Characteristics of β-galactosidase purified from cell suspension cultures of carrot

Five glycosidase activities from cell homogenate of carrot ( Daucus carota L. cv. Kintoki) cell cultures were assayed after extraction successively by phosphate buffer (pH 7.0) and the buffer plus 2 M NaCl. A β-galactosidase (EC 3.2.1.23) was isolated in a highly purified state from the buffer-soluble protein fraction by ammonium sulfate fractionation and chromatography on CM-Sephadex C-50, DEAE-Sephadex A-50 and Sephadex G-200. The molecular weight of this enzyme was ca 104 000 and the isoelectric point was pH 7.8. The optimal activity occurred at pH 4.4 with McIlvaine buffer. The K_m and V_max values were 1.67 m M and 201 units (mg protein)⁻¹, respectively, for p -nitrophenyl β- d -galactopyranoside. The enzyme activity was strongly inhibited by Zn²⁺, Cu²⁺, Hg²⁺ and d -galactono-1,4-lactone. The enzyme acted on the β-1,4-linked galactan prepared from citrus pectin in an exo-fashion. Furthermore, the enzyme was slightly involved in the hydrolysis of the pectic polymer and cell walls purified from carrot cell cultures.     

Purification and properties of an extracellular inulinase-like β-fructosidase from Bacillus stearothermophilus

A novel extracellular β-fructosidase produced by Bacillus stearothermophilus has been identified and purified. The purified enzyme, obtained by using successive QEAE Sepharose fast flow and Sephacryl S300 HR columns, has a 600 kDa relative molecular weight (M_r) and is composed of 60 kDa subunits indicating a multimeric structure. The pH and temperature for optimal activity are 6.5 and 65°C respectively, the enzyme being thermostable at this temperature. The apparent K_m values for sucrose and inulin are 3.56 mmol l^-1 and 1 mmol l^-1 respectively, the total invertase/total inulinase ratio being 4.     

Effect of β-glucanases on Penicillium oxalicum cell wall fractions

The polysaccharidic effect of a purified 1,3- β -glucanase, a purified β -glucosidase, and of partially purified endo-1,3- β -glucanase from autolysed Penicillium oxalicum cultures on cell wall isolate fractions from the same fungus were studied.
Fractionation of 5-day-old cell wall gave rise to a series of fractions that were identified using infrared spectrophotometry. The fractions used were: F₁, an α -glucan; F₃, a β -glucan; F₄, a chitin-glucan; and F_4b, a β -glucan. The fractions were incubated with each of the enzymes and with a mixture of equal parts of the three enzymes and the products of the enzymatic hydrolysis were analyzed after 96 h incubation.
The enzymes were found to degrade fraction F_4b ( β -glucan); the greatest degree of hydrolysis was reached when the three enzymes were used together, suggesting the need for synergic action by these enzymes in the cell wall degradation process.     

Endo‐1,4‐β‐glucanase gene expression and cell wall hydrolase activities during abscission in Valencia orange

The physiological and molecular events of ethylene‐induced abscission in mature fruit calyx, laminar and floral abscission zones of cv. Valencia orange were examined. Continuous exposure of fruit explants to 5 µl 1⁻¹ ethylene for 2 to 40 h resulted in marked increases in endo‐1,4‐β‐glucanase (cellulase) and polygalacturonase (PG) activities in calyx abscission zones. Two abscission‐related cellulases and one PG were found. The major peak of cellulase activity corresponded to a pI of 8.0 and molecular weight of 51 kDa, whereas the minor cellulase peak had a pI of 5.5. The abscission polygalacturonase had a pI of 5.5. Calyx abscission zone RNA was amplified with degenerate primers based on sequence of the purified Valencia orange calyx abscission cellulase, and cloned. The two partial cellulase cDNA clones were 59% identical at the nucleotide level. Genomic Southern analysis suggested that Valencia orange contained two groups of cellulase genes. A full‐length cDNA clone from each group was isolated from a cDNA library prepared from ethylene‐induced calyx abscission zone mRNA. Both genes were expressed in ethylene‐induced calyx, laminar and floral abscission zones, but were not expressed in non‐induced abscission zones or mature leaves treated with or without ethylene, young bark or young fruit of Valencia.     

