Purification and properties of two lactose hydrolases from Trichosporon cutaneum

Two enzymes that hydrolysed lactose were purified essentially to homogeneity from cell extracts of the oleaginous yeast Trichosporon cutaneum. One enzyme of Mr 120,000 had properties typical of a beta-galactosidase (EC 3.2.1.23). It hydrolysed lactose, lactulose and nitrophenyl-beta-D-galactosides. The enzyme required K+ or Rb+ for activity, and other monovalent cations tested were not effective. Enzyme activity was abolished by EDTA and stimulated by Mg2+, Mn2+ and Ca2+. The beta-galactosidase was induced by lactose, galactose, lactulose and lactobionic acid. The other enzyme, a beta-glycosidase (EC 3.2.1.21) of Mr 52,000 showed no ionic requirements and it hydrolysed lactose, nitrophenyl-beta-D-galactosides, 4-nitrophenyl-beta-D-glucoside, cellobiose, laminaribiose, laminaritriose and sophorose, but not gentiobiose, 4-nitrophenyl-beta-D-mannoside or sucrose. This enzyme was induced by lactose, galactose and lactulose, and also by cellobiose.     

Utilization of organic substrates during mixotrophic and heterotrophic cultivation of algae

Pure cultures ofChlorella pyrenoidosa (82) andScenedesmus obliquus (125) were grown in the nutrient medium according to Benson in the presence of 0·05m sugars or 0·025m sodium salts of organic acids. The density of culture was measured throughout the course of growth. Satisfactory heterotrophic sources of nutrition forChlorella pyrenoidosa appear to be galactose, glucose and acetate, whereasScenedesmus utilizes glucose, cellobiose and acetate. The growth ofChlorella in the light is enhanced by galactose, glucose, fructose, cellobiose and maltose, that ofScenedesmus by glucose, fructose, cellobiose, galactose, maltose, acetate and pyruvate. Soluble starch suppresses growth of both cultures. The role of the substrates is discussed. It follows from the results that the growth-promoting sugars and organic acids can act not only as a source of carbon during general carbon shortage but also as ergastic material. The mechanism of utilization of some organic substrates will be taken up in a subsequent paper.     

Pichia norvegensis sp. nov.

A new yeast species Pichia norvegensis Leask et Yarrow is described as the perfect state of Candida norvegensis (Dietrichson) van Uden et Farinha ex van Uden et Buckley. Strains of this species were isolated on 3 occasions from human vaginas. This species differs from other Pichia species that assimilate glucose but not galactose, sucrose, maltose, lactose, D-mannitol and D-glucitol by assimilating cellobiose.     

固囊酵母的一个新种

从中国保定槐茂甜面酱中分离到一株酵母。经鉴定,这株酵母菌属于固囊酵母属(Citeromyces),并为该属中的一个新种,命名为保定团囊酵母(Citeromyces baodingensis zhangsp.Nov.)。保定团囊酵母与固囊酵母生理生化特性有显著区别,保定固囊酵母(Citeromycesbaodingensis)不发酵蔗糖、棉子糖,同化半乳糖和纤维二糖,不同化海藻糖和棉子糖,G+C含量为48.5mol％。     

