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PubMed1977

Inhibition of beta-galactosidase synthesis in Escherichia coli after infection with different DNA and RNA phages.

Infection of Escherichia cooi with T1, T2r+, T3 and T4 phages leads to an immediate inhibition of beta-galactosidase synthesis. Similar results were obtained with the virulent mutant of phage lambda. The degree of inhibition of beta-galactosidase synthesis depends on the time delay between the addition of the inducer and the phage particles, and on the amount of phage DNA, which has penetrated into the host cell. RNA phage MS2 exhibited no inhibitory effect on enzyme synthesis.

Coliphages↗
PubMed2003

Application of selectively acylated glycosides for the alpha-galactosidase-catalyzed synthesis of disaccharides.

4-Nitrophenyl alpha-D-galactopyranosyl-(1-->3)-6-O-acetyl-alpha-D-galactopyranoside was prepared in a transglycosylation reaction catalyzed by alpha-D-galactosidase from Talaromyces flavus using 4-nitrophenyl alpha-D-galactopyranoside as a glycosyl donor and 4-nitrophenyl 6-O-acetyl-alpha-D-galactopyranoside as an acceptor. 4-Nitrophenyl 6-O-acetyl-alpha-D-galactopyranoside and 4-nitrophenyl 6-O-acetyl-beta-D-galactopyranoside were prepared in a regioselective enzymic transesterification in pyridine-acetone catalyzed by the lipase PS from Burkholderia cepacia. A series of water-miscible organic solvents (acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, 1,4-dioxane, 2-methoxyethanol, pyridine, 2-methylpropan-2-ol, tetrahydrofuran, propargyl alcohol) were used as co-solvents in this enzymic reaction. Their influence on the activity and stability of the alpha-galactosidase from T. flavus was established. 2-Methylpropan-2-ol and acetone (increasing the solubility of the modified substrate acceptors and displaying the minimum impairment of the activity and stability of the enzyme) were used as co-solvents in transglycosylation reactions.

Burkholderia cepacia↗
PubMed2002

Food-grade host/vector expression system for Lactobacillus casei based on complementation of plasmid-associated phospho-beta-galactosidase gene lacG.

A new food-grade host/vector system for Lactobacillus casei based on lactose selection was constructed. The wild-type non-starter host Lb. casei strain E utilizes lactose via a plasmid-encoded phosphotransferase system. For food-grade cloning, a stable lactose-deficient mutant was constructed by deleting a 141-bp fragment from the phospho-beta-galactosidase gene lacG via gene replacement. The deletion resulted in an inactive phospho-beta-galactosidase enzyme with an internal in-frame deletion of 47 amino acids. A complementation plasmid was constructed containing a replicon from Lactococcus lactis, the lacG gene from Lb. casei, and the constitutive promoter of pepR for lacG expression from Lb. rhamnosus. The expression of the lacG gene from the resulting food-grade plasmid pLEB600 restored the ability of the lactose-negative mutant strain to grow on lactose to the wild-type level. The vector pLEB600 was used for expression of the proline iminopeptidase gene pepI from Lb. helveticus in Lb. casei. The results show that the food-grade expression system reported in this paper can be used for expression of foreign genes in Lb. casei.

Aminopeptidases↗
PubMed2003

Lactulose production by beta-galactosidase in permeabilized cells of Kluyveromyces lactis.

Lactulose production from lactose and fructose was investigated with several commercial beta-galactosidases. The enzyme from Kluyveromyces lactis exhibited the highest lactulose productivity among the beta-galactosidases tested. The reaction conditions for lactulose production were optimized using cells that had been permeabilized by treatment with 50% (v/v) ethanol: cell concentration, 10.4 g l(-1); concentration of substrates, 40% (w/v) lactose and 20% (w/v) fructose; temperature, 60( degrees )C; pH 7.0. Under these conditions, the permeabilized cells produced approximately 20 g l(-1) lactulose in 3 h with a lactulose productivity of 6.8 g l(-1) h(-1). These results represent 1.3- and 2.1-fold increases in lactulose concentration and productivity compared with untreated washed cells. This is the first reported trial of enzymatic synthesis of lactulose using permeabilized yeast cells.

Cell Membrane Permeability↗
PubMed2000

A comparison of enzyme-aided bleaching of softwood paper pulp using combinations of xylanase, mannanase and alpha-galactosidase.