Isolation, characterization and significance of papaya β‐galactanases to cell wall modification and fruit softening during ripening

β‐Galactosidases (EC 3.2.1.23) from ripe papaya ( Carica papaya L. cv. Eksotika) fruits having galactanase activities were fractionated by a combination of cation exchange and gel‐filtration chromatography into three isoforms, viz., β‐galactosidase I, II and III. The native proteins of the respective isoforms have apparent molecular masses of 67, 67 and 55 kDa, each showing one predominant polypeptide upon SDS‐PAGE of about 31 and 33 kDa for β‐galactosidases I and III, respectively, and of 67 kDa for β‐galactosidase II. The β‐galactosidase I protein, which was undetectable in immature fruits, appeared to be specifically accumulated during ripening. The β‐galactosidase II protein was present in developing fruits, but its level seemed to decrease with ripening. β‐Galactosidase I seemed to be an important softening enzyme; its activity increased dramatically (4‐ to 8‐fold) to a peak early during ripening and correlated closely with differential softening as related to position in the fruit tissue. The inner mesocarp tissue was softer, and its wall pectins were modified earlier and firmness decreased more rapidly during ripening compared to the outer mesocarp tissue. β‐Galactosidase II also may contribute significantly to softening because of its ability to catalyse increased solubility and depolymerization of pectins as well as through its ability to modify the alkali‐soluble hemicellulose fraction of the cell wall. The physiological significance of both β‐galactosidase isoforms may partly be attributed to their functional capacity as β‐(1,4)‐galactanases.     

Purification and characterization of ββ-N-acetylhexosaminidase from the phytopathogenic fungus Bipolaris sorokiniana

N-acetylhexosaminidase (HEX) from the phytopathogenic fungus Bipolaris sorokiniana was isolated and characterized. The production of HEX by B. sorokiniana was not altered by growing on different carbon sources. Enzyme purification was carried out by sequential liquid chromatography on Sephacryl S-200 HR, and p-aminobenzyl-2-acetamido-2-deoxy-β- d -thioglucopyranoside agarose. The purification was about 70-fold, with a yield of 41%, determined with p-nitrophenyl-N-acetylglucosaminide as substrate. The enzyme had pH and temperature optima of 4·5 and 55 °C, respectively. The molecular weight of non-denatured enzyme was estimated as 120 000 Da by gel filtration chromatography, and about 55 000 Da by SDS-PAGE. The fungal HEX had glycosylated residues as evidenced by binding to Concanavalin-A. Bipolaris sorokiniana enzyme was also active with p-nitrophenyl-chitobioside and p-nitrophenyl-N-acetylgalactosaminide as substrates.     

Characterization of a cell wall β-galactosidase of Cicer arietinum epicotyls involved in cell wall autolysis

β-Galactosidase (EC 3.2.1.23) has been established as the main enzyme involved in the autolytic process. The enzyme extracted from cell walls of epicotyls of Cicer arietinum L. cv. Castellana with 3 M LiCl is a 45 kDa protein composed of a single subunit, having an optimum pH of 4; an optimum temperature of 45°C and K_m and V_max of 1.72 m M and 18.5 nkat (mg protein)^–1 respectively, as evaluated against p -nitrophenyl-β- d -galactopyranoside. The enzyme is inhibited by Hg²⁺, d -galactono-1,4-lactone and galactose, substances that also inhibit the autolytic process. Ca²⁺ and EDTA, which do not affect the activity of the β-gaiactosidase, do however modify the hydrolysis of the cell wall mediated by the enzyme, and they also inhibit the autolytic process. Ca²⁺ decreased both processes, whereas EDTA increased them; and when both substances were added together, their individual effects were neutralized. The effects of both agents is probably due to modifications in the cell wall that prevent access of the enzyme to its substrate.     

Purification and characterization of ββ-glucosidase from Aspergillus niger strain 322

An extracellular β-glucosidase enzyme was purified from the fungus Aspergillus niger strain 322 . The molecular mass of the enzyme was estimated to be 64 kDa by SDS gel electrophoresis. Optimal pH and temperature for β-glucosidase were 5·5 and 50 °C, respectively. Purified enzyme was stable up to 50 °C and pH between 2·0 and 5·5. The K_m was 0·1 mmol l⁻¹ for cellobiose. Enzyme activity was inhibited by several divalent metal ions.     

