Isolation and characterization of the enzymes involved in disintegration of the cell wall of Chlorella fusca

Using different chromatographical methods we have isolated an enzyme from the sporangia of Chlorella fusca Shihira et Krauss var. vacuolata , strain SAG 211–8b, which is responsible for the partial disintegration of the sporangium wall. We refer to it as carbohydrate-releasing activity (CRA). It is an endoenzyme and splits oligosaccharides from the inner layer of the cell wall. In appropriate tests it shows β- d -fucosidase activity (EC 3.2.1.,38): The protein has a molecular weight around 45 kDa and an isoelectric point of pH 4.3; maximum activity is found at pH 5.4 and 60°C, although this temperature inactivates the enzyme quickly. β- d -Mannosidase (EC 3.2.1.25) and β- d -glucosidase (EC 3.2.1.21) were also found in the presence of CRA. These glycosidases were identified as exoenzymes. They are involved in the further degradation of the liberated obligosaccharides.     

Cloning of a Thermomonospora fusca xylanase gene and its expression in Escherichia coli and Streptomyces lividans.

Thermomonospora fusca chromosomal DNA was partially digested with EcoRI to obtain 4- to 14-kilobase fragments, which were used to construct a library of recombinant phage by ligation with EcoRI arms of lambda gtWES. lambda B. A recombinant phage coding for xylanase activity which contained a 14-kilobase insert was identified. The xylanase gene was localized to a 2.1-kilobase SalI fragment of the EcoRI insert by subcloning onto pBR322 and derivatives of pBR322 that can also replicate in Streptomyces lividans. The xylanase activity produced by S. lividans transformants was 10- to 20-fold higher than that produced by Escherichia coli transformants but only one-fourth the level produced by induced T. fusca. A 30-kilodalton peptide with activity against both Remazol brilliant blue xylan and xylan was produced in S. lividans transformants that carried the 2.1-kilobase SalI fragment of T. fusca DNA and was not produced by control transformants. T. fusca cultures were found to contain a xylanase of a similar size that was induced by growth on xylan or Solka Floc. Antiserum directed against supernatant proteins isolated from a Solka Floc-grown T. fusca culture inhibited the xylanase activity of S. lividans transformants. The cloned T. fusca xylanase gene was expressed at about the same level in S. lividans grown in minimal medium containing either glucose, cellobiose, or xylan. The xylanase bound to and hydrolyzed insoluble xylan. The cloned xylanase appeared to be the same as the major protein in xylan-induced T. fusca culture supernatants, which also contained at least three additional minor proteins with xylanase activity and having apparent molecular masses of 43, 23, and 20 kilodaltons.     

Possible role of cAMP in the synthesis of β-glucanases and β-xylanases of Bacillus circulans WL-12

Abstract Bacillus circulans WL-12 secretes 1,4-β- d -xylanase and 1,3-β- d - and 1,6-β- d -glucanase activities. All of them are catabolites regulated by glucose and, while xylanase needs xylan as the inducer, the two latter enzyme activities are formed once glucose is depleted. Cyclic nucleotides such as adenosine 3',5'-monophosphate (cAMP) and guanosine 3',5' monophosphate (cGMP) exhibit a negative effect on enzyme synthesis if added to the culture media. Based on the fact that only cAMP is found in cells growing in glucose-rich media we propose a model for B. circulans WL-12 in which cAMP acts as a negative effector for regulating the synthesis of these enzymes. The model is not, however, extrapolated to other Bacillus species and all B. circulans strains.     

Logistics in the life cycle of Photosystem II—lateral movement in the thylakoid membrane and activation of electron transfer

