Glycosidases of moulds

Selection of a large number of different strains of hyphal fungi of the genusAspergillus, capable of production of extracellular mannosidase and mannanase type enzymes, was carried out. Before cultivating the strains on liquid synthetic medium containing 0.5%Saccharomyces cerevisiae mannan as the carbon source, they were adapted by multiple passage on solid synthetic media containingd-mannose,d-mannose and α-mannan and lastly only α-mannan. The extracellular enzymatic preparations of the mould fungi were tested for their ability to hydrolyse three different substrates—Saccharomyces cerevisiae, Torulopsis ingeniosa andTorulopsis colliculosa mannan. The production of α-mannosidase was found to be specifically dependent on the character of the substrate used for cultivation of the fungus.     

Characterization of raffinose synthase from rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L. var. Nipponbare)

The putative raffinose synthase gene from rice was cloned and expressed in Escherichia coli. The enzyme displayed an optimum activity at 45°C and pH 7.0, and a sulfhydryl group was required for its activity. The enzyme was specific for galactinol and p-nitrophenyl-α-d-galactoside as galactosyl donors, and sucrose, lactose, 4−β-galactobiose, N-acetyl-d-lactosamine, trehalose and lacto-N-biose were recognized as galactosyl acceptors.     

Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus <Emphasis Type="Italic">Paecilomyces variotii</Emphasis>

An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and pH were 55 °C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 °C, with a t ₅₀ of 45 min at 60 °C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose, p-nitrophenyl α-d-maltoside, methyl-α-d-glucopyranoside, pullulan, α- and β-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis, analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in α-helix. The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-α-d-glucan glucohydrolase).     

Characterization of a recombinant cellobiose 2-epimerase from <Emphasis Type="BoldItalic">Caldicellulosiruptor saccharolyticus</Emphasis> and its application in the production of mannose from glucose

A putative N-acyl-d-glucosamine 2-epimerase from Caldicellulosiruptor saccharolyticus was cloned and expressed in Escherichia coli. The recombinant enzyme was identified as a cellobiose 2-epimerase by the analysis of the activity for substrates, acid-hydrolyzed products, and amino acid sequence. The cellobiose 2-epimerase was purified with a specific activity of 35 nmol min^–1 mg^–1 for d-glucose with a 47-kDa monomer. The epimerization activity for d-glucose was maximal at pH 7.5 and 75°C. The half-lives of the enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 142, 71, 35, 18, and 4.6 h, respectively. The enzyme catalyzed the epimerization reactions of the aldoses harboring hydroxyl groups oriented in the right-hand configuration at the C2 position and the left-hand configuration at the C3 position, such as d-glucose, d-xylose, l-altrose, l-idose, and l-arabinose, to their C2 epimers, such as d-mannose, d-lyxose, l-allose, l-gulose, and l-ribose, respectively. The enzyme catalyzed also the isomerization reactions. The enzyme exhibited the highest activity for mannose among monosaccharides. Thus, mannose at 75 g l^–1 and fructose at 47.5 g l^–1 were produced from 500 g l^–1 glucose at pH 7.5 and 75°C over 3 h by the enzyme.     

Characterization of ribose-5-phosphate isomerase of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-allose from <Emphasis Type="SmallCaps">d</Emphasis>-psicose

The rpiB gene, encoding ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum, was cloned and expressed in Escherichia coli. RpiB converted d-psicose into d-allose but it did not convert d-xylose, l-rhamnose, d-altrose or d-galactose. The production of d-allose by RpiB was maximal at pH 7.5 and 65°C for 30 min. The half-lives of the enzyme at 50°C and 65°C were 96 h and 4.7 h, respectively. Under stable conditions of pH 7.5 and 50°C, 165 g d-allose l⁻¹ was produced without by-products from 500 g d-psicose l⁻¹ after 6 h.     

