洗涤剂用酶的研究与开发──新型碱性脂肪酶

讨论了碱性脂肪酶的性质、生产及其在洗涤剂中的应用     

Acidic β-mannanase from Penicillium pinophilum C1: Cloning, characterization and assessment of its potential for animal feed application

The β-mannanase gene, man5C1, was cloned from Penicillium pinophilum C1, a strain isolated from the acidic wastewater of a tin mine in Yunnan, China, and expressed in Pichia pastoris. The sequence analysis displayed the gene consists of a 1221-bp open reading frame encoding a protein of 406 amino acids (Man5C1). The deduced amino acid sequence of Man5C1 showed the highest homology of 57.8% (identity) with a characterized β-mannanase from Aspergillus aculeatus belonging to glycoside hydrolase family 5. The purified rMan5C1 had a high specific activity of 1035 U mg^–1 towards locust bean gum (LBG) and showed highest activity at pH 4.0 and 70°C. rMan5C1 was adaptable to a wide range of acidity, retaining > 60% of its maximum activity at pH 3.0–7.0. The enzyme was stable over a broad pH range (3.0 to 10.0) and exhibited good thermostability at 50°C. The K_m and V_max values were 5.6 and 4.8 mg mL^–1, and 2785 and 1608 μmol min^–1 mg^–1, respectively, when LBG and konjac flour were used as substrates. The enzyme had strong resistance to most metal ions and proteases (pepsin and trypsin), and released 8.96 mg g^–1 reducing sugars from LBG in the simulated gastric fluid. All these favorable properties make rMan5C1 a promising candidate for use in animal feed.     

基于气相色谱-离子迁移谱法分析青霉侵染木耳菌丝的挥发性化合物

目的 基于气相色谱-离子迁移谱法(gas chromatography-ion mobility spectrometry, GC-IMS)研究青霉侵染木耳菌丝的挥发性化合物, 发掘潜在的特征标志物。方法 以分离自木耳菌包的桔青霉、歧皱青霉及黑木耳菌丝为研究对象, 采用气相色谱-离子迁移谱法(gas chromatography-ion mobility spectrometry, HS-GC-IMS)分析青霉侵染木耳菌丝不同时期挥发性化合物的种类及变化趋势, 并利用主成分分析(principal component analysis, PCA)比较不同侵染阶段挥发性化合物间的差异。结果 指纹图谱及PCA分析均可以比较明显地区分开青霉侵染木耳菌丝的不同阶段。挥发性化合物主要在桔青霉和歧皱青霉侵染木耳菌丝后的第4~8 d产生, 整体变化趋势基本一致, 共鉴定出41种化合物, 包括酯类、醇类、酮类、醛类及其他类。1-戊醇、乙酸己酯、2,3-戊二酮、2-丁酮、2,3丁二酮、1-丙醇、2-己醇、乙酸乙酯、丁酸乙酯、2-甲基丁酸等挥发性化合物可作为青霉侵染木耳菌丝的潜在标志物, 其中, 1-戊醇、乙酸己酯可作为青霉早期污染木耳菌丝的潜在标志物。3-羟基-2-丁酮可作为桔青霉侵染木耳菌丝后的潜在特征标志物。结论 本研究利用GC-IMS分析获得了两种青霉污染木耳菌丝的特征标志物, 为木耳栽培过程中青霉污染的早期预测提供了一定的理论基础。     

α-松油醇对意大利青霉的抑制作用

测定α-松油醇对意大利青霉菌丝体形态及菌丝体生长的影响,并对其抑菌机制进行初步探讨。结果表明:α-松油醇能明显抑制意大利青霉菌丝体生长,最小抑菌浓度(minimal inhibitory concentration,MIC)和最小杀菌浓度(minimal fungicidal concentration,MFC)分别为2.00μL/mL和8.00μL/mL;经MIC和MFCα-松油醇处理后,意大利青霉菌丝体胞外pH值、胞外电导率及260 nm条件下吸光度显著增加,总脂质含量有显著下降。提示α-松油醇能改变细胞膜的通透性、破坏细胞膜结构的完整性,导致胞内成分泄露,从而抑制意大利青霉菌丝体生长。     