Kinetic study of a cellobiase purified from Neocallimastix frontalis EB188

A cellobiase was purified from the culture supernatant of Neocallimastix frontalis EB188. This enzyme possessed a molecular weight of 85,000 and an isoelectric point of 6.95. The enzyme rapidly hydrolyzed cellobiose, p-nitrophenyl (pNP) beta-D-glucopyranoside (pNPG) and cellotriose and slowly hydrolyzed cellopentaose and salicin. The enzyme did not hydrolyze pNP alpha-D-glucopyranoside or pNP beta-D-cellobioside. Substrate inhibition was observed when cellobiose or pNPG were used as the substrates and glucose production was measured. The kinetic parameters were: K = 0.053 mM, V = 5.88 U/mg of protein and Ki = 0.95 mM for cellobiose; K = 0.36 mM, V = 1.05 U/mg and Ki = 8.86 mM for pNPG. Substrate inhibition was not detected during the hydrolysis of pNPG when pNP production was measured. The kinetic parameters for pNPG were: K = 0.67 mM and V = 1.49 U/mg of protein. The presence of an enzyme.glucose.substrate complex and transglucosylation was evident during the catalysis. Glucose, cellobiose, glucono-delta-lactone, galactose, lactose, maltose and salicin acted as competitive inhibitors during the hydrolysis of pNPG with the apparent inhibition constants (Kis) of 4.8 mM, 0.035 mM, 0.062 mM, 28.5 mM, 0.38 mM, 15.0 mm and 31.0 mM, respectively.     

Carbohydrases of the rumen ciliate Epidinium ecaudatum (Crawley). Hydrolysis of plant hemicellulose fractions and beta-linked glucose polymers

1. Cell-free extracts from Epidinium ecaudatum (Crawley) hydrolysed the three hemicellulose fractions of pasture plants, but at different rates. 2. All of the constituent monosaccharides are released from the hemicellulose fractions, galactose and uronic acids being liberated at much slower rates than pentoses. 3. An arabinofuranosidase, which removes arabinose from highly branched arabinoxylan before the xylan chain can be hydrolysed, was isolated free from other pentosanases. 4. A xylanase hydrolysing xylan (by random cleavage) and xylodextrins of degree of polymerization (D.P.) > 3 to xylotriose and xylobiose was isolated free from other pentosanases. 5. A separate xylodextrinase hydrolysing (by random cleavage) xylodextrins of D.P. > 2 to xylobiose and xylose was also obtained; this enzyme did not hydrolyse xylan or xylobiose and the original extracts themselves possessed very weak xylobiase activity. 6. The epidinial extracts hydrolysed laminaribiose, laminarin, lichenin and cellodextrins of D.P. < 7 rapidly, cellobiose and gentiobiose slowly but cellulose not at all. 7. Polysaccharide glucose associated with plant linear B hemicellulose was liberated with cellobiose and possibly laminaribiose as intermediates. 8. The cellodextrinase hydrolysed cellopentaose initially to cellobiose plus cellotriose and is a distinctly different enzyme from the xylanase and xylodextrinase. 9. Extracts from Entodinium species and Eremoplastron bovis also hydrolysed all three types of plant hemicellose.     

Engineered Escherichia coli capable of co-utilization of cellobiose and xylose

Natural ability to ferment the major sugars (glucose and xylose) of plant biomass is an advantageous feature of Escherichia coli in biofuel production. However, excess glucose completely inhibits xylose utilization in E. coli and decreases yield and productivity of fermentation due to sequential utilization of xylose after glucose. As an approach to overcome this drawback, E. coli MG1655 was engineered for simultaneous glucose (in the form of cellobiose) and xylose utilization by a combination of genetic and evolutionary engineering strategies. The recombinant E. coli was capable of utilizing approximately 6 g/L of cellobiose and 2 g/L of xylose in approximately 36 h, whereas wild-type E. coli was unable to utilize xylose completely in the presence of 6 g/L of glucose even after 75 hours. The engineered strain also co-utilized cellobiose with mannose or galactose; however, it was unable to metabolize cellobiose in the presence of arabinose and glucose. Successful cellobiose and xylose co-fermentation is a vital step for simultaneous saccharification and co-fermentation process and a promising step towards consolidated bioprocessing.     