Carbon and nitrogen utilization and acid production by mycelia of the ectomycorrhizal fungusTricholoma bakamatsutake in vitro

Mycelial growth of an isolate ofT. bakamatsutake was tested in media with C/N ratio ranging from 0 to 50 and with 32 carbon and 12 nitrogen sources. The isolate grew best at the C/N ratio of 30. It utilized the monosaccharidesd-glucose,d-mannose, andd-fructose, the disaccharide trehalose, and polysaccharide pectin among the carbon sources; and yeast extract,l-glutamic acid, and ammonium compounds among the nitrogen sources. The growth of ten isolates and secretion of gluconic and oxalic acids were compared ind-glucose, trehalose, and pectin media. The utilization ofd-glucose, trehalose, and pectin differed among the ten isolates, but all the isolates secreted gluconic acid in thed-glucose media and oxalic acid in the pectin media.     

Leclercia adecarboxylata Gen. Nov., Comb. Nov., formerly known asEscherichia adecarboxylata.

The nameLeclercia adecarboxylata is proposed for a group of the family Enterobacteriacae previously known asEscherichia adecarboxylata. Leclercia adecarboxylata can be phenotypically differentiated from all other species of Enterobacteriaceae. The members of this species are positive for motility, indole production, methyl red, growth in the presence of KCN, malonate, beta-galactosidase, beta-xylosidase, esculin hydrolysis, gas production fromd-glucose, and acid production fromd-cellobiose,d-lactose, melibiose,l-rhamnose, adonitol,d-arabitol, dulcitol, and salicin; the strains were negative for Voges-Proskauer, citrate (Simmons), H₂S (Kligler), lysine and ornithine decarboxylases, arginine dihydrolase, phenylalanine deaminase, gelatinase, DNase, Tween-80 hydrolysis, and acid production from myoinositol and alpha-methyl-d-glucoside. Fermentation ofd-raffinose,d-sucrose, andd-sorbitol is variable with strains. DNA relatedness of 11 strains ofL. adecarboxylata to three strains including the type strain of this species averaged 80% in reactions at 65°C. DNA relatedness to other species in Enterobacteriaceae was 2%–32%, indicating that this species was placed in a new genusLeclercia gen. nov. The type strain ofL. adecarboxylata is ATCC 23216.     

Contents of soluble carbohydrates in yellow lupin seeds maturated at various temperatures

The objective of this paper was to compare the levels of soluble sugars in seeds of yellow lupin cv. Juno matured at different temperatures. The temperature regimes applied were 1). 26 °C for 24 h (high temperature), 2). 24 °C for 12 h and 19 °C for the next 12 h (optimum temperature regime), 3). 26 °C for 16 h and 4 °C for the next 8 h (high-low temperatures). Six soluble carbohydrates (d-galactose, myo-inositol, sucrose, raffinose, stachyose and verbascose) were quantified. Seeds maturing at constant temperature 26 °C accumulated more raffinose (by 100 %) than seeds maturing at optimum temperature regime. Seeds maturing at high temperature accumulated less stachyose and verbascose than those maturing at optimum temperature conditions, the differences being 45 and 24 %, respectively. In seeds maturing at high-low temperature the level of raffinose decreased while the level of stachyose and verbascose increased, compared to those maturing at optimum conditions. The contents of sucrose, d-galactose and myo-inositol in seeds maturing at optimum temperatures was lower than in seeds maturing at both high and high-low temperature regimes. It was shown, that temperature conditions — constant high temperature, or physiologically optimal thermal oscillations (24 °/19 °C) or high-low temperature regime — differently affect the contents of six soluble carbohydrates in maturing seeds of yellow lupin.     

d-Xylose isomerases from a newly isolated strain, Paenibacillus sp., and from Alcaligenes ruhlandii : isolation, characterization and immobilisation to solid supports

