐菌Trametes versicolor漆酶的分离纯化及其预处理对麦草蒸煮性能和物理性能的影响

介绍了白腐菌Trametes versicolor漆酶的分离纯化,利用漆酶在蒸煮前对原料进行预处理,研究漆酶预处理对麦草蒸煮性能和成浆物理性能的影响。结果表明：预处理最适条件为pH值5．0,温度40℃,液比1：6,时间4h,酶用量30lU／g。在上述适宜条件下预处理后浆料得率提高5．46个百分点,Kappa值下降了3．9;裂断长、撕裂指数和白度分别提高25．55％、43．71％、4．6％SBD。     

Improving the nutritional value of wheat bran by a white-rot fungus

Summary  This investigation was concerned with the solid-state cultivation of the white-rot fungus Lentinula edodes on wheat bran and its impact on the nutritional value of the substrate. A considerable reduction (60%) in the concentration of the phytic acid of wheat bran was detected after 7 days of incubation, but no further reduction was observed during the following weeks. Protein digestibility increased gradually during the first 3 weeks of incubation reaching a maximum (82%) at day 20. Hydrolytic and oxidative exoenzymes, such as phytase, endocellulase and polyphenol oxidase, were released by the mycelium during the colonization of the substrate. Total and insoluble dietary fibre and crude protein content in bran increased with fungal growth while the in vitro dry matter enzyme digestibility decreased.     

云芝发酵处理对甘草渣中总黄酮提取的影响

以甘草加工后的残渣为原料,采用固态发酵方式研究云芝发酵作用对甘草渣中总黄酮分离提取的影响,并以甘草总黄酮得率为指标优化工艺条件。结果表明：与乙醇直接提取法相比,云芝发酵处理能有效提高甘草总黄酮的得率;发酵过程受多种因素影响,其中发酵温度、发酵料含水率、发酵时间对总黄酮得率影响较大;最终确定云芝发酵法提取甘草渣中总黄酮的最佳工艺条件为：添加0.2g/L 酒石酸铵作为氮源,将甘草渣发酵料含水率调至55%,接种云芝菌于28℃发酵3d,在该工艺条件下,总黄酮得率达到1.18%,比乙醇直接提取法的总黄酮得率(0.62%)提高了90.32%。     

Isolation of five laccase gene sequences from the white-rot fungus Trametes sanguinea by PCR, and cloning, characterization and expression of the laccase cDNA in yeasts

To obtain laccase-gene-specific sequences from the white-rot fungus Trametes sanguinea M85-2, a PCR screening method was used. Degenerate primers were designed based on highly conserved copper-binding regions I and IV of known laccases and used to amplify laccase sequences from T. sanguinea M85-2 genomic DNA. A single 1.6-kbp DNA band was amplified and cloned into a vector. Partial sequences of 21 clones were classified into five groups (lcc1-5) and the deduced amino acid sequences were all homologous to known laccase sequences. Based on the partial sequence of lcc1, the 5'-end of its cDNA was obtained by a PCR termed 5' rapid amplification of cDNA ends (5'-RACE), and RT-PCR was then carried out using the 5'-primer and the poly-dT primer to obtain the full-length lcc1 cDNA. The obtained cDNA encoded a protein consisting of 518 amino acid residues and its first 21 amino acid residues were predicted to be the signal peptide for secretion. The conserved characteristic structures of laccase, such as copper-binding ligands, N-glycosylation sites, and cysteine residues for disulfide bridges, were observed. The genomic DNA sequence of the lcc1 gene was also cloned by PCR method and the sequence revealed 10 introns. The lcc1 cDNA was inserted into yeast vectors for heterologous expression by Saccharomyces cerevisiae and Pichia pastoris. Phenol-oxidizing activity was detected from transformants of the yeasts, indicating that the obtained cDNA encodes a laccase. Previously, two laccase isozymes were biochemically characterized and purified from T. sanguinea M85-2. Using the sequential PCR method presented here, we have obtained partial sequences of at least five laccase genes and one cDNA clone encoding a protein with laccase activity but without any enzymatic information, suggesting that expressed enzymes under restricted conditions may not represent all the isozymes in target microorganisms. PCR cloning and heterologous expression of the cloned genes can be an alternative method of screening enzymes if these enzymes have conserved sequences.     

