极端微生物及其适应机理的研究进展

极端微生物是生物对极端环境适应的特殊种类 ,研究极端微生物的特性对探索生命的起源、微生物的育种及开发利用等具有重要意义。从嗜热微生物、嗜冷菌和耐冷菌、极端嗜酸微生物、嗜碱微生物、嗜盐微生物、嗜压微生物等方面总结了极端微生物及其适应机理的多样性以及其研究进展 ,旨在为极端微生物的开发利用提供一定的参考依据。     

Antibiotics from Extremophilic Micromycetes

Russian Journal of Bioorganic Chemistry - Extremophilic microorganisms, which are capable of functioning normally at extremely high or low temperatures, pressure, and in other environmental...     

嗜极性木聚糖酶

木聚糖酶内切水解木聚糖主链的1,4-β-D-糖苷键,木聚糖是植物细胞壁中一种主要的多糖。自然界中木聚糖是多种糖类的复合体,这就使得木聚糖酶呈现多态性和多域性,由此需将繁多的木聚糖酶进行归类。木聚糖酶的催化反应属于双置换机制。在已研究的真菌或细菌性木聚糖酶中,大多数在温和的条件下表现出最佳活性,但有很多在极端环境下生长的生物体,为了适应极端环境而产生嗜极性的酶,其中嗜酸的、嗜碱的、嗜热的木聚糖酶,现在已有广泛的研究。对嗜极性木聚糖酶的研究进展作了论述。     

Solvent-Dependent Gating Motions of an Extremophilic Lipase from Pseudomonas aeruginosa

Understanding how organic solvent-stable proteins can function in anhydrous and often complex solutions is essential for the study of the interaction of protein and molecular immiscible interfaces and the design of efficient industrial enzymes in nonaqueous solvents. Using an extremophilic lipase from Pseudomonas aeruginosa as an example, we investigated the conformational dynamics of an organic solvent-tolerant enzyme in complex solvent milieux. Four 100-ns molecular dynamics simulations of the lipase were performed in solvent systems: water, hexane, and two mixtures of hexane and water, 5% and 95% (w/w) hexane. Our results show a solvent-dependent structural change of the protein, especially in the region that regulates the admission of the substrate. We observed that the lipase is much less flexible in hexane than in aqueous solution or at the immiscible interface. Quantified by the size of the accessible channel, the lipase in water has a closed-gate conformation and no access to the active site, while in the hexane-containing systems, the lipase is at various degrees of open-gate state, with the immiscible interface setup being in the widely open conformation ensembles. The composition of explicit solvents in the access channel showed a significant influence on the conformational dynamics of the protein. Interestingly, the slowest step (bottleneck) of the hexane-induced conformational switch seems to be correlated with the slow dehydration dynamics of the channel.     

Evaluation of the LIVE/DEAD BacLight Kit for Detection of Extremophilic Archaea and Visualization of Microorganisms in Environmental Hypersaline Samples

Extremophilic archaea were stained with the LIVE/DEAD BacLight kit under conditions of high ionic strength and over a pH range of 2.0 to 9.3. The reliability of the kit was tested with haloarchaea following permeabilization of the cells. Microorganisms in hypersaline environmental samples were detectable with the kit, which suggests its potential application to future extraterrestrial halites.     

Milk-clotting Enzyme from Microorganisms

The milk-clotting activity of Mucor-rennin obtained from Mucor pusillus Lindt, was not changed by the addition of DFP in the reaction mixture. This finding suggested the probable absence of a serine residue at the active center of the enzyme. Sulfhydryl reagents such as Nekelgon, N-ethyl maleimide, PCMB failed to influence the milk-clotting reaction, indicating that a. reactive sulfhydryl group is not required for the enzymatic activity. The activity was inhibited when Mucor-rennin was treated with iodine at pH higher than 5.0. It was shown that ¹³¹I₂ was incorporated into the enzyme. When Mucor-rennin was photooxidized in the presence of methylene blue, the milk-clotting activity was inactivated. In this case, tyrosine, tryptophan, and histidine residues in the enzyme were oxidized. Among these amino acids, the histidine residue was more rapidly oxidized than other amino acids. A parallel relation was observed between the decrease of the amount of histidine residue and the inactivation of the enzyme. From these results, it is concluded that the histidine residue present in Mucor-rennin has a relation to the active center of this enzyme.     

