Factors affecting the formation of polysaccharide depolymerase and glycoside hydrolyase enzymes by Butyrivibrio fibrisolvens NCDO 2249

Specific activities of hemicellulose-degrading polysaccharide depolymerase and glycoside hydrolase enzymes were measured in batch and continuous cultures of Butyrivibrio fibrisolvens NCDO 2249 grown on cellobiose or a hemicellulosic carbohydrate. Enzyme activities were influenced by the growth substrate and by the rate and stage of growth of the micro-organism. In cellobiose batch cultures specific activities were maximal as the growth rate declined and in the initial stages of the stationary phase. The growth substrate did not affect the range of glycoside hydrolases formed, although specific activities were substrate-dependent, with activity increases (up to 200-fold) occurring in enzymes essential for effective substrate utilization. Appreciable xylanase activity was present only in xylan-grown cultures. The substrate effects were also evident in chemostat cultures. The activity response of the nine enzymes monitored to growth rate changes differed in that while the activity of some enzymes, including xylanase, declined at high dilution rates the activities of others were not growth rate-dependent and were maintained over the range of dilution rates examined. Exocellular activities were detected only in spent media from cultures grown with a polymeric (hemicellulosic) carbohydrate.     

Polysaccharide-degrading enzymes formed by three species of anaerobic rumen fungi grown on a range of carbohydrate substrates

The range of polysaccharide-degrading enzymes formed by three anaerobic rumen fungi (Neocallimastix patriciarum, Piromonas communis, and an unidentified isolate (F] was monitored following growth on seven mono-, di-, and poly-saccharide carbohydrate substrates. Enzymes capable of degrading a variety of alpha- and beta-glucans, beta-galactans, galactomannan, and hemicellulosic arabinoxylans were present in all three isolates. Although reducing saccharides were released from pectin, polygalacturonic acid was not degraded by the preparations. Enzyme activity was present in both the zoospore and vegetative stages of the life cycle and was also detected extracellularly in culture supernatants after vegetative growth. The specific activities of the polysaccharidases were affected by the growth substrate, being lowest in preparations grown on mono- and di-saccharides, whereas polysaccharidic growth substrates resulted in increased activity of the corresponding polysaccharidases. The enzymes were, however, formed after growth on all substrates. Oligomers and monosaccharides were produced as a result of polysaccharide breakdown by the unfractionated enzyme preparations. Studies on hemicellulose (arabinoxylan) breakdown by unfractionated vegetative preparations of the three isolates indicated that their modes of action, pH optima, substrate affinities, and response to potential inhibitors were similar.     

Production of Cell Wall-Degrading Enzymes by the Phytopathogenic Fungus Sclerotinia sclerotiorum

The range of polysaccharide-degrading enzymes and glycosidases formed by the phytopathogenic fungus Sclerotinia sclerotiorum was monitored following growth on 16 carbohydrate substrates. Endo- and exoenzymes capable of degrading cellulosic, hemicellulosic, and pectinolytic polysaccharides were secreted. Pectinolytic activities were produced constitutively on all of the substrates tested. Cellulolytic enzymes were not induced in simple sugar (i.e., glucose or xylose) media. Polysaccharide growth substrates and cellulase inducers increased all of the enzyme activities tested. Gel filtration analysis revealed the appearance of new molecular forms of pectinase, β-xylosidase, and cellobiosidase during induction on pectin and carboxymethyl cellulose media.     

Evidence for isofunctional enzymes in the degradation of phenol, m- and p-toluate, and p-cresol via catechol meta-cleavage pathways in Alcaligenes eutrophus.

