三种白腐菌及其组合菌种木质素降解酶比较研究

朱红栓菌Trametes cinnabarina、糙皮侧耳Pleurotus ostreatus、黄孢原毛平革菌Phanerochaete chrysosporium是产生木质素降解酶能力强的菌株。对三种白腐菌及其组合菌种产生木质素降解酶能力和行为进行了比较分析和研究。结果表明,最佳培养方式为液体振荡培养;最佳培养基为酵母膏液体培养基。在产漆酶(laccases,lacs)方面,Pleurotus ostreatus和Phanerochaete chrysosporium的组合菌种的酶活最强,在第6天出现峰值,酶活达到450U/L;在产锰过氧化物酶(manganese peroxidases,mnps)方面,Trametes cinnabarina和Pleurotus ostreatus的组合菌种的酶活最强,在第10天出现峰值,酶活达到1050U/L;在产木质素过氧化物酶(lignin peroxidases,lips)方面,Trametes cinnabarina和Phanerochaete chrysosporium的组合菌种的酶活最强,在第8天出现产酶峰值,酶活达到2990U/L。筛选结果表明,组合菌种比单菌种产生的三种主要木质素降解酶的活性强,这为白腐菌高效产酶提供了一条新的途径,并为白腐菌研究领域的后续工作奠定基础。     

Defective pyocin particles produced by some mutant strains of Pseudomonas aeruginosa.

Mutants of Pseudomonas aeruginosa, defective in the production of active R-type pyocins, were isolated from pyocinogenic strains and their products were characterized. Polysheath-like structures were found in induced lysates of 29 out of 42 mutants. Two mutants (strain P15-16 and M189) were found to produce special defective particles, which were characterized in detail. The other 11 mutants did not produce significant amounts of any structure visible under an electron microscope. Serum blocking powers were found in lysates from P15-16 and M189 to significant amounts. Defective particle produced by strain P15-16 lacked the sheath component, whereas M189 had morphological defects at the junction between sheath and baseplate, and also in the architecture of baseplate. Both defective particles could adsorb to the surface of bacteria, that were sensitive to pyocin, at the tip of their fibers without killing cells. All M189 particles attached to the bacteria had the extended sheaths. Therefore, attachment to the bacteria by fibers is not sufficient to kill cells, and contraction of sheath must occur after the initial adsorption by fibers for pyocin to express its biological activity. Defective particles of strain P15-16, which was derived from strain P15 (a pyocin R1 producer), could be converted to active forms by an in vitro complementation reaction with extracts from certain mutants originated from strain PAO (a pyocin R2 producer). This result indicated the exchangeability of components between R-type pyocins belonging to the different groups.     

Cold-adapted enzymes produced by fungi from terrestrial and marine Antarctic environments

Antarctica is the coldest, windiest, and driest continent on Earth. In this sense, microorganisms that inhabit Antarctica environments have to be adapted to harsh conditions. Fungal strains affiliated with Ascomycota and Basidiomycota phyla have been recovered from terrestrial and marine Antarctic samples. They have been used for the bioprospecting of molecules, such as enzymes. Many reports have shown that these microorganisms produce cold-adapted enzymes at low or mild temperatures, including hydrolases (e.g. α-amylase, cellulase, chitinase, glucosidase, invertase, lipase, pectinase, phytase, protease, subtilase, tannase, and xylanase) and oxidoreductases (laccase and superoxide dismutase). Most of these enzymes are extracellular and their production in the laboratory has been carried out mainly under submerged culture conditions. Several studies showed that the cold-adapted enzymes exhibit a wide range in optimal pH (1.0–9.0) and temperature (10.0–70.0?°C). A myriad of methods have been applied for cold-adapted enzyme purification, resulting in purification factors and yields ranging from 1.70 to 1568.00-fold and 0.60 to 86.20%, respectively. Additionally, some fungal cold-adapted enzymes have been cloned and expressed in host organisms. Considering the enzyme-producing ability of microorganisms and the properties of cold-adapted enzymes, fungi recovered from Antarctic environments could be a prolific genetic resource for biotechnological processes (industrial and environmental) carried out at low or mild temperatures.     

Characterization and biobleaching effect of hemicellulases produced by thermophilic fungi

The culture supernatant of Thermomyces lanuginosus strains MED 2D and MED 4B1 had high activities of xylanase with low inducible activities of -xylosidase. The crude xylanase was optimally active at 70 °C and at pH 6.0 to 6.5. Subsequently Eucalyptus kraft pulp was treated with MED 2D supernatant at 10 IU per gram pulp resulting in a 10.5% reduction in Kappa number. XECEDED-Refined bleached pulp resulted in handsheets with increased brightness compared to the control (X = xylanase treatment; E = alkaline extraction; C = Cl2 treatment; D = ClO2 treatment).     