Milk-clotting Enzyme from Microorganisms

The milk-clotting activity of Mucor-rennin (Milk clotting enzyme of Mucor pusillus Lindt) was inhibited by reaction of diazo-l-H-tetrazole accompanied with increase of the value of the absorbance of biazo-histidine at 480 nm. The activity did not remain when the absorbance reached 50% of maximum value. It is concluded from these results that one mole of histidine in 2 moles of histidine contained in the enzyme has a relation to active center.     

Studies on Xylanase from Microorganisms

X-Ray diffraction analysis of the α-cyclodextrin complexes with a number of organic guest molecules were carried out. Several different kinds of the X-ray diffraction patterns were obtained. It was found that different guest molecules enclosed within the void of the dextrin cause large changes in the diffraction patterns of the complexes. However, most of the diffraction patterns could be reasonably interpreted in terms of the hexagonal unit cells with minor differences in the unit cell dimensions ranging a = b = 27.0 ~ 27.8 Å and c = 14.7 ~ 16.7 Å. The crystal structure of the complexes could be accounted for by a closest packing of channel cylinders that are made by coaxial alignments of the dextrin molecules and the cage structure in the crystal, in which the dextrin molecules align non-coaxially, may not be plausible.     

Milk Clotting Enzyme from Microorganisms

The purification of the milk clotting enzyme from Mucor pusillus Lindt could be achieved by column chromatography on Amberlite IRC-50 by raising pH from 3.5 to 4.5 and about 70% of activity was recovered after this treatment. After the treatment through the column of DEAE-Sephadex A-25, the trace cellulase activity could be eliminated.

The homogeneity of the purified preparation was proved by ultracentrifugal analysis and electrophoretic patterns at various pH values.

Isoelectric point of this enzyme is considered to lie between pH 3.5 and 3.8.

The enzyme activity was inhibited by Hg⁺⁺ or Fe⁺⁺⁺.

Trypsinogenkinase activity was not contained in this enzyme.

The antiserum against the milk clotting enzyme from Mucor pusillus reacted with the purified and crude enzyme preparations in precipitin test and inhibited their enzyme activities, but did not react with other enzymes such as rennin, pepsin, acid proteases from Aspergillus saitoi and Aspergillus oryzae, or the culture filtrates of some strains of Mucor and Rhizopus.

The antigen-antibody reaction was so specific that it might be possible with this antibody to identify this enzyme and also the strain itself.

Normal sera from some mammals inhibited this enzyme activity too, but the degree was less than that with rennin.     

Milk Clotting Enzyme from Microorganisms

Out of some 800 strains of microorganisms, a potent fungus for milk clotting enzyme was isolated from soil during the course of screening tests and was identified as one of strains of Mucor pusillus Lindt. Satisfactory results were obtained in cheese making experiments with this enzyme which could be produced effectively by solid culture on wheat bran at 30°C for about 70 hrs.

The balance between milk clotting activity and proteolytic activity of this enzyme resembled very much to that of rennet.

Microbial rennet from Mucor pusillus F-27 was obtained with high productivity by solid culture followed by water extraction. The enzyme could be precipitated by salting out with ammonium sulfate and also by mixing with various water-miscible organic solvents such as ethanol, methanol or acetone.

This enzyme is one of acid proteases having its optimal pH for milk casein digestion around 3.5. The ratio of milk clotting activity to proteolytic activity of this enzyme resembled that of calf rennet than those of other proteases of fungal origin. This was more heat stable and more resistant against pH changes than animal rennet. Apparent activity of milk clotting was more affected by Ca ion concentration in milk than that of calf rennet.

The liberation of 12% TCA soluble nitrogen from casein fraction was a little less specific than that of calf rennet. The optimal temperature for milk clotting lay around 56°C.

Electrophoretic patterns of α-peak of casein treated with this enzyme showed the weak proteolysis which resembled that with rennet.     