A study of the degradation of phenol, p-cresol, and m- and p-toluate by Alcaligenes eutrophus 345 has provided evidence that these compounds are metabolized via separate catechol meta-cleavage pathways. Analysis of the enzymes synthesized by wild-type and mutant strains and by strains cured of the plasmid pRA1000, which encodes m- and p-toluate degradation, indicated that two or more isofunctional enzymes mediated several steps in the pathway. The formation of three catechol 2,3-oxygenases and two 2-hydroxymuconic semialdehyde hydrolases was indicated from an examination of the ratio of the specific activities of these enzymes against various substrates. Evidence for two 2-hydroxymuconic semialdehyde dehydrogenases, two 4-oxalocrotonate isomerases and decarboxylases, and three 2-ketopent-4-enoate hydratases was derived from the induction of these enzymes under different growth conditions. Each activity was detected when the wild type was grown in the presence of m-toluate, but not when grown with phenol (except for a hydratase) or p-cresol, whereas in strains cured of pRA1000, growth with phenol or p-cresol, but not with m-toluate, induced these enzymes. Hydroxylation of phenol and p-cresol appears to be mediated by the same enzyme.     

The regulation of xylanolytic enzyme formation by Butyrivibrio fibrisolvens NCFB 2249

Butyrivibrio fibrisolvens NCFB 2249 formed xylan-degrading enzymes on a wide range of carbohydrate growth substrates. The specific activities of α-L-arabinofuranosidase and β-D-xylosidase were increased (up 20-fold) after growth on xylan or xylose-containing saccharides. Xylose was not an effective substrate for xylanase production although its formation was induced on xylobiose and higher DP xylose-containing saccharides. Acetyl esterase activity was also highest after growth on xylan. The synthesis of xylanase and β-xylosidase was repressed by glucose and hemicellulosic pentoses and although α-L-arabinofuranosidase formation was also subject to catabolite regulation, xylose did not repress its synthesis.     

The production of plant cell wall polysaccharide-degrading enzymes by hemicellulolytic rumen bacterial isolates grown on a range of carbohydrate substrates

Several cultures of bacteria, isolated from the rumen, that were able to utilize plant cell wall structural polysaccharides were grown on a range of carbohydrate substrates and the activities of the principal polysaccharide-degrading enzymes determined. The esterase activity was also monitored. The extent of hemicellulose degradation and utilization by the isolates was comparable with that of the hemicellulolytic type strains. Enzyme activities in all of the cultures examined were affected by the carbon source in the growth medium. Many responses were strain specific, although growth on glucose (or cellobiose and maltose to a lesser extent) resulted in reduced activities in most of the organisms examined, whilst polysaccharidic substrates resulted in higher levels of the appropriate polysaccharidase. However, enzyme activity was detectable in some isolates after culture on mono- or disaccharides in the absence of the principal or related polysaccharide substrate.     

Temperature modulates the cell wall mechanical properties of rice coleoptiles by altering the molecular mass of hemicellulosic polysaccharides

The present study was conducted to investigate the mechanism inducing the difference in the cell wall extensibility of rice ( Oryza sativa L. cv. Koshihikari) coleoptiles grown under various temperature (10–50°C) conditions. The growth rate and the cell wall extensibility of rice coleoptiles exhibited the maximum value at 30–40°C, and became smaller as the growth temperature rose or dropped from this temperature range. The amounts of cell wall polysaccharides per unit length of coleoptile increased in coleoptiles grown at 40°C, but not at other temperature conditions. On the other hand, the molecular size of hemicellulosic polysaccharides was small at temperatures where the cell wall extensibility was high (30–40°C). The autolytic activities of cell walls obtained from coleoptiles grown at 30 and 40°C were substantially higher than those grown at 10, 20 and 50°C. Furthermore, the activities of (1→3),(1→4)- β -glucanases extracted from coleoptile cell walls showed a similar tendency. When oat (1→3),(1→4)- β -glucans with high molecular mass were incubated with the cell wall enzyme preparations from coleoptiles grown at various temperature conditions, the extensive molecular mass downshifts were brought about only by the cell wall enzymes obtained from coleoptiles grown at 30–40°C. There were close correlations between the cell wall extensibility and the molecular mass of hemicellulosic polysaccharides or the activity of β -glucanases. These results suggest that the environmental temperature regulates the cell wall extensibility of rice coleoptiles by modifying mainly the molecular mass of hemicellulosic polysaccharides. Modulation of the activity of β -glucanases under various temperature conditions may be involved in the alteration of the molecular size of hemicellulosic polysaccharides.     