Cellulase induction by lactose in Trichoderma reesei PC-3-7

In an attempt to clarify the function of lactose in cellulase induction, experiments were carried out on cellulase formation by lactose along with other sugars in a resting cell system of Trichoderma reesei PC-3-7, a hypercellulase-producing mutant. Although lactose alone induces little cellulase under the conditions used, a synergistic effect on cellulase formation was observed following the respective addition of sophorose, cellobiose or galactose to lactose. The lactose consumption was more rapid when these sugars were added than in their absence. Furthermore, following lactose addition 10 h after the beginning of cultivation in the presence of cellobiose, cellulase formation was initiated with only a little lag, and lactose consumption started immediately, being complete in 14 h. \-Galactosidase induction experiments suggested that the rapid consumption of lactose is possibly not dependent on lactose degradation by the enzyme. From these results, it is suggested that lactose may function as an inducer for cellulase formation if it is taken up in the mycelium of T. reesei PC-3-7, and that sophorose, cellobiose or galactose may induce a putative lactose permease. *** DIRECT SUPPORT *** AG903066 00005     

Purification and characterization of a human lectin specific for penultimate galactose residues

A novel lectin has been found in human plasma. The lectin was purified by affinity chromatography using an adsorbent in which 2-O-alpha-D-glucopyranosyl-O-beta-D-galactopyranosylhydroxylysine (Glc-Gal-Hyl) was coupled to Sepharose. The molecular weight of the lectin was determined by gradient gel electrophoresis to be approximately 240,000. On polyacrylamide gel electrophoresis in sodium dodecyl sulphate, the subunit had the molecular weight of 29,500. Composition analysis has shown the lectin is a glycoprotein in which 12% of the molecule consists of carbohydrate. Native human, horse, calf, sheep, rabbit, and rat erythrocytes were agglutinated by the lectin in the presence of calcium. Glc-Gal-Hyl, N-acetylated Glc-Gal-Hyl, and stachyose inhibited the hemagglutination, whereas monosaccharides, maltose, cellobiose, lactose, raffinose, galactosylhydroxylysine, and N-acetylated galactosylhydroxylysine were not inhibitory. The lectin is strongly inhibited by the desialylated bovine erythrocyte glycoprotein, which contains galactose beta 1-3galactose beta-sequence at the nonreducing termini of the sugar chains, whereas disialylated orosomucoid did not inhibit the lectin. These results indicate that the lectin recognizes the penultimate galactose residue in a hapten molecule in contrast to usual galactose-binding proteins or galactose-specific lectins, which recognize exposed, terminal galactose residues of sugar chains.     

The mechanism of removal of leukocytes by cellulose columns

Cellulose columns efficiently remove leukocytes from whole blood. Interaction of leukocytes with cellulose particles is not affected by glucose, galactose, fructose, mannose, or cellobiose. Although red cells normally pass through cellulose columns, they are retained after fixation in glutaraldehyde. We conclude that the leukocyte-removing activity of cellulose columns is due to mechanical filtration rather than to specific adherence of leukocytes to the cellulose particles.     

Exopolysaccharide Biosynthesis in Lactococcus lactis NIZO B40: Functional Analysis of the Glycosyltransferase Genes Involved in Synthesis of the Polysaccharide Backbone

We used homologous and heterologous expression of the glycosyltransferase genes of the Lactococcus lactis NIZO B40 eps gene cluster to determine the activity and substrate specificities of the encoded enzymes and established the order of assembly of the trisaccharide backbone of the exopolysaccharide repeating unit. EpsD links glucose-1-phosphate from UDP-glucose to a lipid carrier, EpsE and EpsF link glucose from UDP-glucose to lipid-linked glucose, and EpsG links galactose from UDP-galactose to lipid-linked cellobiose. Furthermore, EpsJ appeared to be involved in EPS biosynthesis as a galactosyl phosphotransferase or an enzyme which releases the backbone oligosaccharide from the lipid carrier.     