Studies on Xylanase from Microorganisms

As xylanase-producing microorganisms, 64 strains belonging to the genus Streptomyces were isolated from the barn-yard manures, silages and litters collected in Hokkaido district. Among these isolates the strain 102–1–4, which was found to be a new species under taxonomical studies and named Streptomyces xylophagus nov. sp., had the most outstanding ability for the enzyme production. In addition to the isolates, 38 strains of Streptomyces and 480 strains of filamentous fungi which have been preserved in our culture collection were also examined on their ability to produce the enzyme. 1) Among the strains of Streptomyces tested, only two strains, St. albogriseolus IAM 0031 and St. olivaceus IAM 0025 were found to have the ability, but their abilities were less than that of St. xylophagus nov. sp. 2) Out of 480 strains of fungi tested, Chaetomium, Schyzophyllum, Trametes, Echinodontium, Alternaria, Cepharosporium, Cercospora, Gibberella, Glomerella and Macrosporium produced the enzyme. Especially, Ch. trilateral 2264 was the most excellent.     

九种蕨类植物多糖提取物抗动植物病原菌活性

从9种蕨类植物中提取多糖,以苯酚-硫酸比色法测定其含量,并采用纸片法进行抗动植物病原菌实验。结果表明,9种蕨类多糖含量差别较大,其多糖提取物表现出不同程度的抑制动植物病原菌活性。     

食用菌多糖的研究

食用菌多糖具有独特的生理特性和极高的药用价值,已成为当前药物研发的热点。从食用菌多糖的化学结构、提取分离、药用价值和应用等几个方面对其进行了全面的总结和展望。     

Extremophilic yeasts: plasma-membrane fluidity as determinant of stress tolerance

Our aim was to investigate the response of selected yeasts and yeast-like fungi from extreme?environments to various temperatures at the level of their plasma membranes, in order to elucidate the connections between their plasma-membrane fluidity (measured by electron paramagnetic resonance spectroscopy - EPR), growth temperature range, stress tolerance, and ecological distribution. Although all studied fungi can be considered mesophilic according to their growth temperature profiles, their plasma-membrane fluidity indicated otherwise. Arctic yeast Rhodosporidium diobovatum could be classified as psychrotolerant?due to its higher average membrane fluidity. Extremely halotolerant black yeast-like fungus Hortaea werneckii isolated from solar salterns, on the other hand, is not adapted to low temperature, which is reflected in the higher average rigidity of its plasma membrane and as a consequence its inability to grow at temperatures lower than 10°C. The plasma membrane of Aureobasidium sp. isolated so far exclusively from an Arctic glacier with its intermediate fluidity and high fluidity variation at different temperatures may indicate the specialization of this yeast-like fungus to the specific glacial environment. Similar behaviour of plasma membrane was detected in the reference yeast, non-extremophilic Saccharomyces cerevisiae. Its membranes of intermediate fluidity and with high fluidity?fluctuation at different temperatures may reflect the specialization of this yeast to mesophilic environments and prevent its colonization of extreme environments. Halotolerant Aureobasidium pullulans from salterns, and Arctic Cryptococcus liquefaciens and Rhodotorula?mucilaginosa with moderately fluctuating plasma membranes of intermediate fluidity are representatives of globally distributed generalistic and stress-tolerant species that can thrive in a variety of environments. Keeping the membranes stable and flexible is one of the necessities for the microorganisms to survive changes in extreme habitats. Our data suggest that plasma-membrane fluidity can be used as an indicator of fitness for survival in the extreme environments. In addition to the average fluidity of plasma membrane, the fluctuation of fluidity is an important determinant of stress tolerance: high absolute fluidity fluctuation is tied to decreased survival. The fluidity and its variation therefore reflect survival strategy and fitness in extreme environments and are good indicators?of the adaptability of microorganisms.     

Phytotoxic Diterpenoids from Plants and Microorganisms

Phytotoxic natural products with either unique or various structures are one of the most abundant sources for the discovery of potential allelochemicals, natural herbicides, and plant growth regulators. Phytotoxic diterpenoids, a relatively large class of natural products, play an important role in the plant‐plant or plant‐microorganism interactions. This article argues that the phytotoxic diterpenoids isolated from the plants and microorganisms can either inhibit the seed germination and the growth of plant seedlings or lead to some disease symptoms on the tested plant tissues and plant seedlings.     

Glyphosate-Degrading Microorganisms from Industrial Activated Sludge

A plating medium was developed to isolate N-phosphonomethylglycine (glyphosate)-degrading microorganisms, with glyphosate as the sole phosphorus source. Two industrial biosystems treating glyphosate wastes contained elevated microbial counts on the medium. One purified isolate metabolized glyphosate to aminomethylphosphonic acid, mineralizing this accumulating intermediate during log growth. This microorganism has been identified as a Flavobacterium species.