Decrease of the chlorogenic acid content in commercial sunflower meal using a polyphenol oxidase preparation secreted by the white‐rot fungus Trametes versicolor ATCC 42530

BACKGROUND: Sunflower meal (SFM) is a by‐product from the oil extraction process from sunflower seeds. The meal is used as a protein supplement in the livestock diet. However, relatively high levels of polyphenols, among which chlorogenic (CGA) and caffeic acids are in larger amounts, in the meal compromises its use for animal feed and human consumption. The aim of this work was to investigate an enzymatic process for upgrading the quality of SFM by decreasing its CGA content using an enzyme preparation from the white‐rot fungus Trametes versicolor. RESULTS: The effects of pH, temperature, enzyme and meal concentrations, and mass transfer on the decrease of the CGA content in SFM were investigated. It was found that: (1) the optimum pH and temperature were 3.4 and 45 °C, respectively. (2) The system was saturated with the enzyme when its concentration was 5 nkat/mL of liquid phase; (3) the agitation speed of the system influenced the extraction of CGA from the meal; and (4) the conversion of CGA in the SFM system increased in the presence of larger volumes of liquid phase. CONCLUSIONS: The enzyme preparation used in the experiments is able to decrease successfully the CGA content in SFM. Copyright © 2007 Society of Chemical Industry     

白腐菌发酵对麸皮性质的影响

为了提高麸皮的附加值,探究了白腐菌发酵对麸皮性质的影响。以白腐菌发酵麸皮为研究对象,以漆酶酶解和未处理麸皮为对照,测定溶胀性、持水性、膳食纤维含量、植酸含量等多种理化指标。结果表明:经白腐菌发酵和漆酶酶解的麸皮可溶纤维含量明显提高,持水性和持油性升高,溶胀性和植酸含量降低。白腐菌发酵后麸皮表面结构变得疏松多孔。为麸皮资源的开发利用提供理论依据。     

Determination of co-metabolism for 1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane (DDT) degradation with enzymes from Trametes versicolor U97

Trametes versicolor U97 isolated from nature degraded 73% of the 1,1,1-trichloro-2,2-bis(4-chlorophenyl) ethane (DDT) in a malt extract liquid medium after a 40-d incubation period. This paper presents a kinetic study of microbial growth using the Monod equation. T. versicolor U97 degraded DDT during an exponential growth phase, using glucose as a carbon source for growth. The growth of T. versicolor U97 was not affected by DDT. DDT was degraded by T. versicolor U97 only when the secondary metabolism coincided with the production of several enzymes. Furthermore, modeling of several inhibitors using the partial least squares function in Minitab 15, revealed lignin peroxidase (98.7 U/l) plays a role in the degradation of DDT. T. versicolor U97 produced several metabolites included a single-ring aromatic compound, 4-chlorobenzoic acid.     

Effect of some metal nanoparticles on the spectroscopy analysis of Paulownia wood exposed to white-rot fungus

The present study was conducted to investigate the effects of impregnation with silver, copper, and zinc oxide aqueous nanofluids on the chemical properties of Paulownia fortunei decayed with the white-rot fungus Trametes versicolor. Wood specimens with a dry density of 0.37 g/cm³ were impregnated with a 400 ppm aqueous suspension of nanoparticles in a pressure vessel under 2.5 bars for 20 min. Specimens were inoculated with the fungus and incubated for 16 weeks at 26 °C and 65 % relative humidity in accordance with EN113. The spectra of specimens impregnated with nanosilver, nanocopper, or nanozinc-oxide showed no significant changes in their relative peak intensities of lignin and carbohydrates after exposure to T. versicolor. This can indicate fungicide effects of the metal nanoparticles on T. versicolor.     

Family 3 beta-glucosidase from cellulose-degrading culture of the white-rot fungus Phanerochaete chrysosporium is a glucan 1,3-beta-glucosidase

The substrate specificity of an extracellular beta-glucosidase (BGL) from cellulose-degrading culture of the white-rot fungus Phanerochaete chrysosporium was investigated, using a variety of compounds with beta-glucosidic linkages. Amino acid sequencing data for the purified BGL showed that the enzyme is identical to the glycoside hydrolase (GH) family 3 BGL of the same fungus previously reported [Li, B. and Renganathan, V, Appl. Environ. Microbiol., 64, 2748-2754 (1998)]. The BGL can hydrolyze both cellobiose and cellobionolactone, but cellobionolactone was hydrolyzed very much more slowly than cellobiose. Moreover, cellobionolactone inhibited cellobiose hydrolysis by the BGL, suggesting that this enzyme cannot cooperate with cellobiose dehydrogenase (CDH) in cellulose degradation by P. chrysosporium. In addition to cellobiose, BGL utilized various glucosyl-beta-glucosides, such as sophorose, laminaribiose and gentiobiose, as substrates. Among the four substrates, laminaribiose (beta-1,3-glucosidic linkage) was hydrolyzed most effectively. Moreover, the hydrolytic rate of laminarioligosaccharides increased proportionally to the degree of polymerization (DP), and the activity of BGL even towards laminarin with an average DP of 25 was similar to that towards laminaripentaose (DP 5). Therefore, we conclude that the extracellular BGL from P. chrysosporium is primarily a glucan 1,3-beta-glucosidase (EC 3.2.1.58), which might play a role on fungal cell wall metabolism, rather than a beta-glucosidase (EC 3.2.1.21), which might be involved in the hydrolysis of beta-1,4-glucosidic compounds during cellulose degradation.     

