粘细菌产生的水解酶类研究进展

粘细菌隶属于δ变形菌纲(Deltaproteobacteria),是重要的药源微生物类群,但是其分离培养困难,严重限制了粘细菌资源的发掘和开发利用。粘细菌是微生物捕食者,通过产生丰富多样的胞外水解酶,如淀粉酶、蛋白酶、几丁质酶、纤维素酶、磷酸酶、蛋白酶等来裂解其他微生物或分解纤维素等作为营养物质来源。目前,粘细菌分离纯化技术主要是利用被捕食菌或纤维素诱导法。可以说,粘细菌胞外水解酶是研究其分离培养方法的物质基础。然而,目前研究者们对粘细菌产生的水解酶类关注较少。本文主要对粘细菌产生的水解酶种类、性质及其功能进行归纳总结,为今后粘细菌分离培养技术和开发利用等相关研究提供参考。     

Extracellular enzymes produced by microorganisms isolated from maritime Antarctica

Antarctic environments can sustain a great diversity of well-adapted microorganisms known as psychrophiles or psychrotrophs. The potential of these microorganisms as a resource of enzymes able to maintain their activity and stability at low temperature for technological applications has stimulated interest in exploration and isolation of microbes from this extreme environment. Enzymes produced by these organisms have a considerable potential for technological applications because they are known to have higher enzymatic activities at lower temperatures than their mesophilic and thermophilic counterparts. A total of 518 Antarctic microorganisms, were isolated during Antarctic expeditions organized by the Instituto Antártico Uruguayo. Samples of particules suspended in air, ice, sea and freshwater, soil, sediment, bird and marine animal faeces, dead animals, algae, plants, rocks and microbial mats were collected from different sites in maritime Antarctica. We report enzymatic activities present in 161 microorganisms (120 bacteria, 31 yeasts and 10 filamentous fungi) isolated from these locations. Enzymatic performance was evaluated at 4 and 20°C. Most of yeasts and bacteria grew better at 20°C than at 4°C, however the opposite was observed with the fungi. Amylase, lipase and protease activities were frequently found in bacterial strains. Yeasts and fungal isolates typically exhibited lipase, celullase and gelatinase activities. Bacterial isolates with highest enzymatic activities were identified by 16S rDNA sequence analysis as Pseudomonas spp., Psychrobacter sp., Arthrobacter spp., Bacillus sp. and Carnobacterium sp. Yeasts and fungal strains, with multiple enzymatic activities, belonged to Cryptococcus victoriae, Trichosporon pullulans and Geomyces pannorum.     

Effect of hydrolytic and oxidative enzymes produced by solid-state fermentation on greige linen fabric

Solid state fermentation (SSF) was applied for production of fungal enzyme preparations from Phanerochaete chrysosporium, Aspergillus oryzae, Aspergillus giganteus and Trichoderma virens using cotton seed-coat fragment waste as a carbon source and enzyme inducer. Lignin-holocellulose matrix of cotton seed coat fragment proved to be effective in inducing production of ligninolytic, cellulolytic and xylanolytic enzymes in solid-state fermentation. The effect of the enzymes produced by SSF on greige linen fabric is discussed and evaluated. In the first experiment the hydrolytic and accompanying oxidative enzymes in the buffer extract of the whole SSF cultures were used for fabric treatment. In the second trial, the enzymes produced in situ (whole SSF material—mixture of fungal biomass, residual substrate and enzymes) were used for the treatment. Weight loss, reducing sugar liberation and removal of colouring materials were measured. The results showed that at equal enzyme charges the intact SSF materials were more efficient than the enzyme extracts. Of the six strains evaluated, Ph. chrysosporium VKM F-1767 was the most effective in removing colouring matters from greige linen fabric.     

Productivity of hydrolytic enzymes by mycorrhizal mushrooms

To survey the potential for production of extracellular hydrolytic enzymes by mycorrhizal mushrooms, productivities of these exo-enzymes from mycelia on potato-dextrose liquid medium were determined.Tricholoma matsutake produced relatively high levels of CM-cellulase and avicelase activities in all test strains. It also produced higher activity of acid proteinase than neutral proteinase. Its xylanase activities seemed to be higher than those of the other carbohydrases. The productivities ofLyophyllum shimeji strains were at similar levels to those ofT. matsutake strains. CM-cellulase and avicelase activities ofL. shimeji were higher than those ofT. matsutake. Neutral proteinase inL. shimeji strains showed higher activity levels than acid proteinase. The relative productivities of hydrolytic enzymes between the groups of mycorrhizal mushrooms and wood-rotting mushrooms were also examined.T. matsutake andL. shimeji both produce many kinds of hydrolytic enzymes in their culture broth, and the potential for production of hydrolytic enzymes by mycorrhizal mushrooms was judged to be relatively high.     