The metabolism and intracellular localization of salicylic acid (SA) was investigated in soybean ( Glycine max [L.] cv Williams 82) cell suspension cultures. [7–¹⁴C]SA was added to the cell cultures, the metabolites were extracted from the cells at various time points and analysed by TLC and HPLC. The [7–¹⁴C]SA was taken up rapidly from the culture media and converted primarily to SA 2- O -β- d -glucose (SAG). Lower levels of glucosylated 2,5-dihydroxbenzoic acid (gentisic acid) and methyl salicylate 2- O -β- d -glucose were also formed. Examination of the intracellular localization of the glucose conjugates revealed that all of the conjugates associated with the protoplasts were found in the vacuoles. An SA glucosyltransferase (SAGT) that could catalyse the formation of SAG from SA and UDP-glucose could be extracted from soybean cells and assayed in vitro. Increasing concentrations of SA added to the culture media induced the SAGT activity. The highest levels of SAGT activity were observed in cells treated with 0.5 m M SA. The SAGT activity in these cells was 88-fold greater than the SAGT activity in the untreated cells. The intracellular localization of the SAGT activity was also examined and it was determined that the majority of the SAGT activity in the protoplasts was located outside the vacuole. Therefore, it appears as if SAG is formed from SA outside the vacuole, presumably in the cytoplasm, and then subsequently transported into the vacuole where it accumulates.     

A constitutive, plasma-membrane bound β-glucosidase in Trichoderma reesei

Abstract Plasma membranes of Trichoderma reesei QM 9414, isolated from protoplasts by means of the concanavalin A procedure, contained β-glucosidase activity, which appeared constitutively upon growth on glucose. The enzyme had a pH optimum around 6, and was active on p -nitrophenyl-β- d -glucoside, cellobiose and sophorose ( K _m 0.7, 3.9 and 3.1 mM, respectively). Glucose was only weakly inhibitory ( K _i 7 mM). Treatment of the plasma membranes with Triton X-100, Tween 80 or digitonin solubilized more than 60% of the membrane-bound β-glucosidase activity. The enzyme so solubilized exhibited an M _r of 70 000 ± 5000 and an isoelectric point at pH 8.2 ± 0.3.     

Uptake and metabolism of taurine in the green alga Chlorella fusca

Taurine entered the alga Chlorella fusca Shihira et Krauss strain 21l-8b via a pH and energy-dependent system ("permease"). Transport followed triphasic kinetics from 10⁻⁶ to 10⁻² M with K_m values for taurine of 5.4 × 10⁻⁵, 4.1 × l0⁻⁴ and l.5 × 10⁻³ M. This uptake system was specific for sulfonic acids and showed no affinity for α- and β -amino acids or Na⁺; thus the permease of C. fusca is different from all known taurine transport systems with respect to structural specificity and lack of Na⁺ -dependence. Uptake was not observed in sulfate-grown algae but developed as a response to sulfate limitation within 2 h. Sulfate addition caused a rapid decline in taurine transport capacity. Labeled taurine was rapidly metabolized in C. fusca to sulfate and ethanolamine, suggesting oxidative hydrolysis as the mechanism of C-S bond cleavage. Further incorporation of these catabolic products in C - and S -metabolism was demonstrated. Taurine catabolism was also detected in other green algae and some cyanobacteria.     

Hydrolytic and phosphorolytic metabolism of cellobiose by the marine aerobic bacterium Saccharophagus degradans 2-40T

Saccharophagus degradans 2-40 is a marine gamma proteobacterium that can produce polyhydroxyalkanoates from lignocellulosic biomass using a complex cellulolytic system. This bacterium has been annotated to express three surface-associated β-glucosidases (Bgl3C, Ced3A, and Ced3B), two cytoplasmic β-glucosidases (Bgl1A and Bgl1B), and unusual for an aerobic bacterium, two cytoplasmic cellobiose/cellodextrin phosphorylases (Cep94A and Cep94B). Expression of the genes for each of the above enzymes was induced when cells were transferred into a medium containing Avicel as the major carbon source except for Bgl1B. Both hydrolytic and phosphorolytic degradation of cellobiose by crude cell lysates obtained from cellulose-grown cells were demonstrated and all of these activities were cell-associated. With the exception of Cep94B, each purified enzyme exhibited their annotated activity upon cloning and expression in E. coli. The five β-glucosidases hydrolyzed a variety of glucose derivatives containing β-1, (2, 4, or 6) linkages but did not act on any α-linked glucose derivatives. All but one β-glucosidases exhibited transglycosylation activity consistent with the formation of an enzyme-substrate intermediate. The biochemistry and expression of these cellobiases indicate that external hydrolysis by surface-associated β-glucosidases coupled with internal hydrolysis and phosphorolysis are all involved in the metabolism of cellobiose by this bacterium.     