Biosynthesis of p-anisaldehyde by the white-rot basidiomycete Pleurotus ostreatus

The white-rot basidiomycete Pleurotus ostreatus produced sweet flavor compounds on a liquid medium. The major and minor compounds identified by GC-MS analysis were p-anisaldehyde (4-methoxybenzaldehyde) and 3-chloro-p-anisaldehyde (3-chloro-4-methoxybenzaldehyde), respectively. p-Anisaldehyde was only produced under static culture conditions. Differences in the type and quantity of flavor compounds produced among wild strains of P. ostreatus were observed. Aryl alcohol oxidase and manganese peroxidase activities increased parallel to the production of p-anisaldehyde. These results indicated that the biosynthesis of p-anisaldehyde is concerned in generating H2O2-activated peroxidase in the lignin-degradation system. Addition of L-tyrosine to the culture led to higher production of p-anisaldehyde. The flavor extract, which contains p-anisaldehyde, exhibited antimicrobial activity against Bacillus subtilis, Pseudomonas aeruginosa, Aspergillus niger and Fusarium oxysporum.     

Azo dyes decolourization by the laccase from Trametes trogii

Dye decolourizing potential of laccase obtained from the white rot fungus Trametes trogii was studied for two reactive dyes; namely Reactive Black 5 (diazoic) and Reactive Violet 5 (monoazoic). The presence of a redox mediator as 1-hydroxybenzotriazole (HBT) was found to be essential for the decolourization of the said two dyes. The optimization of the decolourization process using experimental design was studied with three variables: dye (25, 50, 100 mg/L), enzyme (0.1; 0.5; 1 U/mL) and redox mediator (0.1; 0.5; 1 mM) concentrations. Results of this investigation revealed that the optimum concentrations of dye, enzyme and HBT for the degradation of the Reactive Black 5 (RB5) were 25 mg/L, 1U/mL and 1 mM, respectively, for a maximum decolourization about 93%. However, the optimum concentrations for Reactive Violet 5 (RV5) were found to be 25 mg/L, 0.5 U/mL and 0.5 mM, for a total removal of the dye. The apparent capacity of this laccase to decolourize azo dyes make it a suitable candidate for further applications in biobleaching and the treatment of textile effluents.     

Effect of exposure history on microbial herbicide degradation in an aerobic aquifer affected by a point source

The effects of in situ exposure to low concentrations (micrograms per liter) of herbicides on aerobic degradation of herbicides in aquifers were studied by laboratory batch experiments. Aquifer material and groundwater were collected from a point source with known exposure histories to the herbicides mecoprop (MCPP), dichlorprop, BAM, bentazone, isoproturon, and DNOC. Degradation of the phenoxy acids, mecoprop and dichlorprop, was observed in five of six sampling points from within the plume. Mecoprop was mineralized, and up to 70% was recovered as 14CO2. DNOC was degraded in only two of six sampling points from within the plume, and neither BAM, bentazone, nor isoproturon was degraded in any sampling point. A linear correlation (R2 > or = 0.83) between pre-exposure and amount of herbicide degraded within 50 days was observed for the phenoxy acids, mecoprop and dichlorprop. An improved model fit was obtained from using Monod degradation kinetics compared to zero- and first-order degradation kinetics. An exponential correlation (R2 > or = 0.85) was also found between numbers of specific phenoxy acid degrading bacteria and pre-exposure. Combination of these results strongly indicates that the low concentration exposure to phenoxy acids in the aquifer resulted in the presence of acclimated microbial communities, illustrated bythe elevated numbers of specific degraders as well as the enhanced degradation capability. The findings support application of natural attenuation to remediate aerobic aquifers contaminated by phenoxy acids from point sources.     

Decolorization of azo dye by the white-rot basidiomycete Phanerochaete sordida and by its manganese peroxidase

We investigated the decolorization of an azo-reactive dye, Reactive Red 120, by a white-rot basidiomycete, Phanerochaete sordida strain YK-624. In liquid culture of P. sordida in a medium containing 3% malt extract and 200 mg/l of the dye, the dye was 90.6% decolorized after 7 d. Manganese peroxidase (MnP) activity was detected during the decolorization process. The dye could be decolorized by purified MnP of P. sordida in the presence of Mn(II) and Tween 80. The involvement of lipid peroxidation during decolorization with MnP was considered. With shaking, the dye could be decolorized without the addition of hydrogen peroxide. The decolorization did not occur under anaerobic conditions, suggesting that dye decolorization by MnP is influenced by dissolved oxygen. Since catalase did not inhibit the decolorization with MnP, we inferred that the MnP catalytic cycle would be promoted by hydroperoxides formed from the decomposition of malonate or from lipid peroxidation.     