d-Xylose/d-glucose isomerases from two strains, a newly isolated strain, Paenibacillus sp., and from Alcaligenes ruhlandii are described herein. The enzymes were purified to apparent homogeneity. Both of these d-xylose isomerases are homotetramers with relative subunit molecular masses of 45 000 and 53 000, respectively, as estimated by sodium dodecylsulphate-polyacrylamide gel electrophoresis. The native molecular masses determined on Superose 12 gel chromatography are 181 kDa for the enzyme from Paenibacillus sp. and 199 kDa for that from A. ruhlandii. The activity of both enzymes shows a requirement for divalent metal ions; the d-xylose isomerase from Paenibacillus sp. has the highest activity with Mn²⁺, while the enzyme from A. ruhlandii prefers Mg²⁺. Both enzymes also accept Co²⁺ with a somewhat lower efficiency, while Cu²⁺ inhibits the enzyme reaction. The binding of the metal ions obeys a biphasic characteristic, indicating the presence of two non-identical binding sites per subunit. d-Glucose is converted to d-fructose at a rate that is two- to three-fold slower than for the d-xylose isomerisation. d-Xylitol and d-lyxose are competitive inhibitors of both enzymes. Both enzymes have a pH optimum between 6.5 and 7.0, and they are active up to 60 °C. The enzyme from Paenibacillus sp. retained 50% of its activity after 4 days at 55 °C, whereas that from A. ruhlandii still retained 50% of its activity after 6 days at 55 °C. Polyacrylamide entrapment and immobilisation to both controlled pore glass and cyanogen-bromide-activated Sepharose were achieved for both enzymes with high efficiency. Received: 14 May 1998 / Received last revision: 29 July 1998 / Accepted: 29 July 1998     

Increased <Emphasis Type="SmallCaps">d</Emphasis>-allose production by the R132E mutant of ribose-5-phosphate isomerase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Ribose-5-phosphate isomerase from Clostridium thermocellum converted d-psicose to d-allose, which may be useful as a pharmaceutical compound, with no by-product. The 12 active-site residues, which were obtained by molecular modeling on the basis of the solved three-dimensional structure of the enzyme, were substituted individually with Ala. Among the 12 Ala-substituted mutants, only the R132A mutant exhibited an increase in d-psicose isomerization activity. The R132E mutant showed the highest activity when the residue at position 132 was substituted with Ala, Gln, Ile, Lys, Glu, or Asp. The maximal activity of the wild-type and R132E mutant enzymes for d-psicose was observed at pH 7.5 and 80°C. The half-lives of the wild-type enzyme at 60°C, 65°C, 70°C, 75°C, and 80°C were 11, 7.0, 4.2, 1.5, and 0.6 h, respectively, whereas those of the R132E mutant enzymes were 13, 8.2, 5.1, 3.1, and 0.9 h, respectively. The specific activity and catalytic efficiency (k _cat/K _m) of the R132E mutant for d-psicose were 1.4- and 1.5-fold higher than those of the wild-type enzyme, respectively. When the same amount of enzyme was used, the conversion yield of d-psicose to d-allose was 32% for the R132E mutant enzyme and 25% for the wild-type enzyme after 80 min.     

Some features of carbohydrate metabolism inRhodotorula glutinis

The yeastRhodotorula glutinis converts bothd-glucose andd-xylose up to 40% to trehalose, the final intracellular level reaching as much as 65 mg trehalose/g dry wt. The reaction ofd-xylose is inhibited byd-glucose both at the transport and the metabolic level. The formation of CO₂ both from endogenous and from externally added trehalose is low. Uncouplers of oxidative phosphorylation (2,4-dinitrophenol and carbonylcyanidem-chlorophenylhydrazone) increase the endogenous production of CO₂ together with a decrease of the intracellular level of trehalose. It is likely that trehalose can serve as a reserve substance forRhodotorula glutinis and that its degradation is blocked during the stationary phase of growth.     

Numerical taxonomy ofKlebsiella pneumoniae strains isolated from clinical and nonclinical sources

A total of 180 clinical and nonclinical isolates ofKlebsiella pneumoniae, for which 99 characteristics were recorded, were subjected to numerical taxonomy analysis. Of these strains, 172 clustered into five major groups, with an overall similarity of 64%. Intragroup similarities ranged from 77 to 82%, with the subgroups corresponding to the speciesK. pneumoniae sensu stricto, K. oxytoca, andKlebsiella spp. 1, 2, and 3. Biochemical tests useful in distinguishing the species included production of indole, degradation of pectate, growth at 10°C, fecal coliform response, production of urease, fermentation of inulin andd-tartrate, utilization ofl-arginine and gentisate andm-hydroxybenzoate, and pigment formation ond-gluconate ferric citrate agar.     