Formaldehyde kills spores of Bacillus subtilis by DNA damage and small, acid-soluble spore proteins of the ααααααα/βββββββ-type protect spores against this DNA damage

Killing of wild-type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed α⁻β⁻) lacking the two major α/β-type small, acid-soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis. A recA mutation sensitized both wild-type and α⁻β⁻ spores to formaldehyde treatment, which caused significant expression of a recA - lacZ fusion when the treated spores germinated. Formaldehyde also caused protein–DNA cross-linking in both wild-type and α⁻β⁻ spores. These results indicate that: (i) formaldehyde kills B. subtilis spores at least in part by DNA damage and (b) α/β-type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.     

Molecular cloning and characterization of a Candida albicans gene (EFB1) coding for the elongation factor EF-1β

Abstract The formation of H₂ by chemolithoautrophically growing Oligotropha carboxidovorans has been identified as the result of the oxidation of CO mediated by the cytoplasmic species of the molybdenum-containing CO dehydrogenase multienzyme complex as follows: CO + H ₂ O → CO ₂+ H ₂. Purified CO dehydrogenase was shown to carry hydrogen uptake and formation activities in addition to its catabolic function which is the oxidation of CO. Among the electron donors supporting H₂ formation were CO, NADH, reduced flavins and reduced viologen dyes. The reduction of protons to H₂ by cytoplasmic CO dehydrogenase is interpreted as a detoxification reaction for electrons to prevent cell damage in O. carboxidovorans .     

Partial purification of cell wall β-galactosidases from Cicer arietinum epicotyls. Relationship with cell wall autolytic processes

The protein extracted from the cell wall of the epicotyls of Cicer arietinum L. cv. Castellana was separated by ion exchange chromatography in four different fractions with β-D-galactosidase (EC 3.2.1.23) activity. These were called βI, βII, βIII and βIV, according to their order of elution. βII was associated with a particularly high β-D-glucosidase (EC 3.2.1.21) activity. Gel filtration chromatography of each of the fractions gave further subdivision of fractions βI and βIII. Subfractions 1 βI, 1 βII and 1 βIV have glucosidase activity and subfractions 2 βI and 2 βIII have galactosidase activity.
The studies on the hydrolytic capacity of these fractions and its relationship with the autolytic process seem to show that subfraction 2 βIII is responsible for autolysis. The release of total and reducing sugars is very similar for autolysis and hydrolysis by 2 βIII. The sugars released are mainly galactose and, to a lesser extent arabinose and glucose. Galactose is released as a monosaccharide, while arabinose remains associated to a polysaccharide component together with glucose and small amounts of galactose.     

Cloning and characterization of β-galactoside and β-glucoside hydrolysing enzymes of Thermotoga maritima

Abstract A gene library of the hyperthermophilic bacterium Thermotoga maritima strain MSB8 was constructed in Escherichia coli . Two non-related T. maritima chromosomal DNA fragments were physically characterized. They conferred the synthesis of thermostable X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside)-hydrolysing activity upon the host organism. The biochemical properties of the recombinant enzymes indicated that genes for a β-galactosidase (BgaA) and a broad-specificity β-glucosidase (Bg1A) had been isolated. The genes were desiignted bgaA and bglA , respectively. According to analytical size exclusion chromatography data, BgaA and BglA had native molecular masses of approximately 240 kDa and 95 kDa, respectively. Both enzymes apparently have dimeric subunit structure. An additional β-glucosidase (designated BglB) activity, clearly distinct from BglA in terms of substrate specificity, could be detected in a crude extract of T. maritima .     