Enzymatic pretreatment of softwood kraft pulp was investigated using xylanase A (XylA) from Neocallimastix patriciarum in combination with mannanase and alpha-galactosidase. Mannanase A (ManA) from Pseudomonas fluorescens subsp. cellulosa and ManA from Clostridium thermocellum, both family 26 glycosyl hydrolases, are structurally diverse and exhibit different pH and temperature optima. Although neither mannanase was effective in pretreating softwood pulp alone, both enzymes were able to enhance the production of reducing sugar and the reduction of single-stage bleached kappa number when used with the xylanase. Sequential incubations with XylA and P. fluorescens ManA produced the largest final kappa number reduction in comparison to control pretreated pulp. The release of galactose from softwood pulp by alpha-galactosidase A (AgaA) from P. fluorescens was enhanced by the presence of ManA from the same microorganism, and a single pretreatment with these enzymes, in combination with XylA. gave the most effective kappa number reduction using a single incubation. Results indicated that mixtures of hemicellulase activities can be chosen to enhance pulp bleachability.

Glycoside Hydrolases↗
PubMed2000

Construction of a flocculent brewer's yeast strain secreting Aspergillus niger beta-galactosidase.

One way of improving heterologous protein production is to use high cell density systems, one of the most attractive being the flocculating yeast production system. Also, lactose is available in large amounts as a waste product from cheese production processes. The construction of flocculent and non-flocculent brewer's yeast strains secreting beta-galactosidase and growing on lactose is presented. A plasmid was constructed coding for an extracellular beta-galactosidase of Aspergillus niger and having, as selective marker, the yeast CUP1 gene conferring resistance to copper. This selective marker allows for the transformation of wild-type yeasts. This work represents an important step towards the study of heterologous protein secretion by flocculent cells.

Aspergillus niger↗
PubMed2001

Electroinduced extraction of beta-galactosidase from Kluyveromyces lactis.

A new methodology for the extraction of beta-galactosidase from the yeast Kluyveromyces lactis was obtained by electropulsation. The application of a series of electric pulses (2 ms duration, 1 Hz frequency, and 4-4.5 kV/cm field strength) to fresh cells suspended in deionized water, followed by incubation in PBS, led to a spontaneous slow release of enzyme at a yield of 75-80% without any further treatment. Most of the enzyme was extracted within 8 h after electropulsation. This release was dependent on the growth phase. The specific activity of beta-galactosidase in the supernatant of pulsed cells was higher by a factor of 1.5-1.7 in comparison with crude extract.

Biotechnology↗
PubMed2000

Nucleotide sequence of alpha-galactosidase MEL gene from Zygosaccharomyces mrakii.

The region encompassing the alpha-galactosidase MELr gene was amplified from Zygosaccharomyces mrakii IFO 1835(T) by inverse-PCR and then sequenced. The nucleotide sequence of this region revealed a single open reading frame of 1410 bp encoding a 470 amino acid protein with a molecular weight of 51,909. The similarity of the deduced mature protein to other yeast alpha-galactosidases was 63.3% to Zygosaccharomyces cidri, 71.5% to Torulaspora delbrueckii, and 70. 7-73.9% to Saccharomyces species. The nucleotide and amino acid sequences are deposited in the DDBJ/EMBL/GenBank database under Accession Number AB030209.

Fungal Proteins↗
PubMed1998

Isolation of a beta-galactosidase-encoding gene from Bacillus licheniformis: purification and characterization of the recombinant enzyme expressed in Escherichia coli.

The Bacillus licheniformis beta-galactosidase gene, lacBl, was cloned on a 5.8-kb HindIII fragment into pBR322 and expressed by its own promoter in Escherichia coli. Deletion and complementation analysis showed that the enzyme-encoding region was located on a 4. 1-kb HindIII-ClaI fragment. The transcription region for the lacBl was identified on this fragment with promoter- and terminator-probe plasmids. The deduced sequence of 149 aa of the N-terminal part of lacBl showed aa sequence homology with beta-Gal from B. stearothermophilus, B. circulans, Haloferax alicantei, Clostridium perfringens, Arthrobacter sp.. No significant homology was shared with those found in the lacZ and lacS families. The recombinant beta-galactosidase (LacB1) was purified by FPLC. The molecular mass of the enzyme (80 kDa) and its optimal pH (5.7) and temperature (45 degrees C) were determined.

Bacillus↗
PubMed1999

Two-color detection of mRNA transcript localizations in fish and fly embryos using alkaline phosphatase and beta-galactosidase conjugated antibodies.

A fast and simplified two-color in situ hybridization procedure for the simultaneous detection of two different mRNAs in whole-mounted zebrafish and Drosophila embryos is presented. Transcript distributions are detected in a single incubation step using a mixture of alkaline phosphatase and beta-galactosidase coupled antibodies. The different transcripts are visualized in contrasting colors by the use of beta-galactosidase substrates that develop color precipitates (magenta, blue) easily distinguishable from those of the standard alkaline phosphatase substrates. This protocol can be followed by standard immunohistochemistry to detect the expression of a third gene (at the protein level) in a third color.