Microbial fluxes of free monosaccharides and total carbohydrates in freshwater determined by PAD-HPLC

Abstract A new sensitive pulsed amperometric detection (PAD) method for measurements of mono- and disaccharides in nM concentrations was used in combination with high performance liquid chromatography (HPLC) to study fluxes of dissolved free and combined carbohydrates (DFCHO and DCCHO) in lake water. In a diel study concentrations of individual free saccharides typically were 5–50 nM, while total DFCHO concentrations ranged from 67 to 224 nM. No diel trends in concentration changes were obvious. At in situ light-dark conditions, dominant DFCHO were galactose, glucose, fructose and mannose/xylose. In addition to these saccharides, an increased abundance of melibiose and arabinose was measured in a parallel dark incubation. In a 118 h laboratory incubation of 1.0 μm filtered lake water, concentrations of DFCHO decreased from 194 nM (at 12 h) to a minimum of 54 nM (at 73 h). Dominant DFCHO were glucose, fructose and cellobiose. During the incubation DCCHO varied from 1.27 to 2.20 μM. Glucose, galactose and cellobiose made up 40, 30 and 10 mol-%, respectively, of the DCCHO. Fructose was degraded during hydrolysis of the DCCHO. A decline of DCCHO at 55 h was reflected in a simultaneous increase of DFCHO, but otherwise no similarities between the two saccharide pools were found. Incrased DCCHO concentrations and a high assimilation of glucose and fructose that was not reflected in a decline of their concentrations, both indicate that carbohydrates were produced during the experiment. Polysaccharides were probably excreted by the bacteria. Net assimilation of glucose and fructose sustained 14–19% (diel study) and 32% (long-term study) of the net bacterial carbon requirement.     

Non-osmotic effects of external sugars on the cell water content and transport of cations across the plasmalemma inHydrodictyon reticulatum

Saccharides added to the cultivation medium influence the properties of the cell wall of the strictly autotrophic green algaHydrodictyon reticulatum, most probably in such a way that they interfere with the processes of growth and repair of microfibrils. Natural monosaccharides (glucose, fructose, galactose, mannose, sucrose, cellobiose, raffinose) reduce the cell water content, increase the intracellular concentration mostly of both potassium and sodium cations and reduce the ohmic resistance of the cell membrane.     

A rapid method for gas chromatographic analysis of mono- and disaccharide mixtures

A technique is described for the rapid gas-liquid chromatographic analysis of mixtures of carbohydrate trimethylsilyl ethers prepared at mutarotation equilibrium. A mixture containing arabinose, ribose, xylose, fructose, glucose, galactose, mannose, sucrose, maltose, and cellobiose can be determined in less than 16 min using high-rate temperature programming.     

The release of fermentable carbohydrate from peat by steam explosion and its use in the microbial production of solvents

Steam treatment of peat at 200 degrees C for 3 min, followed by instantaneous decompression (steam explosion), solubilized up to 28% of the dry matter. Seventy-five percent of the solubilized material was carbohydrate, 33% of which was composed of mono- and disaccharides, including galactose, glucose, xylose, mannose, arabinose, and cellobiose, in order of decreasing concentration. The solubilized materials served as the sole source of carbohydrate for growth and solvent production by Clostridium acetobutylicum and C. butylicum which utilized up to 40% of the carbohydrate. Of the saccharides in this mixture, galactose was the least readily utilized. Approximately 30% of the fermentable carbohydrate used was converted to fatty acids and solvents, with the primary fermentation product being butyrate. Clostridium thermohydrosulfuricum was able to utilize ca. 50% of the carbohydrate, and simultaneously produced slightly more than 1 mol ethanol/mol saccharide metabolized. This organism, like other strains tested, used galactose less readily than the other sugars. The residue from the steam explosion process contained 24% cellulose, but it could not serve as a source of carbohydrate for the growth of either Bacteroides succinogenes or Clostridium thermocellum, suggesting that inhibitors were released during the steam treatment.     

Utilisation of carbon substrates by multiple genotypes of ericoid mycorrhizal fungal endophytes from eastern Australian Ericaceae

The abilities of six genotypes of two putative Helotiales ascomycete ericoid mycorrhizal fungal taxa from Woollsia pungens and Leucopogon parviflorus (Ericaceae) to utilise glucose, galactose, mannose, cellobiose, carboxymethylcellulose, crystalline cellulose, starch and xylan as sole carbon sources were tested in axenic liquid culture. With the exception of all taxon II isolates on carboxymethylcellulose, all genotypes of both taxa produced measurable biomass on all substrates. Significant intraspecific variation was observed in biomass production on all substrates. While pooled data for all genotypes of each taxon revealed significant interspecific differences in biomass production on carboxymethylcellulose, glucose, cellobiose, and starch, mean biomass production for each taxon on the latter three substrates differed less than threefold, suggesting that the saprotrophic abilities of the two taxa are broadly similar.     

