Comparative study of some microbial arabinan-degrading enzymes

Summary A variety of thermophilic organisms andBacillus species were screened in shake flask culture for arabanase andp-nitrophenyl--l-arabinosidase activities. Highest arabanase activity was produced by strains ofThielavia terrestris andSporotrichum cellulophilum. Thermoascus aurantiacus and severalBacillus species were most active producers of arabinosidase. Arabinosidases fromBacillus strains had pH optima in the range 5.9–6.7. pH optima of fungal arabinosidases ranged from 2.9 to 6.7.Bacillus arabanases had neutral pH optima, whereas fungal arabanases had pH optima in the range 3.7–5.1. In general, arabinosidases were found to be relatively thermostable, retaining >70% activity for 3 h at 60°C. TheT. aurantiacus enzyme retained 98% activity at 70°C after 3 h.Bacillus arabanases were relatively unstable. All fungal arabanases except theT. aurantiacus enzyme were fully denatured at 70°C after 3 h.     

Hemicellulases of Bacillus species: preliminary comparative studies on production and properties of mannanases and galactanases

A range of Bacillus subtilis strains and other Bacillus species were screened for mannanase, β-mannosidase and galactanase activities. Maximum mannanase activity, 106.2 units/ml, was produced by B. subtilis NRRL 356. β-Mannosidase and galactanase activities from all strains were relatively low. The effect of carbon and nitrogen source on mannanase and galactanase production by B. brevis ATCC 8186, B. licheniformis ATCC 27811, B. polymyxa NRRL 842 and B. subtilis NRRL 356 was investigated. Highest mannanase production was observed in the four strains tested when the mannan substrate, locust bean gum, was used as carbon source. Induction was most dramatic in the case of B. subtilis NRRL 356 where only basal enzyme levels were produced in the presence of other carbon sources. β-Mannosidase was induced in the four Bacillus cultures by locust bean gum. Results indicated that galactose acted as an inducer for production of galactanase. Organic and inorganic nitrogen sources resulted in induction of high mannanase titres in B. subtilis. Highest galactanase activity was produced by each organism in media containing sodium nitrate as nitrogen source. Mannanases from B. brevis, B. licheniformis, B. polymyxa and B. subtilis retained 100% residual activity after a 3 h incubation at 65°C, 65°C, 60°C and 55°C respectively. Galactanases retained more than 95% activity at 55°C after 3 h. The pH optima of mannanases ranged from 6.5–6.8 whereas galactanases ranged from 5.1 in the case of B. brevis to 7.0 for B. polymyxa.     

Production of D-xylanases by thermophilic fungi using different methods of culture

Summary Seven thermophilic strains of fungi were examined for their ability to produce D-xylanase in Liquid and solid-state fermentations. It was confirmed that the best producers of xylanase, among microorganisms used, wereH. Lanuginosa andS. thermophile in liquid fermentation, andT. aurantiacus andH. lanuginosa in solid-state fermentations. The higher productivity of xylanase, namely 18,72 IU/ml, was obtained in liquid culture ofH. lanuginosa. The pH and temperature optima of enzymes from liquid and solid-state cultures of fungi used were also presented.     

Materials for the fungus flora of Japan (49)

Two interesting microfungi are described as new to Japan:Talaromyces galapagensis (anam.Penicillium galapagense), isolated from soil in Shizuoka; andPenicillium megasporum, isolated from marine sludge in Nagasaki. Some observations are recorded, particularly on ascomatal initials ofT. galapagensis, which are similar to those described forTalaromyces flavus.(48): Kaneko, S., Mycoscience 36: 359–360, 1995.     