Sensitivity of Penicillium expansum field isolates to tebuconazole, iprodione, fludioxonil and cyprodinil and characterization of fitness parameters and patulin production

A total of 236 Penicillium expansum field isolates from decayed apple fruit collected from packinghouses and processing industries located in the region of Imathia, Northern Greece were tested for their sensitivity to tebuconazole, fludioxonil, iprodione and cyprodinil. Preliminary fungitoxicity tests on the response of the isolates showed several phenotypes, distinguished according to their sensitivity to fungicides tested. The EC₅₀ values ranged from 0.64 to 5 (average = 0.98) μg/ml for iprodione, 0.9 to 7.3 (average = 2.66) μg/ml for tebuconazole, 0.008 to 1.28 (average = 0.55) μg/ml for cyprodinil and from 0.013 to 0.47 (average = 0.08) μg/ml for fludioxonil. A bimodal distribution of the EC₅₀ values of isolates with distinct sensitive and resistant populations to fludioxonil and tebuconazole were observed. In the case of cyprodinil, a much broader, hundred-fold, range of sensitivity was found, probably indicating that some isolates are relatively insensitive to cyprodinil compared to the most sensitive ones. Isolates exhibiting simultaneously reduced sensitivity to tebuconazole and fludioxonil or tebuconazole and iprodione or to tebuconazole and cyprodinil were also observed at low frequencies. A small portion of the population (7.5%) showed multiple resistance to tebuconazole, fludioxonil and iprodione. Study of fitness determining parameters showed that the resistance to tebuconazole, fludioxonil and iprodione had a significant adverse effect on mycelial growth rate and pathogenicity. Contrary to that, these fitness parameters were not affected in the isolates showing reduced sensitivity to cyprodinil. Analysis of patulin production on YES-agar growth medium and on artificially inoculated apple fruit showed that all isolates were mycotoxigenic. Most of the cyprodinil-insensitive isolates produced patulin at concentrations similar to or relatively higher (up to 1.5-fold on growth medium) than the sensitive ones. In contrast, a significant reduction (up to 98% of multiple resistant isolates) in patulin production was observed in all other phenotypes, indicating an adverse effect of fitness penalties on the mycotoxigenic ability of resistant isolates. The above mentioned data clearly show a considerable risk for the selection of P. expansum isolates resistant to fludioxonil, iprodione, tebuconazole and cyprodinil. The potential risk of increased patulin contamination of apples and their byproducts by the appearance and predominance of highly mycotoxigenic isolates of P. expansum resistant to the anilinopyrimidines is discussed.     

A kinetic model for the decrease in lipase activity of rhizopus arrhizus and penicillium cyclopium mycelia in organic media

The decrease in Lipase activity of Rhizopus arrhizus and Penicillium cyclopium mycelia (RAM and PCM) were studied in alkanes, ethers and chloroalkanes. The two biocatalysts showed similar behaviour in organic media, although RAM was slightly more stable. The kinetic model obtained indicated that the decrease in activity followed a succession of two first-order reactions. Both RAM and PCM were more stable in hydrophobic solvents. These two mycelia were used to synthesize N-octyl-myristylamide; yield of the amide was linked in a linear relationship to the average activity of biocatalyst in the solvent employed.     