Extracellular RNase produced by Yarrowia lipolytica

Production of extracellular RNase(s) by Yarrowia lipolytica CX161-1B was examined in media between pHs 5 and 7. RNase production occurred during the exponential growth phase. High-molecular-weight nitrogen compounds supported the highest levels of RNase production. Several RNases were detected in the supernatant medium. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the RNases had estimated molecular weights of 45,000, 43,000, and 34,000. It was found that Y. lipolytica secretes only one RNase (the 45,000-molecular-weight RNase) and that the 43,000 and 34,000-molecular-weight RNases are degradation products of this RNase. The alkaline extracellular protease secreted by Y. lipolytica was shown to have a major role in the 45,000- to 43,000-molecular-weight conversion, and it was demonstrated that the 45,000-molecular-weight RNase could be purified from a mutant which does not produce the alkaline extracellular protease. Purification of the RNase from a wild-type strain resulted in purification of the 43,000-molecular-weight RNase. This RNase was a glycoprotein with a molecular weight of 44,000 as estimated by gel filtration, an isoelectric point of pH 4.8, and a pH optimum between 6.5 and 7.0.     

Assay of diadenosine tetraphosphate hydrolytic enzymes by boronate chromatography

A new procedure was described for assay of diadenosine tetraphosphate (Ap4A) hydrolases based on boronate chromatography. Potential reaction products, AMP, ADP, and ATP, of the hydrolysis of Ap4A were separated from residual substrate by chromatography on a boronate-derivatized cation-exchange resin, Bio-Rex 70. Separation was achieved by changing the concentrations of ethanol and ammonium acetate in the elution buffers. Picomole masses of products were detectable, blank dpm values were less than 0.5% of the total dpm, and auxiliary enzymes were not required. The procedure was specifically described for Ap4A pyrophosphohydrolase from Physarum polycephalum. The assay is generally applicable for dinucleoside polyphosphate hydrolases which hydrolyze other substrates such as Ap3A, Ap5A, Ap6A, and Gp4G. Dinucleotide polyphosphates are readily purified by chromatography on this boronate resin in a volatile buffer. Tes, Tricine, and Tris buffers significantly interfered with the chromatography of ATP.     

Contribution of hydrolytic enzymes produced by saprophytic fungi to the decrease in plant toxicity caused by water-soluble substances in olive mill dry residue

We studied the influence of saprophytic fungi on the toxic effect that the water-soluble substances in dry residues from olive (ADOR) have on the growth of plants. All saprophytic fungi were able to decrease the phytotoxicity of ADOR, although the toxicity of this residue did not decrease in the same way. Penicillium chrysogenum was able to reduce the toxicity of ADOR when this residue was applied at the highest dose of 15%. Fusarium lateritum, F. graminearum and Mucor racemosus were able to reduce the toxicity of ADOR when this residue was applied at the intermediate doses. However, F. oxysporum decreased the phytotoxicity of ADOR only when the residue was applied at the lowest dose of 2.5%. All saprophytic fungi tested produce endoglucanase, endopolymetylgalacturonase and endoxiloglucanase when grown in the presence of ADOR. A close relationship was found between the decrease in the phytotoxicity of ADOR and the amount of hydrolytic enzymes produced by the saprophytic fungi. These results shows that hydrolytic enzymes can be important in the degradation of phytotoxic substances present in olive mill dry residue.     

Extracellular glycoconjugates produced by cyanobacterium Wollea saccata

In order to survive in a highly competitive environment, freshwater or marine phototrophic microorganisms have to develop defense strategies that result in a tremendous diversity of compounds from different metabolic pathways. Recent trends in drug research from natural sources have shown that algae and cyanobacteria are promising organisms to furnish novel biochemically active compounds. In this study, we have analysed the extracellular mucilaginous proteoglycan produced by fresh-water heterocytous filamentous cyanobacterium Wollea saccata, strain Hindák 2000/18. This mucilaginous material is an acidic proteoglycan containing 30% protein and 52% carbohydrates on the basis of fraction dry weight. The constituent sugars of the carbohydrate component include glucose, fucose, 3-O-methylfucose, xylose, galactose, 3-O-methylgalactose, mannose, rhamnose, arabinose and glucuronic acid. The extracellular proteoglycan has been separated into five fractions (WF1-WF5) by anion exchange chromatography. Individual polymeric fractions varied in protein (16-57%) and carbohydrate (31-66%) contents, and in the composition of constituent monosaccharides.     