Xylan-degrading activity in yeasts: Growth on xylose,xylan and hemicelluloses

The ability to grow in liquid media withD-xylose, xylan from deciduous trees, and hemicelluloses from conifers was tested in 95 strains of 35 genera of yeasts and yeast-like organisms. Of 54 strains thriving on xylose, only 13 (generaAureobasidium, Cryptococcus andTrichosporon) utilized xylan and hemicelluloses as growth substrates. The árowth media of these strains were found to contain xylandegrading enzymes splitting the substrate to xylose and a mixture of xylose oligosaccharides. The ability of these yeasts to utilize the wood components (hitherto unknown in the genusCryptococeus) makes them potential producers of microbial proteins from industrial wood wastes containing xylose oligosaccharides, xylan, and hemicelluloses as the major saccharide components without previous saccharification.     

Predominance of malolactic fermentation overd-glucose utilization inLactobacillus plantarum andLactobacillus curvatus wine strains

Summary Two selected wine strains of the genusLactobacillus (L. plantarum 197 andL. curvatus 783) were tested for their ability to complete malolactic fermentation (MLF) in a synthetic medium (PBM-broth) supplemented withL-malic acid (7.5–74.6 mM) andD-glucose (5.5–55 mM). The 24 directed fermentation assays, 12 for each bacterial strain, were carried out at 20°C and pH 3.5. MLF was completed (residualL-malic acid 0.2 mM) in eight days in 19 of the 24 fermentation assays, even in the presence of 74.6 mML-malic acid or 55.5 mMD-Glucose utilization was generally simultaneous to MLF but was completed (residual concentrations 0.2 mM) only in 6 of the 24 fermentation assays. These results support the use of these strains in directed MLF assays at the very differentL-malic acid andD-glucose concentrations tested.     

Effect of temperature on <Emphasis Type="SmallCaps">d</Emphasis>-arabitol production from lactose by <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis>

d-Arabitol production from lactose by Kluyveromyces lactis NBRC 1903 has been studied by following the time courses of concentrations of cell mass, lactose, d-arabitol, ethanol, and glycerol at different temperatures. It was found that temperature is a key factor in d-arabitol production. Within temperatures ranging from 25 to 39°C, the highest d-arabitol concentration of 99.2 mmol l⁻¹ was obtained from 555 mmol l⁻¹ of lactose after 120 h of batch cultivation at 37°C. The yield of d-arabitol production on cell mass growth increased drastically at temperatures higher than 35°C, and the yield reached 1.07 at 39°C. Increasing the cell mass concentration two-fold after 24 h of culture growth at 37°C, the d-arabitol concentration further increased to 168 mmol l⁻¹. According to the distribution of the metabolic products, metabolic changes related to growth phase were also discussed. The stationary-phase K. lactis cells in the batch culture that is started with exposing the precultured inoculum to high osmotic stress, high oxidative stress, and high heat stress are found to be preferable for d-arabitol production.     

Production of trehalose synthase from a basidiomycete, Grifola frondosa, in Escherichia coli

The genomic DNA and cDNA for a gene encoding a novel trehalose synthase (TSase) catalyzing trehalose synthesis from α-d-glucose 1-phosphate and d-glucose were cloned from a basidiomycete, Grifola frondosa. Nucleotide sequencing showed that the 732-amino-acid TSase-encoding region was separated by eight introns. Consistent with the novelty of TSase, there were no homologous proteins registered in the databases. Recombinant TSase with a histidine tag at the NH₂-terminal end, produced in Escherichia coli, showed enzyme activity similar to that purified from the original G. frondosa strain. Incubation of α-d-glucose 1-phosphate and d-glucose in the presence of recombinant TSase generated trehalose, in agreement with the enzymatic property of TSase that the equilibrium lay far in the direction of trehalose synthesis. Received: 12 January 1998 / Received revision: 20 February 1998 / Accepted: 20 March 1998     

Enhancement of the thermostability and hydrolytic activity of GH10 xylanase by module shuffling between <Emphasis Type="Italic">Cellulomonas fimi</Emphasis> Cex and <Emphasis Type="Italic">Thermomonospora alba</Emphasis> XylA