Studies on Pectic Enzymes of Microorganisms

To pick out potent strains which specifically produce one of several pectic enzymes, endo- and exo-polygalacturonase, pectin esterase, macerating, and apple juice clarifying activities were examined with regard to 344 strains of mold (containing 71 strains of phytopathogenic mold) grown on a bran culture medium and 56 strains of shakingly cultured yeast. As the result of screening, Asper gillus saitoi and Penicillium islandicum were isolated as potent specific producers of endo-polygalacturonase. And the composition of pectic enzymes of mold was found to be rather genus or species specific. So far as examined in crude enzyme systems, there was no parallelism between anyone of pectic enzyme activities and apple juice clarifying or macerating activities.     

Production of xylanolytic enzymes by strains of Aspergillus

Summary Production of hemicellulolytic enzymes required in the hydrolysis of different xylans was investigated using strains of seven species of Aspergillus. Of the strains producing highest levels of xylanolytic activities, a. foetidus VTT-D-71002 was apparently non-cellulolytic and could therefore be a possible source of cellulase-free hemicellulase for applications in the pulping industry. The non-metabolizable synthetic xylobiose analogue -methyl-D-xyloside was the best xylanase inducer of the materials tested. Batches of hemicellulase produced in laboratory scale fermentations on practical media were tested in the hydrolysis of both cellulosic and hemicellulosic substrates.     

The isolation and characterization of lysosomal particles from myxamoebae of the cellular slime mould Dictyostelium discoideum

1. The myxamoebae of the cellular slime mould Dictyostelium discoideum possess several typically lysosomal enzyme activities. 2. These enzymes are present in the cell in association and in a lysosome-like particle. 3. The lysosomes of myxamoebae grown axenically have a different enzymic composition and a different density from those grown on bacteria. 4. During cell differentiation the specific activities of the lysosomal enzymes change. 5. It is suggested that both during growth and differentiation the amounts of lysosomal enzymes present in the cell are regulated.     

Properties of Candida lipolytica mutants with the modified glyoxylate cycle and their ability to produce citric and isocitric acid

Summary Enzyme activities of the tricarboxylic acid (TCA) cycle and the anaplerotic pathways, as well as the cell cytology of two C. lipolytica mutants with the modified glyoxylate cycle and their parent strain were studied during the exponential growth phase on glucose or hexadecane.Among the TCA cycle enzymes, the key enzyme citrate synthase had the highest activity in all three strains grown on both substrates. NAD-dependent isocitrate dehydrogenase had the minimum activity. All strains had well-developed mitochondria.Pyruvate carboxylation was active in the wild strain and mutant 2 grown on glucose, where this reaction is the basic anaplerotic pathway for oxal-acetate synthesis; mutant 1 had actively functioning enzymes for both anaplerotic pathways — pyruvate carboxylase, isocitrate lyase and malate synthase.During hexadecane assimilation, the number of peroxisomes in all strains increased sharply, accompanied by a simultaneous increase in isocitrate lyase activity.The low activities of both isocitrate lyase and pyruvate carboxylase in mutant 2 give reason to believe that this strain has an additional pathway for oxalacetic acid synthesis during the assimilation of n-alkane.     

In vitro water activity and pH dependence of mycelial growth and extracellular enzyme activities of Trichoderma strains with biocontrol potential