An enzyme system hydrolysing the polysaccharides of Xanthomonas species

Enzymes hydrolysing the exopolysaccharides of Xanthomonas campestris and related species (xanthan) have been obtained from a Bacillus species isolated by enrichment culture. Growth on xanthan induced a number of enzymes acting on the xanthan molecule. These included one or more β-glucanohydrolases and β-glucosidases, together with mannosidases. The former activities were also present in cultures grown in the presence of laminaran or scleroglucan, but not in simple synthetic media with glucose as substrate. Partial purification of the enzymes active on glucans was achieved by ammonium sulphate precipitation and chromatography on DEAE-sepharose and CM-sepharose. The specificity of the β-glucosidase and the β-glucanohydrolase were investigated. Several β-glucans were hydrolysed to glucose and disaccharides, but there was no activity against β→ 6 linked polymers, cellulose azure or microcrystalline cellulose. Carboxymethylcellulose was hydrolysed, as were laminaran, scleroglucan and pachyman. Activity was greater against the β→ 4 linked glucans than against the β→ 3 linked glucans tested. As periodate-oxidized laminarin was also hydrolysed, it was concluded that the glucanohydrolase acted as an endo enzyme. The β-glucosidase had a pH optimum at about 8–2 and a temperature optimum at 45°C; it showed higher activity against o -nitrophenyl-D-glucopyranoside, cellobiose, trehalose and sophorose than against gentibiose.     

β-Glucan synthetase activity in Golgi vesicles ofPetunia hybrida

-Glucan synthetase activity has been demonstrated in a Golgi vesicle fraction isolated from pollen tubes ofPetunia hybrida. This-glucan synthetase activity differs from that of most other higher plants in its inability to incorporate [¹⁴C]glucose from GDP-[¹⁴C]glucose. UDP-[¹⁴C]glucose, however, is an appropriate glucose donor for this enzyme. The optimum conditions for this-glucan synthetase activity are: 1 mg Golgi vesicle protein/ml reaction mixture; pH=±8 and a temperature of 25°C. The newly synthesized alkali-insoluble glucan contains-1,3- as well as -1,4-glucosidic linkages.     

Use of perfused cores for evaluating extracellular enzyme activity in stream-bed sediments

Abstract β-Glucosidase activity was investigated in stream-bed sediments using 4-methylumbelliferyl-β- d -glucopyranoside (MUF-β-Glc) as a model substrate. In a perfused core technique, water containing MUF-β-Glc was perfused up through sediment cores. β-glucosidase activity quantified from the release of fluorescent MUF in water discharge from the cores. At low rates of perfusion, maximum β-glucosidase activity ( V _max) in perfused sediments was similar to that in suspended (unperfused) sediments. Substrate affinity( K m)was higher in the suspended sediments. V _maxand K _m both increased when the perfusion rate was raised, although naturally-low substrate concentrations could mean that variability in perfusion rates has little effect on enzyme activity in the field. V _max was uninfluenced by whether ground or stream water was perfused through the sediments, but K _m was higher in cores perfused with groundwater. Increasing concentrations of glucose in the perfusion water resulted in a progressive inhibition of β-glucosidase activity. Although natural concentrations of glucose were low, the high turnover of enzymatically-released glucose probably means that β-glucosidase activity could be regulated by product concentration.     

Extracellular β-D-glucosidase of the Penicillium canescens marine fungus

Extracellular β-D-glucosidase was isolated in a homogeneous state from the Penicillium canescens marine fungus. According to SDS-electrophoresis, the molecular weight of the enzyme was 64 kDa and the maximal activity was observed at pH 5.2 and 70°C. Glucosidase catalyzed the hydrolysis of β-glycosidic bonds both in glycosides and in glucose disaccharides and had transglycosylation activity. The enzyme can be used for the deglycosylation of natural glycosides and in enzymatic synthesis of new carbohydrate—containing compounds.     

Production of β-glucosidase (cellobiase) by Curvularia sp.