Purification and some properties of the mannanases fromThielavia terrestris

Summary Thielavia terrestris NRRL 8126 cell free supernatants contained mannanase and -mannosidase when cultured on a complex media containing locust bean gum. Using acetone precipitation, SP-Sephadex C50 ion exchange chromatography and preparative gel electrophoresis, the crude enzyme was resolved into one -d-mannosidase and four -d-mannanase components. -d-mannosidase had a specific activity of 0.02 (U/mg) onp-nitrophenyl--d-mannopyranoside substrate. Mannanase components M1, M2, M3 and M4 had specific activities of 28.2, 38.7, 52.8 and 4.17 (U/mg) respectively on purified locust bean galactomannan substrate. pH optima for the enzymes were in the range 4.5–5.5. Mannanase component M4 manifested the greatest thermostability, retaining full activity for 3 h at 60°C. Molecular weights determined by SDS-PAGE were 72 000 for -mannosidase and 52 000, 30 000, 55 000 and 89 000 for M1, M2, M3 and M4 respectively. Carbohydrate contents of the enzymes ranged from 6–36%. Preliminary studies indicate that enzyme components hydrolyse the mannan substrate in a synergistic manner.     

Phylogenetic relationships inSecale (Poaceae): An isozymatic study

The genetic frequencies of 9 isozyme loci have been estimated in 23 samples of 4 species ofSecale by means of starch gel electrophoresis. The populations ofS. silvestre andS. vavilovii were monomorphic and uniform within each species, those ofS. montanum andS. cereale were polymorphic for most of the isozyme loci. On the basis of isozyme patterns as well as allelic and genotypic frequencies of isozyme loci,S. silvestre can be distinguished fromS. vavilovii, and both fromS. cereale andS. montanum; but there is no clear differentiation between the two latter species. Clusters constructed from genetic distances separateS. silvestre andS. vavilovii, whereasS. cereale andS. montanum were grouped together. The isozymatic data presented here, along with cytogenetic and life habit data, agree with the generally admitted existence of 4 species inSecale, and support the relationships suggested byKhush & Stebbins (1961).     

Purification and characterization of a <Emphasis Type="Italic">N</Emphasis>-acetylglucosaminidase produced by <Emphasis Type="Italic">Talaromyces emersonii</Emphasis> during growth on algal fucoidan

A β-N-acetylglucosaminidase produced by a novel fungal source, the moderately thermophilic aerobic ascomycete Talaromyces emersonii, was purified to apparent homogeneity. Submerged fermentation of T. emersonii, in liquid medium containing algal fucoidan as the main carbon source, yielded significant amounts of extracellular N-acetylglucosaminidase activity. The N-acetylglucosaminidase present in the culture-supernatant was purified by hydrophobic interaction chromatography and preparative electrophoresis. The enzyme is a dimer with molecular weight and pI values of 140 and 3.85, respectively. Substrate specificity studies confirmed the glycan specificity of the enzyme for N-acetylglucosamine. Michaelis-Menten kinetics were observed during enzyme-catalyzed hydrolysis of the fluorescent substrate methylumbelliferyl-β-D-N-acetylglucosaminide at 50°C, pH 5.0 (K_m value of 0.5 mM). The purified N-acetylglucosaminidase displayed activity over broad ranges of pH and temperature, yielding respective optimum values of pH 5.0 and 75°C. The T. emersonii enzyme was less susceptible to inhibition by N-acetylglucosamine and other related sugars than orthologs from other sources. The enzyme was sensitive to Hg²⁺, Co²⁺ and Fe³⁺.     

Selection and characterization of mannanase-producing bacteria useful for the formation of prebiotic manno-oligosaccharides from copra meal