Cloning and characterization of the glucooligosaccharide catabolic pathway beta-glucan glucohydrolase and cellobiose phosphorylase in the marine hyperthermophile Thermotoga neapolitana

Characterization in Thermotoga neapolitana of a catabolic gene cluster encoding two glycosyl hydrolases, 1,4-beta-D-glucan glucohydrolase (GghA) and cellobiose phosphorylase (CbpA), and the apparent absence of a cellobiohydrolase (Cbh) suggest a nonconventional pathway for glucan utilization in Thermotogales. GghA purified from T. neapolitana is a 52.5-kDa family 1 glycosyl hydrolase with optimal activity at pH 6.5 and 95 degrees C. GghA releases glucose from soluble glucooligomers, with a preference for longer oligomers: k(cat)/K(m) values are 155.2, 76.0, and 9.9 mM(-1) s(-1) for cellotetraose, cellotriose, and cellobiose, respectively. GghA has broad substrate specificity, with specific activities of 236 U/mg towards cellobiose and 251 U/mg towards lactose. With p-nitrophenyl-beta-glucoside as the substrate, GghA exhibits biphasic kinetic behavior, involving both substrate- and end product-directed activation. Its capacity for transglycosylation is a factor in this activation. Cloning of gghA revealed a contiguous upstream gene (cbpA) encoding a 93.5-kDa cellobiose phosphorylase. Recombinant CbpA has optimal activity at pH 5.0 and 85 degrees C. It has specific activity of 11.8 U/mg and a K(m) of 1.42 mM for cellobiose, but shows no activity towards other disaccharides or cellotriose. With its single substrate specificity and low K(m) for cellobiose (compared to GghA's K(m) of 28.6 mM), CbpA may be the primary enzyme for attacking cellobiose in Thermotoga spp. By phosphorolysis of cellobiose, CbpA releases one activated glucosyl molecule while conserving one ATP molecule per disaccharide. CbpA is the first hyperthermophilic cellobiose phosphorylase to be characterized.     

Glycosyltransferases in the Golgi membranes of onion stem

Cell fractions consisting largely of Golgi membranes were prepared from the meristematic region of the onion. Several enzyme activities were found to be localized in these fractions: inosine diphosphatase, galactosyltransferases and glucosyltransferases. The fractions catalysed the transfer of [(14)C]galactose from UDP-galactose to endogenous and cell-sap acceptors, to N-acetylglucosamine and to ovalbumin. In the presence of bovine alpha-lactalbumin, transfer to glucose (lactose synthesis) was catalysed. [(14)C]Glucose was transferred from UDP-glucose to endogenous and cell-sap acceptors, to cellobiose and to fructose (sucrose synthesis). All these activities were latent, being potentiated by detergents (Triton X-100 or sodium deoxycholate). The characteristics of some of these enzyme activities are described and their biological significance is discussed.     

Carbohydrate assimilation by clinical and environmental Rhodotorula glutinis strains

This study was carried to determine the carbohydrate assimilation patterns of Rhodotorula strains isolated from clinical and environmental specimens. We have tested the commercial system ID 32C (bioMerieux, France) on 80 different strains of Rhodotorula glutinis: 47 strains from clinical samples and 33 strains from environmental samples. The assimilation percentages obtained in our study for galactose, cellobiose, gluconate and sorbose were lower than those showed in the identification table of the method. However, the assimilation percentages for mannitol and esculin were higher. According to our results, we conclude that the numerical profiles and the identification software of the commercial system present limitations for the characterization of some R. glutinis strains.