Comparison of proteolytic activities produced by entomopathogenic Photorhabdus bacteria: strain- and phase-dependent heterogeneity in composition and activity of four enzymes

Twenty strains (including eight phase variant pairs) of nematode-symbiotic and insect-pathogenic Photorhabdus bacteria were examined for the production of proteolytic enzymes by using a combination of several methods, including gelatin liquefaction, zymography coupled to native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and activity measurement with two chromogen substrate types. Four protease activities (approximately 74, approximately 55, approximately 54, and approximately 37 kDa) could be separated. The N-terminal sequences of three of the proteases were determined, and a comparison with sequences in databases allowed identification of these proteases as HEXXH metallopeptidases. Thus, the 74-kDa protease (described formerly as Php-B [J. Marokhazi, G. Koczan, F. Hudecz, L. Graf, A. Fodor, and I. Venekei, Biochem. J. 379:633-640, 2004) is an ortholog of OpdA, a member the thimet oligopeptidase family, and the 55-kDa protease is an ortholog of PrtA, a HEXXH+H peptidase in clan MB (metzincins), while the 37-kDa protease (Php-C) belongs to the HEXXH+E peptidases in clan MA. The 54-kDa protease (Php-D) is a nonmetalloenzyme. PrtA and Php-C were zymographically detected, and they occurred in several smaller forms as well. OpdA could not be detected by zymography. PrtA, Php-C, and Php-D were secreted proteases; OpdA, in contrast, was an intracellular enzyme. OpdA activity was found in every strain tested, while Php-D was detected only in the Brecon/1 strain. There was significant strain variation in the secretion of PrtA and Php-C activities, but reduced activity or a lack of activity was not specific to secondary-phase variants. The presence of PrtA, OpdA, and Php-C activities could be detected in the hemolymph of Galleria melonella larvae 20 to 40 h postinfection. These proteases appear not to be directly involved in the pathogenicity of Photorhabdus, since strains or phase variants lacking any of these proteases do not show reduced virulence when they are injected into G. melonella larvae.     

Production of hydrolytic enzymes by oral isolates of Eikenella corrodens

Abstract Thermus thermophilus cells harboring an expression plasmid for the aqualysin I gene secrete the mature enzyme into the medium. In an Escherichia coli expression system, a precursor of the enzyme with the C-terminal pro-sequence is accumulated in the cells, and upon treatment at 65°C the active enzyme is produced. One- to 10-amino acid residue deletions, as well as complete 105-residue deletion of the C-terminal pro-sequence from the C-terminus, did not affect the production of the enzyme in T. coli cells. T. thermophilus cells harboring plasmids for mutant precursors with one- and three-residue deletions secreted the enzyme extracellularly. However, transformants harboring plasmids for mutant precursors with deletions of five or more amino acid residues could not be obtained. These results suggest that the C-terminal pro-sequence plays an important role in the extracellular secretion of the enzyme in T. thermophilus cells.     

Bacteriolytic enzymes produced by Myxococcus xanthus

The bacteriolytic activities in the culture fluid of Myxococcus xanthus were purified and separated into six active fractions by the use of Bio-Gel CM-2 and Bio-Gel P-60. These fractions were identified as: (i) an amidase, (ii) a glucosaminidase, (iii) a glucosaminidase and an amidase, (iv) a protease with probable amidase activity, (v) another protease with probable amidase activity, and (vi) a peptidase active on both d-alanyl-diaminopimelate and d-alanyl-lysine peptide bonds. On one occasion, another amidase was eluted from Bio-Gel CM. Preliminary studies on some characteristics of the enzymes and their production during growth are reported.     

Extracellular isoamylase produced by Bacillus circulans MIR-137

A strain designated MIR-137 was selected after a screening programme for amylolytic enzymes and was characterized as Bacillus circulans. Batch cultures showed the highest amylolytic activities in the stationary phase. Extracellular isoamylase activity exhibited an optical temperature of 60°C at pH 5.0. These properties would allow its use in normal saccharification processes in the starch industries.     

Extracellular iron reductase activity produced by Listeria monocytogenes

Little is known about how pathogenic microorganisms that do not produce low-molecular-weight iron-chelating agents, termed siderophores, acquire iron from their environment. We have identified an extracellular enzyme produced by Listeria monocytogenes that can mobilize iron from a variety of iron-chelate complexes via reduction of the metal. The iron reductase requires Mg²⁺, flavin mononucleotide (FMN), and reduced nicotinamide adenine dinucleotide (NADH) for activity. Saturation kinetics were found when initial velocity studies of iron reduction were carried out as a function of variable FMN concentrations in the presence of 100 μM NADH and 10 mM Mg²⁺. Hyperbolic kinetics were also found when these studies were repeated as a function of variable NADH concentrations along with 20 μM FMN and 10 mM Mg²⁺. This process of extracellular reduction, in all likelihood, could be involved in the mobilization of iron from soils and aqueous environments and from host tissues in pathogenic processes. This is the first report of the extracellular enzymic reduction of iron by microorganisms. Received: 12 March 1996 / Accepted: 16 April 1996