In order to select bacterial strains effectively secreting mannanase activity for the production of prebiotic mannooligosaccharides, a two-step screening procedure was performed. Enriched cultures from isolation medium containing copra meal were primary screened on an isolation agar medium containing 1% locust bean gum (LBG), which resulted in 48 mannanase-producing bacterial isolates with significant clearing zones on the mannan-containing agar. However, only nine isolates showed appreciable mannanase activities against copra meal in their culture supernatants (0.054–0.185 U/mg of protein) as determined in a standard assay based on the detection of reducing sugars released from this substrate. The isolates CW2-3 and ST1-1 displayed the highest activity against LBG and copra meal, respectively. Copra mannan hydrolysates that were obtained by using crude mannanase from these nine isolates were further used for a secondary screening towards a growth-enhancing activity on Lactobacillus reuteri and inhibitory activity against Escherichia coli as well as Salmonella Enteritidis, resulting in 0.09–2.15 log CFU/ml enhancing activity and low inhibitory activity of 0.46–1.78 log CFU/ml as well as 0.37–1.72 log CFU/ml, respectively. The hydrolysate of CW2-3 mannanase showed the highest enhancing activity of 2.15 log CFU/ml while isolate ST1-1 was most effective with respect to growth inhibition against E. coli E010 and S. Enteritidis S003 with 0.76 and 1.61 log CFU/ml, respectively. Based on morphological, physical, biochemical and genetics properties, isolates CW2-3 and ST1-1 were identified as Klebsiella oxytoca and Acinetobacter sp., respectively. Crude mannanase activity from these two strains was characterized preliminarily. The pH optima of mannanase activity from Klebsiella oxytoca CW2-3 and Acinetobacter sp. ST1-1 were 7 and 6, respectively. The enzymes were stable at 4°C over a pH range of 3–6 and 3–10, respectively.     

Studies on ectomycorrhiza

Summary Plants ofPicea abies (L.) Karst were grown in mycorrhizal association withTelephora terrestris (Pers. ex Fr.) andPisolithus tinctorius (Mich. ex Pers.) Coker and Couch on sphagnum peat in petri dishes or Perspex chambers. After 1 yearT. terrestris had formed prominent rhizomorphs which were characterized by light microscopy and investigated for³²P-orthophosphate uptake. The absorbed phosphate was transported to sinks throughout the rhizomorphal system as well as into the plant. The calculated translocation velocity and flux rate in the rhizomorph were in the range of 1–3 cm/h and 0.5–4.0 × 10^-10 mol cm^-2 s^-1, respectively. Label was observed to accumulate in the needles 2–3 days after application. Feeding a non-mycorrhized root with³²P-orthophosphate led to an accumulation of label in needles within 1 h, but no radioactivity appeared in the associatedT. terrestris rhizomorphs. The rhizomorphs ofP. tinctorius revealed a higher structural differentiation than those ofT. terrestris. Translocation of labelled phosphorus through rhizomorphs ofP. tinctorius into spruce needles was also demonstrated.     

Cloning, characterisation and expression analysis of α-glucuronidase from the thermophilic fungus Talaromyces emersonii

The aguA gene encoding α-glucuronidase was isolated from the thermophilic fungus Talaromyces emersonii by degenerate PCR. AguA has no introns and consists of an open reading frame of 2511 bp, encoding a putative protein of 837 amino acids. The N-terminus of the protein contains a putative signal peptide of 17 amino acids yielding a mature protein of 820 amino acids with a predicted molecular mass of 91.6 kDa. Twenty putative N-glycosylation sites and four O-glycosylation were identified. The T. emersonii α-glucuronidase falls into glycosyl hydrolase family 67, showing approximately 63% identity to similar enzymes from other fungi. Analysis of the aguA promoter revealed several possible regulatory motifs including two XlnR and a CreA binding site. Enzyme activity was optimal at 50 °C and pH 5. Enzyme production was investigated on a range of carbon sources and showed induction on beechwood, oat spelt and birchwood xylan, and repression by glucose or glucuronic acid.     

Synthesis of sterigmatocystin on a chemically defined medium by species ofAspergillus andChaetomium

Sterigmatocystin (ST) is a secondary metabolite and a principal mycotoxin known to be produced by over 30 species of filamentous fungi. It is also one of the late intermediates in aflatoxin biosynthesis. We have tested the ability of 7 species ofAspergillus, including 4 strains ofA. versicolor, one species ofBipolaris, and two species ofChaetomium, to produce ST on a sucrose-salts-phenylalanine defined medium as well as on three complex substrates. Highest ST production in our survey was by a strain ofA. versicolor grown on wheat, whereas, the highest ST production on defined medium was byC. cellulolyticum. To our knowledge, this is the first report of ST production byC. cellulolyticum on any substrate. In precursor feeding studies, resting cultures of wild typeA. nidulans andA. versicolor were unable to biotransform O-methylsterigmatocystin (OMST), the last known intermediate in aflatoxin biosynthesis. These results suggest that ST is the end product of polyketide metabolism in the strains tested.     