AIMS: Water activity (aw) and pH are probably the most important environmental parameters affecting the activities of mycoparasitic Trichoderma strains. Therefore it is important to collect information on the effects of these factors on mycelial growth and on the in vitro activities of extracellular enzymes involved in nutrient competition (e.g. beta-glucosidase, cellobiohydrolase and beta-xylosidase) and mycoparasitism (e.g. N-acetyl-beta-glucosaminidase, trypsin-like protease and chymotrypsin-like protease) of Trichoderma strains with biocontrol potential. METHODS AND RESULTS: Water activity and pH dependence of the linear mycelial growth of five examined Trichoderma strains belonging to three different species groups was examined on yeast extract and soil extract media. Maximal growth rates were observed at aw 0.997 and pH 4.0 in the case of all strains. The activities of the examined extracellular enzymes at different aw and pH values were determined spectrophotometrically after incubation with chromogenic p-nitrophenyl and p-nitroaniline substrates. Maximal enzyme activities were measured at aw 0.950 for beta-glucosidase, trypsin-like protease and chymotrypsin-like protease, at 0.910 for cellobiohydrolase and at 0.993 for beta-xylosidase and N-acetyl-beta-glucosaminidase enzymes. Optimal pH values are suggested to be at 5.0 for beta-glucosidase, cellobiohydrolase and N-acetyl-beta-glucosaminidase, at 3.0 for beta-xylosidase, at 6.0 for trypsin-like protease and between 6.0 and 7.0 for chymotrypsin-like protease activities, respectively. CONCLUSIONS: Extracellular enzymes of the examined mycoparasitic Trichoderma strains are able to display activities under a wider range of aw and pH values than those allowing mycelial growth. SIGNIFICANCE AND IMPACT OF THE STUDY: Data about the effects of aw and pH on mycelial growth and extracellular enzyme activities of Trichoderma reveal useful information about the applicability of biocontrol strains in agricultural soils with specific water and pH relations.     

Ecophysiological variabilities in ectohydrolytic enzyme activities of some Pseudoalteromonas species,P. citrea,P. issachenkonii,and P. nigrifaciens

The ecophysiological variabilities in the ectohydrolytic enzyme profiles of the three species of Pseudoalteromonas, P. citrea, P. issachenkonii, and P. nigrifaciens, have been investigated. Forty-one bacteria isolated from several invertebrates, macroalgae, sea grass, and the surrounding water exhibited different patterns of hydrolytic enzyme activities measured as the hydrolysis of either native biopolymers or fluorogenic substrates. The activities of the following enzymes were assayed: proteinase, tyrosinase, lipase, amylase, chitinase, agarase, fucoidan hydrolase, laminaranase, alginase, pustulanase, cellulase, beta-glucosidase, alpha- and beta-galactosidases, beta-N-acetylglucosaminidase, beta-glucosaminidase, beta-xylosidase, and alpha-mannosidase. The occurrence and cell-specific activities of all enzymes varied over a broad range (from 0 to 44 micromol EU per hour) and depended not only on taxonomic affiliation of the strain, but also on the source/place of its isolation. This suggests 'specialization' of different species for different types of polymeric substrates as, for example, all strains of P. citrea and P. issachenkonii hydrolyzed alginate and laminaran, while strains of P. nigrifaciens were lacking the ability to hydrolyze most of the algal polysaccharides. The incidence of certain enzymes such as fucoidan hydrolases, alginate lyases, agarases, and alpha-galactosidases might be strain specific and reflect its particular ecological habitat.     

Differential induction of enzymes involved in anaerobic metabolism of aromatic compounds in the denitrifying bacterium Thauera aromatica

Differential induction of enzymes involved in anaerobic metabolism of aromatic substrates was studied in the denitrifying bacterium Thauera aromatica. This metabolism is divided into (1) peripheral reactions transforming the aromatic growth substrates to the common intermediate benzoyl-CoA, (2) the central benzoyl-CoA pathway comprising ring-reduction of benzoyl-CoA and subsequent β-oxidation to 3-hydroxypimelyl-CoA, and (3) the pathway of β-oxidation of 3-hydroxypimelyl-CoA to three acetyl-CoA and CO₂. Regulation was studied by three methods. 1. Determination of protein patterns of cells grown on different substrates. This revealed several strongly substrate-induced polypeptides that were missing in cells grown on benzoate or other intermediates of the respective metabolic pathways. 2. Measurement of activities of known enzymes involved in this metabolism in cells grown on different substrates. The enzyme pattern found is consistent with the regulatory pattern deduced from simultaneous adaptation of cells to utilisation of other aromatic substrates. 3. Immunological detection of catabolic enzymes in cells grown on different substrates. Benzoate-CoA ligase and 4-hydroxybenzoate-CoA ligase were detected only in cells yielding the respective enzyme activity. However, presence of the subunits of benzoyl-CoA reductase and 4-hydroxybenzoyl-CoA reductase was also recorded in some cell batches lacking enzyme activity. This possibly indicates an additional level of regulation on protein level for these two reductases. Received: 22 December 1997 / Accepted: 12 May 1998     

Bacterial cellulases and saccharification of lignocellulosic materials

Five locally isolated bacterial strains produced extracellular cellulase enzymes, primarily CMCase, when grown on different natural and commercial cellulosic substrates. Extracellular CMCase and avicelase activity was higher with the strain CLS-32, a Cytophaga sp., compared to four other strains. The whole-cell preparations of these isolates were found to saccharify cellulosic substrates to reducing sugars. Maximum release of reducing sugar (5.75 mg ml⁻¹) was obtained with CLS-32 using sugar cane bagasse as growth and hydrolysis substrates.     