诱导物及种间诱导对白腐菌的锰过氧化物酶(MnP)的诱导

本实验通过添加外源诱导物诱导和不同白腐菌种间相互诱导两种途径来研究Trametes versicolor、Pleurotus ostreatus、Dichomitus squalens、Bjerkandera adusta四种白腐菌产Mn P活性,发现这些白腐菌在一些诱导条件下可产生大量Mn P。结果表明,外源诱导物中,2,5-二甲基苯胺是有效的Mn P诱导剂,对四种菌产Mn P均有不同程度的诱导作用。异丙醇能提高T.versicolor、P.ostreatus和D.squalens产Mn P活性,且诱导效果优于乙醇。Cu2+能显著提高T.versicolor和D.squalens产Mn P活性。Mn2+能显著提高P.ostreatus和B.adusta产Mn P活性。不同白腐菌相互作用时,T.versicolor、P.ostreatus和D.squalens三种菌两两共培养均有明显的Mn P种间诱导效果。本研究为大量产Mn P并将其应用于工业提供可能。     

诱导物及种间诱导对白腐菌的锰过氧化物酶(MnP)的诱导

本实验通过添加外源诱导物诱导和不同白腐菌种间相互诱导两种途径来研究Trametes versicolor、Pleurotus ostreatus、Dichomitus squalens、Bjerkandera adusta四种白腐菌产Mn P活性,发现这些白腐菌在一些诱导条件下可产生大量Mn P。结果表明,外源诱导物中,2,5-二甲基苯胺是有效的Mn P诱导剂,对四种菌产Mn P均有不同程度的诱导作用。异丙醇能提高T.versicolor、P.ostreatus和D.squalens产Mn P活性,且诱导效果优于乙醇。Cu2+能显著提高T.versicolor和D.squalens产Mn P活性。Mn2+能显著提高P.ostreatus和B.adusta产Mn P活性。不同白腐菌相互作用时,T.versicolor、P.ostreatus和D.squalens三种菌两两共培养均有明显的Mn P种间诱导效果。本研究为大量产Mn P并将其应用于工业提供可能。      

Enzymatic degradation of cellulose acetate plastic by Novel degrading bacterium Bacillus sp. S2055

Cellulose acetate (CA)-degrading bacteria were isolated from samples obtained from environments at a population size of 6.7 x 10(1) to 1.0 x 10(8) halo-forming cfu/ml-water or g-solid, suggesting their ubiquitous presence. The classification of 35 isolated strains of CA-degrading bacteria into 15 genera indicates that CA-degrading activity is over a wide range of taxonomical groups. From these isolates, Bacillus sp. S2055 was found to be the most efficient CA-degrading bacterium, and its CA-degrading enzyme(s) was partially characterized. The weight loss of CA plastic film (degree of substitution (DS)=1.7) in the culture of S2055 was less than 12% after a 35-d culture while that in the crude enzyme solution extracted from the culture supernatant reached 62% after the same period. Lipase and cellulase activities were detected in the culture supernatant of strain S2055. The crude enzyme solution contained three major protein fractions that have different mean molecular weights (MWs). Fraction I with the highest MW exhibited both lipase and cellulase activities, while fraction II and III exhibited only lipase activity. Fraction I significantly deacetylated CA (DS 1.5) and fragmented CA plastic film into pieces while the other fractions failed to do so even when used in combination with commercially-available cellulases and lipases. The lipase activity of fraction I against various substrates differed considerably from those of known lipases. It was thus suggested that deacetylation of CA mediated by an enzyme with such a peculiar lipase-like activity is a requisite for the efficient biodegradation of CA plastics.     

酶法制备云芝菌丝体肽的研究

通过分析三种蛋白酶对云芝菌丝体的酶解情况,确定以中性蛋白酶作为水解酶用酶,用单因素实验考察了其相应的酶解条件,正交实验得出最佳水解条件为:pH8.0,温度50℃,底物浓度10%,酶浓度0.6mg/g,水解时间2h,在此条件下的水解度为33.28%.将酶解液通过10、5、3kDa截流分子量的超滤膜,测定不同分子量段肽溶液的肽得率和清除羟基自由基的IC50值,结果表明:分子量在3kDa以下的肽得率最高,清除羟基自由基的能力最强.