Enzymology of the thermophilic ascomycetous fungus Thermoascus aurantiacus

Different strains of the thermophilic ascomycetous fungus Thermoascus aurantiacus have been reported in the literature to produce high levels of a variety of industrial interest enzymes (i.e. amylases, cellulases, pectinases and xylanases), which have been shown to be remarkably stable over a wide range of temperatures and appear to have tremendous commercial potential. Most studies on enzyme production by T. aurantiacus are carried out in chemically defined liquid medium, under conditions suitable for induction of a particular enzyme. A few studies have investigated the production of some enzymes by T. aurantiacus by solid-state fermentation, using lignocellulosic materials. The present review focuses on the enzymes produced by T. aurantiacus, their main kinetic parameters, and the effect of different culture conditions on production and enzyme activity. It also provides a view of the possible applications of T. aurantiacus enzymes, considering that this thermophilic fungus could comprise a potential source of thermostable enzymes.     

The 5S DNA units of bread wheat (Triticum aestivum)

A collection of 44 cloned 5S DNA units fromTriticum aestivum cv. Chinese Spring were grouped into 12 sequence-types based on sequence similarity and the respective consensus sequences were then produced. The relationship between these 12 consensus sequences (T. aestivum S 1-S 8 andT. aestivum L 1-L 4), together with two clones sequenced byGerlach andDyer, and the 5S DNA consensus sequences from diploidTriticum spp. were then determined by numerical methods. Both phenetic and cladistic analyses were carried out. The following wheat 5S DNA sequences were found to group with respective sequences from diploidTriticum spp.:T. aestivum S 4, S 6 withT. tauschii S;T. aestivum S 3 withT. monococcum S andT. monococcum S-Rus 7;T. aestivum L 1 andT. aestivum L-G&D withT. speltoides L;T. aestivum L 2, L 3 withT. tauschii L;T. aestivum L 4 withT. monococcum L andT. monococcum L-Rus 12. The analyses suggested that 5 out of the 65S Dna loci present in wheat were identified at the sequence level. The locus that could not be identified in this analysis was the5S Dna-B 1 locus. A group ofT. aestivum sequences (T. aestivum S 1, S 7, S 8, S-G&D) were found to be distinct from the other 5S DNA sequences in the data base. The existence of the distinct group of 5S DNA sequences suggests that there is a gap in our current understanding of wheat evolution with respect to the5S Dna loci.     

Characterization of the Bacillus subtilis WL-3 mannanase from a recombinant Escherichia coli

A mannanase was purified from a cell-free extract of the recombinant Escherichia coli carrying a Bacillus subtilis WL-3 mannanase gene. The molecular mass of the purified mannanase was 38 kDa as estimated by SDS-PAGE. Optimal conditions for the purified enzyme occurred at pH 6.0 and 60 degrees C. The specific activity of the purified mannanase was 5,900 U/mg on locust bean gum (LBG) galactomannan at pH 6.0 and 50 degrees C. The activity of the enzyme was slightly inhibited by Mg(2+), Ca(2+), EDTA and SDS, and noticeably enhanced by Fe(2+). When the enzyme was incubated at 4 degrees C for one day in the presence of 3 mM Fe(2+), no residual activity of the mannanase was observed. The enzyme showed higher activity on LBG and konjac glucomannan than on guar gum galactomannan. Furthermore, it could hydrolyze xylans such as arabinoxylan, birchwood xylan and oat spelt xylan, while it did not exhibit any activities towards carboxymethylcellulose and para-nitrophenyl-beta-mannopyranoside. The predominant products resulting from the mannanase hydrolysis were mannose, mannobiose and mannotriose for LBG or mannooligosaccharides including mannotriose, mannotetraose, mannopentaose and mannohexaose. The enzyme could hydrolyze mannooligosaccharides larger than mannobiose.     