Induction of lytic enzymes by the interaction of Ustilago maydis with Zea mays tissues

The nonpathogenic (FB-2) and pathogenic (FB-D12) strains of Ustilago maydis were grown in medium supplemented with different carbon sources including monosaccharides, polysaccharides, and plant tissues. Both strains were able to grow on all substrates, with doubling times varying from 2 to 25 h depending on the carbon source. Plant tissues supplied as carbon source induced lytic enzymes differentially; pectate lyase and cellulase activities were induced preferentially by apical stem meristem in strain FB-D12, whereas leaves preferentially induced xylanase and cellulase activities in strain FB2. Stems induced polygalacturonase activity in both strains. All enzyme activities, except cellulase in the FB-D12 strain, were detected at a low level when U. maydis was grown on glucose. In planta, chlorosis and production of teliospores were paralleled by an increase in pectate lyase activity. Anthocyanin production and formation of galls and teliospores correlated with polygalacturonase expression whereas cellulase activity increased only during the stage of anthocyanin production and gall formation. Expression of xylanase activity coincided with the last stage of teliospore formation.     

Increased molecular mass of hemicellulosic polysaccharides is involved in growth inhibition of maize coleoptiles and mesocotyls under hypergravity conditions.

Elongation growth of dark grown maize (Zea mays L cv. Cross Bantam T51) coleoptiles and mesocotyls was suppressed by hypergravity at 30 g and above. Acceleration at 300 g significantly decreased the mechanical extensibility of cell walls of both organs. Hypergravity increased the amounts of hemicellulose and cellulose per unit length in mesocotyl walls, but not in coleoptile walls. The weight average molecular masses of hemicellulosic polysaccharides were also increased by hypergravity in both organs. On the other hand, the activities of beta-glucanases extracted from coleoptile and mesocotyl cell walls were decreased by hypergravity. These results suggest that the decreased activities of beta-glucanases by hypergravity cause an increase in the molecular mass of hemicellulosic polysaccharides of both organs. The upshift of molecular mass of hemicellulosic polysaccharides as well as the thickening of cell walls under hypergravity conditions seems to be involved in making the cell wall mechanically rigid, thereby inhibiting elongation growth of maize coleoptiles and mesocotyls.     

Phenotypic Characterization of Polysaccharidases Produced by Four Prevotella Type Strains

Four ruminal Prevotella type strains, P. ruminicola JCM8958^T, P. bryantii B₁4^T, P. albensis M384^T, and P. brevis ATCC19188^T, were characterized for polysaccharide-degrading activities with the reducing sugar release assay and zymogram analyses. Carboxymethylcellulase, xylanase, and polygalacturonate (PG)-degrading enzyme activities were determined in cultures grown on oat spelt xylan, xylose, arabinose, cellobiose, and glucose as sole growth substrates. P. ruminicola and P. albensis showed carboxymethylcellulase induction patterns. When xylan was supplied as a sole growth substrate, xylanase activities produced by P. bryantii and P. albensis were at least 18- and 11-fold higher, respectively, than during growth on other carbohydrates, suggesting that the regulation of the xylanases was highly specific to xylan. All strains constitutively produced PG-degrading enzymes. The corresponding activity of P. bryantii was more than 40-fold higher than in other strains. Zymogram analyses routinely detected the presence of high-molecular-weight (100–170 kDa) polysaccharide-degrading enzymes in ruminal Prevotella. Characteristics of the polysaccharide-degrading activities showed diversity of ruminal Prevotella species. Received: 29 November 1999 / Accepted: 1 February 2000