Two family GH10 xylanases with different thermostability, the Cex (optimum temperature 40°C) from Cellulomonas fimi and the XylA (optimum temperature 80°C) from Thermomonospora alba, were used to construct a chimeric xylanase by module shuffling for investigating the structural determinants responsible for the difference. The parent genes were shuffled by crossovers at selected module borders using self-priming Polymerase Chain Reaction (PCR)s. The shuffled construct, designated as CXC-X4,5, was cloned and its nucleotide sequence was confirmed. The chimera CXC-X4,5 showed activity against 4-O-methyl-d-glucurono-d-xylan–Remazol Brilliant Blue R (RBB-xylan) and over-expressed as His-tag fusion proteins. The homogeneous chimeric protein CXC-X4,5 showed significantly improved thermal profiles (optimum temperature 65°C) compared to those of one of the parents, Cex. This was apparently due to the influence of amino acids in the modules M4 and M5 inherited from thermophilic XylA. Measured K _m and k _cat values for the substrate p-nitrophenyl-β-d-cellobioside (PNP-G2) were closer to those of the other parent, Cex; however the K _m and k _cat values for the substrate p-nitrophenyl-β-d-xylobioside (PNP-X2) were between two parental xylanases. The ability of the chimeric enzyme to produce reducing sugar from xylan was enhanced in comparison with the parental enzymes. These results indicated that the amino acid residues in the modules M4 and M5 of XylA play an important role in determining enzyme characteristics such as thermal stability, and xylanases with improved properties can be prepared by manipulating this segment.     

Characterization of yeasts isolated from red wine surface film

We have isolated 6 morphologically different axenic yeast cultures from the film surface of red wine. Based on morphological, physiological and biochemical characteristics we have identified the strains as follows: isolates1–4 are morphologically different strains of the anamorph basidiomycetous film-forming yeastCandida humicola (Daszewska) Diddens etLodder, syn.Apiotrichum humicola (Daszewska) von Arx Weijman. Isolates5 and6 belong to the genusSaccharomyces of the associated speciesS. cerevisiœ (isolate5 originallyS. bayanus, isolate6 S. capensis). These do not participate in the surface film formation.     

Characterization of the catalytic domains of <Emphasis Type="Italic">Trichoderma reesei</Emphasis> endoglucanase I,II, and III,expressed in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The genes encoding the catalytic domains (CD) of the three endoglucanases (EG I; Cel7B, EG II; Cel5A, and EG III; Cel12A) from Trichoderma reesei QM9414 were expressed in Escherichia coli strains Rosetta-gami B (DE3) pLacI or Origami B (DE3) pLacI and were found to produce functional intracellular proteins. Protein production by the three endoglucanase transformants was evaluated as a function of growth temperature. Maximal productivity of EG I-CD at 15°C, EG II-CD at 20°C and EG III at 37°C resulted in yields of 6.9, 72, and 50 mg/l, respectively. The endoglucanases were purified using a simple purification method based on removing E. coli proteins by isoelectric point precipitation. Specific activity toward carboxymethyl cellulose was found to be 65, 49, and 15 U/mg for EG I-CD, EG II-CD, and EG III, respectively. EG II-CD was able to cleave 1,3–1,4-β-d-glucan and soluble cellulose derivatives. EG III was found to be active against cellulose, 1,3–1,4-β-d-glucan and xyloglucan, while EG I-CD was active against cellulose, 1,3–1,4-β-d-glucan, xyloglucan, xylan, and mannan.     

D-Aminoacylase from a novel producer: Stenotrophomonas maltophilia ITV-0595

A novel bacterial strain producing D-aminoacylase was isolated from organic waste and identified as Stenotrophomonas maltophilia ITV-0595. The isolation was performed using N-acetyl-D-phenylglycine (NAcDPG) as the sole source of C and N. The optimum pH for enzyme expression was 8 at 37°C. Using N-Ac-DPG concentrations from 0.5 up to 3% w/v, it was observed that at the 1% level, the microorganism showed acceptable responses in both enzyme activities and cell growth. From the different tested compounds N-acetyl-D-methionine (1%) was the best enzyme inducer (Sp. act. = 4.14 U mg⁻¹ protein, Vol. act. = 0.17 U ml⁻¹) and the only one that increased cell growth. Received 13 June 1997/ Accepted in revised form 29 October 1998