Isolation of mannan-utilizing bacteria and the culture conditions for mannanase production

A locally isolated strain, Bacillus subtilis NM-39, was selected as an active mannan-utilizing bacterium based on high saccharifying activities on coconut residue and locust bean gum galactomannan. The optimal pH and temperature ranges for activity of the crude enzyme were 5.0 to 6.0 and 50 to 60°C, respectively. The organism gave maximum mannanase activity when grown in liquid mineral salts medium containing 1% (w/v) each of coconut residue and soybean flour, as carbon and nitrogen sources, respectively, at pH 7.0 and in aerobic growth for 28 h at 37°C. High saccharifying activity on coconut mannan was also observed.The authors are with the Industrial Technology Development Institute, Department of Science and Technology, Manila, Philippines. M. Arai and T. Kawaguchi are also currently with the Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 593, Japan; T. Yoshida is also with the Faculty of Engineering, Osaka University, Suita, Osaka 565, Japan.     

Temperature response of trehalase from a mesophilic (Neurospora crassa) and a thermophilic (Thermomyces lanuginosus) fungus

The purified trehalases of the mesophilic fungus, Neurospora crassa, and the thermophilic fungus, Thermomyces lanuginosus, had similar temperature and pH optima for activity, but differed in molecular weight, electrophoretic mobility and Michaelis constant. At lower concentration, trehalases from both fungi were inactivated to similar extent at 60°C. While purified trehalase of T. lanuginosus was afforded protection against heat-inactivation by proteinaceous protective factor(s) present in mycelial extracts, by bovine serum albumin and by casein, these did not afford protection to N. crassa trehalase against heat inactivation. Both trehalases exhibited discontinuous Arrhenius plots with temperature of discontinuity at 40°C. The activation energy calculated from the slope of the Arrhenius plot was higher for the T. lanuginosus enzyme. The plots of apparent K _m versus 1/T for trehalases of N. crassa and T. lanuginosus were linear from 30° to 60°C.The results show that purified trehalases of the mesophilic and the thermophilic fungus are distinct. Although, these exhibit similar thermostability of their catalytic function at low concentration, distinctive thermal stability characteristics of thermophilic enzyme become apparent at high protein concentration. This could be brought about in the cell by the enzyme itself, or by other proteins.     

Cyclodextrin production and genetic characterization of cyclodextrin glucanotranferase of <Emphasis Type="Italic">Paenibacillus graminis</Emphasis>

Paenibacillus graminis strains were described recently as cyclodextrin (CD) producers. Cyclodextrins are produced by cyclodextrin glucanotransferase (CGTase) which has not been characterized in P. graminis. Similar amounts of α- and β-CDs were produced by P. graminis (MC22.13) and P. macerans (LMD24.10^T). Primers were designed to sequence the gene encoding CGTase from P. graminis. A phylogenetic tree was constructed and P. graminis CGTase protein showed to be closer (79.4% protein identity) to P. macerans |P31835|. Hybridization studies suggested that the gene encoding CGTase is located in different positions in the genomes of P. macerans and P. graminis.     

Small enzymes with esterase activities from two thermophilic fungi, Emericella nidulans and Talaromyces emersonii

Extracellular esterase activities in Emericella nidulans and Talaromyces emersonii are attributed to small enzymes with molecular weights less than 10 kDa (microenzymes). A 1.6 kDa esterase accounted for most of the esterase activity observed in both organisms and one of them also contained a 4.1 kDa microenzyme with weaker esterase activity. These esterases were growth-associated and active towards fluorescein dibutyrate and -naphthyl acetate as well as tributyrin.