A Curvularia sp. isolated from soil was found to produce extracellular β-glucosidase activity when grown in yeast extract, peptone, carboxymethylcellulose (YPC) medium. An initial medium pH of 6·5 and cultivation temperature of 30°C were found to be most suitable for high enzyme productivity. The pH and temperature optima for the enzyme were 4·0 and 70°C, respectively. Under these conditions, the enzyme exhibited a K_m (0-nitrophenyl-β- d -glucoside) value of 0.20 mmol/l. Several divalent metal ions inhibited enzyme activity at high concentration. EDTA. also inhibited β-glucosidase activity.     

An exo-β-d-glucanase derived from Zea coleoptile walls with a capacity to elicit cell elongation

Cell wall proteins were extracted from maize coleoptiles, Zea mays L. B37 x MO 17, with high concentrations of LiCl. Ion-exchange, chromatofocusing and gel-filtration chromatography were employed extensively to purify exo-β-glucanase activity from the extract. The purified enzyme functioned as an exo-(1→3)-β-glucanase (E.C. 3.2.1.58) and as a glucosidase (E.C. 3.2.1.21) capable of extensive hydrolysis of the native Zea wall (1→3), (1→4)-β- d -glucan, yielding glucose as the final product. The exoglucanase also enhances elongation of maize coleoptile sections in both the presence and absence of exogenous IAA.     

Effects of 9-cis- and all-trans-retinoic acids on blood glucose homeostasis in the fiddler crab, Uca pugilator

9-cis-Retinoic acid (9CRA) and all-trans-retinoic acid (ATRA) are known to be involved in the regulation of glucose homeostasis in vertebrates by inducing insulin release and expression of glucose transporter proteins. In view of the fact that both 9CRA and ATRA are endogenous to the fiddler crab, Uca pugilator, that a retinoid X receptor exists in this fiddler crab and that activities of insulin-like and insulin-like growth factor-like peptides have been reported for crustaceans, we investigated whether 9CRA and ATRA also play a role in glucose homeostasis in U. pugilator. Neither 9CRA nor ATRA was found to produce hypoglycemic effects at a dose of 10 microg/g live mass. However, 9CRA, but not ATRA, induced hyperglycemia. Such 9CRA-induced hyperglycemia was apparently mediated by the eyestalk hormone CHH since injection of 9CRA into eyestalk-ablated crabs did not result in hyperglycemia. ATRA was found to have an inhibitory effect on the recovery of blood glucose concentration following ATRA administration. Discussion on the possible mechanisms for the actions of 9CRA and ATRA was presented.     

The ftsQ1p gearbox promoter of Escherichia coli is a major sigma S-dependent promoter in the ddlB–ftsA region

We have isolated the ypfP gene (accession number P54166) from genomic DNA of Bacillus subtilis Marburg strain 60015 ( Freese and Fortnagel, 1967 ) using PCR. After cloning and expression in E. coli , SDS–PAGE showed strong expression of a protein that had the predicted size of 43.6 kDa. Chromatographic analysis of the lipids extracted from the transformed E. coli revealed several new glycolipids. These glycolipids were isolated and their structures determined by nuclear magnetic resonance (NMR) and mass spectrometry. They were identified as 3-[ O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl]-1,2-diacylglycerol, 3-[ O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl]-1,2-diacylglycerol and 3-[ O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl-(1→6)- O -β- D -glucopyranosyl]-1,2-diacylglycerol. The enzymatic activity expected to catalyse the synthesis of these compounds was confirmed by in vitro assays with radioactive substrates. In these assays, one additional glycolipid was formed and tentatively identified as 3-[ O -β- D -glucopyranosyl]-1,2-diacylglycerol, which was not detected in the lipid extract of transformed cells. Experiments with some of the above-described glycolipids as ¹⁴C-labelled sugar acceptors and unlabelled UDP-glucose as glucose donor suggest that the ypfP gene codes for a new processive UDP-glucose: 1,2-diacylglycerol-3-β- D -glucosyl transferase. This glucosyltransferase can use diacylglycerol, monoglucosyl-diacylglycerol, diglucosyldiacylglycerol or triglucosyldiacylglycerol as sugar acceptor, which, apart from the first member, are formed by repetitive addition of a glucopyranosyl residue in β (1→6) linkage to the product of the preceding reaction.     