Molecular evolution of plant β-glucan endohydrolases

The evolutionary relationships of two classes of plant β-glucan endohydrolases have been examined by comparison of their substrate specificities, their three-dimensional conformations and the structural features of their corresponding genes. These comparative studies provide compelling evidence that the (1→3)-β-glucanases and (1→3,1→4)-β-glucanases from higher plants share a common ancestry and, in all likelihood, that the (1→3,1→4)-β-glucanases diverged from the (1→3)-β-glucanases during the appearance of the graminaceous monocotyledons. The evolution of (1→3,1→4)-β-glucanases from (1→3)-β-glucanases does not appear to have invoked ‘modular’ mechanisms of change, such as those caused by exon shuffling or recombination. Instead, the shift in specificity has been acquired through a limited number of point mutations that have resulted in amino acid substitutions along the substrate-binding cleft. This is consistent with current theories that the evolution of new enzymic activity is often achieved through duplication of the gene encoding an existing enzyme which is capable of performing the required chemistry, in this case the hydrolysis of a glycosidic linkage, followed by the mutational alteration and fine-tuning of substrate specificity. The evolution of a new specificity has enabled a dramatic shift in the functional capabilities of the enzymes. (1→3)-β-Glucanases that play a major role, inter alia, in the protection of the plant against pathogenic microorganisms through their ability to hydrolyse the (1→3)-β-glucans of fungal cell walls, appear to have been recruited to generate (1→3,1→4)-β-glucanases, which quite specifically hydrolyse plant cell wall (1→3,1→4)-β-glucans in the graminaecous monocotyledons during normal wall metabolism. Thus, one class of β-glucan endohydrolase can degrade β-glucans in fungal walls, while the other hydrolyses structurally distinct β-glucans of plant cell walls. Detailed information on the three-dimensional structures of the enzymes and the identification of catalytic amino acids now present opportunities to explore the precise molecular and atomic details of substrate-binding, catalytic mechanisms and the sequence of molecular events that resulted in the evolution of the substrate specificities of the two classes of enzyme.     

Molecular evolution of ubiquitous β-lactamases towards extended-spectrum enzymes active against newer β-lactam antibiotics

Production of beta-lactamases, and of the plasmid-encoded TEM- and SHV-type enzymes in particular, is the most common mechanism of resistance against beta-lactam antibiotics in Gram-negative bacteria. The two ubiquitous types of enzyme have a large spectrum of activity and preferentially hydrolyse the penicillins as well as some first- and second-generation cephalosporins. Recently, point mutations in the corresponding genes have been observed, apparently selected for, in the clinical setting, by originally 'beta-lactamase-stable' third-generation cephalosporins or by monobactams, which fall into the substrate range of the mutant or 'extended-spectrum' beta-lactamases. The point mutations are clustered in three areas, each adjacent to one of the seven evolutionarily conserved boxes described by Joris et al. (1988). The substituted amino acids at positions 102 (adjacent to the alpha-3 helix), 162 (adjacent to the alpha-7 helix) and 235, 236 and 237 (on the beta-3 strand) are located in close proximity to the active-site cavity and are thought to open up novel enzyme-substrate interactions, involving, in particular, the oxyimino moieties of the newer beta-lactam compounds.     

Immunohistochemical Localization of G Protein β1, β2, β3, β4, β5, and γ3 Subunits in the Adult Rat Brain

Abstract: The regional distributions of the G protein β subunits (Gβ1–β5) and of the Gγ3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gβ and Gγ3 subunits were widely distributed throughout the brain, with most regions containing several Gβ subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gβ immunostaining. Negative immunostaining was observed in cortical layer I for Gβ1 and layer IV for Gβ4. The hippocampal dentate granular and CA1–CA3 pyramidal cells displayed little or no positive immunostaining for Gβ2 or Gβ4. No anti-Gβ4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gβ1 was absent from the cerebellar molecular layer, and Gβ2 was not detected in the Purkinje cells. No positive Gγ3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Gγ3 antibody and individual anti-Gβ1–β5 antibodies displayed regional selectivity with Gβ1 (cortical layers V–VI) and Gβ2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gβ1–β5 with Gγ3, specific dimeric associations in situ were observed within discrete brain regions.     

Molecular characterization of In50, a class 1 integron encoding the gene for the extended-spectrum β-lactamase VEB-1 in Pseudomonas aeruginosa

A clinical isolate of Pseudomonas aeruginosa, JES, was resistant to extended-spectrum cephalosporins with a marked synergistic effect with clavulanic acid on a routine antibiogram. Preliminary PCR analysis revealed the presence of blaVEB-1, an integron-located gene encoding an extended-spectrum beta-lactamase previously identified in Escherichia coli MG-1. Using class 1 integron primers and blaVEB-1 intragenic primers, the insert region of the blaVEB-1 containing integron along with some flanking sequence from P. aeruginosa JES was amplified and subsequently sequenced. In50 contains within its variable region, in addition to qacE delta 1 and sull genes commonly found in class 1 integrons, two gene cassettes, veb1 and aadB. In50 is peculiar since its attI1 site is interrupted by two novel insertion sequences, IS1999 and IS2000. P. aeruginosa JES and Escherichia coli MG-1 strains were isolated from patients previously hospitalized in south east Asian countries. The finding of blaVEB-1 in these strains and on different integrons underlines the interspecies spread of this integron-located extended-spectrum beta-lactamase gene.