Characterization of the intermediate product of coal solubilization by Penicillium simplicissimum

Penicillium simplicissimum has previously been shown to solubilize pre-oxidized alkali-extracted sub-bituminous coal. The product of solubilization, a soluble acid-precipitable coal polymer, was isolated and characterized. The effects of oxidation pretreatments on the ability to solubilize coal were also examined. The intermediate product, which comprised 30% of the original coal, was readily recovered from the growth medium by acid precipitation and possibly consisted of a heterogeneous mixture of high-molecular-weight compounds of approximately 2·7 × 10⁴ molecular weight. Further characterization by elemental analyses revealed that the bioproduct was enriched in inorganic materials, oxygen, nitrogen but lower in carbon, hydrogen and sulphur when compared with the original coal. A 14% loss of carbon atoms occurred during the biodegradation. The product had a featureless visible light spectrum and a shoulder in the ultraviolet range at 290 nm. Infrared analyses showed a decrease in aromatic carbons, methylenic bonds and etheric oxygen. Experimental results suggested that solubilization changes appear to be largely oxidative and may involve cleavage of intermonomeric linkages in coal.     

Biosorption of cadmium(II), zinc(II) and lead(II) by Penicillium simplicissimum: Isotherms, kinetics and thermodynamics

The isotherms, kinetics and thermodynamics of Cd(II), Zn(II) and Pb(II) biosorption by Penicillium simplicissimum were investigated in a batch system. The effects of pH, initial metal ions concentration, biomass dose, contact time, temperature and co-ions on the biosorption were studied. Adsorption data were well described by both the Redlich–Peterson and Langmuir model. Chemical ion-exchange was found to be an important process based on free energy value from Dubini–Radushkevich isotherm for all metal ions. The results of the kinetic studies of all metal ions at different temperature showed that the rate of adsorption followed the pseudo second-order kinetics well. The thermodynamics constants ΔG°, ΔH° and ΔS° of the adsorption process showed that biosorption of Cd(II), Zn(II) and Pb(II) ions on Penicillium simplicissimum were endothermic and spontaneous.     

Enhanced settleability and dewaterability of fungal treated domestic wastewater sludge by liquid state bioconversion process

A study was conducted to evaluate the settleability and dewaterability of fungal treated and untreated sludge using liquid state bioconversion process. The fungal mixed culture of Aspergillus niger and Penicillium corylophilum was used for fungal pretreatment of wastewater sludge. The fungal strains immobilized/entrapped on sludge particles with the formation of pellets and enhanced the separation process. The results presented in this study showed that the sludge particles (pellets) size of 2-5mm of diameter were formed with the microbial treatment of sludge after 2 days of fermentation that contained maximum 33.7% of total particles with 3-3.5mm of diameter. The settling rate (measured as total suspended solids (TSS) concentration, 130 mg/l) was faster in treated sludge than untreated sludge (TSS concentration, 440 mg/l) after 1 min of settling time. In 1 min of settling operation, 86.45% of TSS was settled in treated sludge while 4.35% of TSS settled in raw sludge. Lower turbidity was observed in treated sludge as compared to untreated sludge. The results to specific resistance to filtration (SRF) revealed that the fungal inoculum had significant potentiality to reduce SRF by 99.8% and 98.7% for 1% w/w and 4% w/w of TSS sludge, respectively. The optimum fermentation period recorded was 3 days for 1% w/w sludge and 6 days for 4% w/w sludge, respectively, for dewaterability test.     

一株药用红树尖瓣海莲内生真菌Penicillium sp.化学成分研究

综合运用硅胶柱层析、Sephadex LH-20凝胶柱层析等色谱分离技术,对一株来源于药用红树尖瓣海莲（Bruguiera sexangula var.rhynchopetala）的青霉属真菌Penicillium sp.的发酵产物的化学成分进行分离和纯化.结合理化性质和NMR等技术,并通过与文献对照,从该菌发酵产物中分离鉴定了5个化合物,分别为过氧化麦角甾醇（1）、豆甾-5-烯-3β-醇-7-酮（2）、（3β,5α,6β,22E）-麦角甾-7,22-二烯-3,5,6-三醇（3）、（20S,22E,24R）-麦角甾-7,22-二烯-3β,5α,6β,9α-四醇（4）和β-胡萝卜苷（5）.化合物2-5为首次从尖瓣海莲内生真菌中分离得到.