Inhibition of some mycotoxigenic fungi by iturin A,a peptidolipid produced by Bacillus subtilis

Bacillus subtilis produces peptidolipid compounds of the iturin group that have been shown to have antifungal properties, but not all fungal species are sensitive to these compounds. In this study, the activity of iturin A, produced by B. subtilis strain B-3, was tested. Paper disks impregnated with various concentrations of iturin A were placed on agar plates seeded with conidia of toxigenic species of Fusarium, Gerlacia, Penicillium or Aspergillus. Most isolates were inhibited at iturin A concentrations as low as 4 g/disk. Penicillium italicum, P. vindicatum, A. ochraceus and A. versicolor were most strongly inhibited by the iturin whereas P. citrinum and A. parasiticus were least sensitive to iturin A.Mention of a trademark or proprietary product does not constitute a guarantee or warranty by the US Department of Agriculture and does not imply approval to the exclusion of other products that may also be suitable.     

Tremorgenic toxins produced by soil fungi.

Penitrem A or an unknown tremorgenic toxin, "X," was produced by 10 of 60 fungal isolates obtained from a pasture involved in an outbreak in cattle and sheep resembling migram and ryegrass staggers. Tremorgenic properties of extracts containing penitrem A or toxin X were confirmed by bioassay.     

Hydrolytic properties of crude alpha-L-rhamnosidases produced by several wild strains of mesophilic fungi

AIMS: Observation of the dependence of alpha-L-rhamnosidase activity on pH and temperature and the capability to hydrolyse concentrated naringin solutions and hesperidin suspensions of enzyme complexes produced by several fungi. METHODS AND RESULTS: The enzymes were produced by several wild strains of mesophilic fungi grown in liquid media containing rhamnose as sole carbon source. The properties and their ranges of values measured were as follows: (i) optimum pH, 3.5-6.5; (ii) optimum temperature, 50-65 degrees C; (iii) hydrolysis of supersaturated 100 g l(-1) naringin solutions, 45-100% and (iv) hydrolysis of hesperidin suspensions, 6-35%. CONCLUSIONS: Some alpha-L-rhamnosidase enzymes hydrolysed supersaturated naringin solutions with a high yield. The enzyme produced by Fusarium sambucinum 310 showed good activity even at pH 10. SIGNIFICANCE AND IMPACT OF THE STUDY: Crude enzymes with possible utilization as catalysts for the manufacture of hydrolysis products of the flavonoid glycosides were found.     

Fermentation products and plant cell wall-degrading enzymes produced by monocentric and polycentric anaerobic ruminal fungi.

Five anaerobic fungal isolates from the bovine rumen were grown on Coastal Bermuda grass (CBG) leaf blades and monitored over a 9-day period for substrate utilization, fermentation products, cellulase, and xylanase activities. Two of the fungal isolates showed monocentric growth patterns; one (isolate MC-1) had monoflagellated zoospores and morphologically resembled members of the genus Piromyces; the other (isolate MC-2) had multiflagellated zoospores and resembled members of the genus Neocallimastix. Three other isolates (PC-1, PC-2, and PC-3) exhibited polycentric growth and have not yet been described in the literature; these isolates were characterized by differences in morphology. All of the isolates degraded CBG to approximately the same extent (70% [dry weight]) in 9 days. Fermentation product accumulation was concurrent with substrate utilization. The major fermentation products for all isolates were formate, acetate, D-(-)-lactate, L-(+)-lactate, ethanol, carbon dioxide, and hydrogen. Succinate was produced by all cultures, with the exception of MC-1. Fermentation balances revealed different profiles for each isolate. As a group, monocentric isolates produced a greater ratio of oxidized to reduced products when grown on glucose or CBG than did the polycentric isolates, which produced a nearly equal ratio of these products. All isolates exhibited cellulolytic and xylanolytic activities, including endoglucanase, exoglucanase, beta-glucosidase, xylanase, and beta-xylosidase activities. Increasing enzyme activity correlated with the accumulation of fermentation products and substrate utilization. The optimum pH for the enzymatic activity of polycentric isolates was within a more narrow range (pH 6.4 to 7.0) than that of the monocentric isolates (pH 5.5 to 7.5). Activity toward cellulosic substrates was not detected until after the disappearance of reducing sugars. Xylanase activity was found to be five to seven times that of carboxymethyl cellulase activity for all cultures grown on CBG.     