Cloning, expression and characterization of a family-74 xyloglucanase from Thermobifida fusca.

Thermobifida fusca xyloglucan-specific endo-beta-1,4-glucanase (Xeg)74 and the Xeg74 catalytic domain (CD) were cloned, expressed in Escherichia coli, purified and characterized. This enzyme has a glycohydrolase family-74 CD that is a specific xyloglucanase followed by a family-2 carbohydrate binding module at the C terminus. The Michaelis constant (Km) and maximal rate (Vmax) values for hydrolysis of tamarind seed xyloglucan (tamXG) are 2.4 micro m and 966 micro mol xyloglucan oligosaccharides (XGOs) min-1. micro mol protein-1. More than 75% of the activity was retained after a 16-h incubation at temperatures up to 60 degrees C. The enzyme was most active at pH 6.0-9.4. NMR analysis showed that its catalytic mechanism is inverting. The oligosaccharide products from hydrolysis of tamXG were determined by MS analysis. Cel9B, an active carboxymethylcellulose (CMC)ase from T. fusca, was also found to have activity on xyloglucan (XG) at 49 micro mol.min-1. micro mol protein-1, but it could not hydrolyze XG units containing galactose. An XG/cellulose composite was prepared by growing Gluconacetobacterxylinus on glucose with tamXG in the medium. Although a mixture of purified cellulases was unable to degrade this material, the composite material was fully hydrolyzed when Xeg74 was added. T. fusca was not able to grow on tamXG, but Xeg74 was found in the culture supernatant at the same level as was found in cultures grown on Solka Floc. The function of this enzyme appears to be to break down the XG surrounding cellulose fibrils found in biomass so that T. fusca can utilize the cellulose as a carbon source.     

Chicoric acid, a new compound able to enhance insulin release and glucose uptake

Caffeic acid and chlorogenic acid (CGA), a mono-caffeoyl ester, have been described as potential antidiabetic agents. Using in vitro studies, we report the effects of a dicaffeoyl ester, chicoric acid (CRA) purified from Cichorium intybus, on glucose uptake and insulin secretion. Our results show that CRA and CGA increased glucose uptake in L6 muscular cells, an effect only observed in the presence of stimulating concentrations of insulin. Moreover, we found that both CRA and CGA were able to stimulate insulin secretion from the INS-1E insulin-secreting cell line and rat islets of Langerhans. In the later case, the effect of CRA is only observed in the presence of subnormal glucose levels. Patch clamps studies show that the mechanism of CRA and CGA was different from that of sulfonylureas, as they did not close K_ATP channels. Chicoric acid is a new potential antidiabetic agent carrying both insulin sensitizing and insulin-secreting properties.     

Cloning, expression, and characterization of an aldehyde dehydrogenase from Escherichia coli K-12 that utilizes 3-Hydroxypropionaldehyde as a substrate

For efficient production of isoflavone aglycones from soybean isoflavones, we isolated three novel types of β-glucosidase (BGL1, BGL3, and BGL5) from the filamentous fungi Aspergillus oryzae. Three enzymes were independently displayed on the cell surface of a yeast Saccharomyces cerevisiae as a fusion protein with α-agglutinin. Three β-glucosidase-displaying yeast strains hydrolyzed isoflavone glycosides efficiently but exhibited different substrate specificities. Among these β-glucosidases, BGL1 exhibited the highest activity and also broad substrate specificity to isoflavone glycosides. Although glucose released from isoflavone glycosides are generally known to inhibit β-glucosidase, the residual ratio of isoflavone glycosides in the reaction mixture with BGL1-displaying yeast strain (Sc-BGL1) reached approximately 6.2%, and the glucose concentration in the reaction mixture was maintained at lower level. This result indicated that Sc-BGL1 assimilated the glucose before they inhibited the hydrolysis reaction, and efficient production of isoflavone aglycones was achieved by engineered yeast cells displaying β-glucosidase.