Composition of lipids produced by some strains ofCandida species. Production of single-cell oil in a chemostat culture

Fatty acid composition of the lipids produced by four strains ofCandida species was studied. Oleic acid was the principal fatty acid. Cellular lipids ofCandida sp. andC. pulcherima were rich in palmitic acid. Lipids fromC. lipolytica contained a significant amount of palmitoleic acid, whereasC. farinosa produced oil rich in stearis and α-linolenic acid. Analysis of cellular lipids ofCandida sp. andC. pulcherima during growth on a nitrogen-limited medium showed that oils accumulated in the exponential growth phase were more unsaturated than those accumulated in the decelerating and stationary phases. In a chemostat culture,Candida sp. accumulated about 40% of lipid. The specific rate of lipid formation, at a dilution rate ofD=0.09/h, was 35 mg of lipid per g of biomass per h and the yield of lipid on glucose was 11.4%.     

Hemicellulolytic enzymes of the ligninolytic white-rot fungus Phlebia radiata. Influence of phenolic compounds on the synthesis of hemicellulolytic enzymes

The ligninolytic fungus Phlebia radiata growing in a low-nitrogen medium with wheat bran as the sole carbon source was induced by some lignin monomers, e.g. vanillic, veratric and ferulic acids. In the medium these substances showed a mainly stimulating influence on the hemicellulolytic enzymes activity except for arabinofuranosidase and ferulic acid esterase.     

Characterization of a beta-lactamase produced by Pseudomonas paucimobilis.

A novel beta-lactamase enzyme produced by a strain of Pseudomonas paucimobilis is described. The enzyme differs from other recorded beta-lactamases from Gram-negative aerobic bacteria. It was constitutive, and had the characteristics of a penicillinase. One single band of beta-lactamase activity at pI 4.6 was seen on iso-electric focusing. The enzyme had a molecular mass of 30 kDa. The beta-lactamase was strongly inhibited by tazobactam, sulbactam and clavulanic acid but not by the thiol residue inhibitors p-chloromercuribenzoate and p-chloromercuriphenylsulphonic acid, or by metallo-enzyme inhibitors. Plasmid DNA was not demonstrable, suggesting that the enzyme was chromosomally encoded.     

Differences between pectic enzymes produced by laboratory and wild-type strains of Saccharomyces cerevisiae

The laboratory strain of S. cerevisiae, IM1-8b, showed pectolytic activity in the presence of either glucose, fructose, or sucrose as the carbon source, but not with galactose. The enzyme activity was rapidly lost with shaking. The optimum pH and temperature for activity were 4.5 and 45°C, respectively. The enzyme was an endopolygalacturonase, since it preferentially hydrolysed pectate over pectin and decreased the viscosity of a 5% polygalacturonic solution by about 30% in 30min producing oligogalacturonic acid and digalacturonic acid as end-products.     

Ruminal metagenomic libraries as a source of relevant hemicellulolytic enzymes for biofuel production

The success of second‐generation (2G) ethanol technology relies on the efficient transformation of hemicellulose into monosaccharides and, particularly, on the full conversion of xylans into xylose for over 18% of fermentable sugars. We sought new hemicellulases using ruminal liquid, after enrichment of microbes with industrial lignocellulosic substrates and preparation of metagenomic libraries. Among 150 000 fosmid clones tested, we identified 22 clones with endoxylanase activity and 125 with β‐xylosidase activity. These positive clones were sequenced en masse, and the analysis revealed open reading frames with a low degree of similarity with known glycosyl hydrolases families. Among them, we searched for enzymes that were thermostable (activity at > 50°C) and that operate at high rate at pH around 5. Upon a wide series of assays, the clones exhibiting the highest endoxylanase and β‐xylosidase activities were identified. The fosmids were sequenced, and the corresponding genes cloned, expressed and proteins purified. We found that the activity of the most active β‐xylosidase was at least 10‐fold higher than that in commercial enzymatic fungal cocktails. Endoxylanase activity was in the range of fungal enzymes. Fungal enzymatic cocktails supplemented with the bacterial hemicellulases exhibited enhanced release of sugars from pretreated sugar cane straw, a relevant agricultural residue.     

Stimulation of growth and glucose catabolite enzymes by succinate in some thermophilic fungi

Thermophilic Humicola lanuginosa, Penicillium duponti, Sporotrichum thermophile and Mucor pusillus required succinate in addition to glucose for optimal growth. The requirement for succinate was concentration-dependent and the concentration needed for one half of the maximal growth was 6.14 mM. In the presence of succinate, glucose utilization from the medium was markedly increased and this was associated with increased levels of the enzymes of the glycolytic and Krebs cycle pathways. Addition of succinate to cultures growing in glucose at any stage of growth stimulated the growth with the resulting rate of growth remaining high if the addition was made within 3 days of inoculation. Cycloheximide (71.4 M) prevented the succinate-mediated derepression of the enzymes suggesting that succinate may remove the catabolite repression in the presence of glucose.A preliminary part of this work was presented at the 17th annual meeting of the Association of Microbiologists of India at Manipal (India) held from Dec. 13 to 15, 1976