Alkaline Phosphatase↗
PubMed2002

Potential of beta-galactosidase-expressing Toxoplasma gondii for in situ localization and observation of rare stages of the parasite life cycle.

A cyst-forming strain of Toxoplasma gondii was transfected with the Escherichia coli LacZ gene and expressed beta-galactosidase constitutively. This strain has been used to localize and analyze the early stages of development and reactivation of T. gondii in mice. The chromogenic detection of the enzyme allows an easy detection of the parasites after light fixation and therefore allows a submacroscopic analysis of tissue distribution within the organism. Also, it allows further embedding and retrieval of rare stages for electron microscopic observation. that detect the presence of the parasite and initiate the response, and (2) the early stages of reactivation, when the cysts are supposed to break open and release the infectious bradyzoïtes. We have taken advantage of the possibility of detecting the enzymatic activity of beta-galactosidase (beta-gal) in transfected parasites to show that one could perform a semi-macroscopic detection and that this was compatible with further analysis by histological or electron microscopic techniques, being therefore able to detect the rare events and then to analyze them further with more refined morphological techniques.

Animals↗
PubMed1996

Fluorescence-assisted mismatch analysis (FAMA) for exhaustive screening of the alpha-galactosidase A gene and detection of carriers in Fabry disease.

We used the fluorescence-assisted mismatch analysis (FAMA) method to screen rapidly the alpha-galactosidase A gene in patients with Fabry disease in order to identify unknown mutations and help define genotype-phenotype correlations in this X-linked lysosomal storage disorder. Chemical cleavage at mismatches on heteroduplex DNA end-labeled with strand-specific fluorescent dyes, reliably detects sequence changes in DNA fragments of up to 1.5 kb and locates them precisely. Exhaustive scanning of the alpha-galactosidase gene was accomplished on four polymerase chain reaction-generated amplicons, covering all seven exons, the exon-intron boundaries, and 700 bp of 5'-flanking sequence. Mutations were identified in each of the 15 patients studied from nine unrelated kindreds. Among the seven previously undescribed sequence changes, three are obviously pathogenic because they lead to premature protein termination. The other four, a splicesite mutation and three missense mutations, were the only changes found upon complete scanning of the gene and its promoter. In addition, FAMA also detects female heterozygous carriers more dependably than direct sequencing, and thus provides a valuable diagnostic test. In Fabry disease, this molecular criterion is especially important for genetic counseling since heterozygotes can be asymptomatic and their enzymatic values within the normal range.

Adult↗
PubMed2004

Bounded water kinetic model of beta-galactosidase in reverse micelles.

In the present investigation, beta-galactosidase was solubilized into Aerosol OT (AOT)/isooctane reverse micelles. Kinetic data for the hydrolysis of o-nitrophenyl-beta-D-galactopyranoside (ONPG) at different pH values and molar ratios of water to AOT (Wo) were collected. It was observed that the usual kinetic model used for beta-galactosidase catalysis in aqueous systems failed to represent the experimental data. A bounded water model, however, showed a better correlation between enzymatic activity and Wo. In contrast to the aqueous system, controlling the water concentration in the reverse micelles allows the rate constants for the reaction between water molecules and glycosyl-enzyme complexes to be evaluated.

Catalysis↗
PubMed2005

The ratio of alpha-galactosidase to beta-glucuronidase activities in dried blood for the identification of female Fabry disease patients.

Female heterozygous patients with Fabry disease are difficult to identify because of the relatively high residual activity of alpha-galactosidase. We systematically evaluated the activities of various lysosomal enzymes in dried blood samples from Fabry patients and found that the beta-glucuronidase activity was frequently elevated. The ratio of alpha-galactosidase to beta-glucuronidase proved to be a helpful tool for the diagnosis of female Fabry disease patients.

Blood↗
PubMed2004

A novel alkaline alpha-galactosidase gene is involved in rice leaf senescence.

We previously isolated and identified numerous senescence-associated genes (SAGs) in rice leaves. Here we characterized the structure and function of an SAG- Osh69 encoding alkaline alpha-galactosidase that belongs to a novel family of glycosyl hydrolases. Osh69 is a single-copy gene composed of 13 exons located on rice chromosome 8. The expression level of Osh69 is not only up-regulated during natural leaf senescence but also induced rapidly by darkness, hormones (methyl jasmonic acid, salicylic acid), and stresses (H2O2 and wounding). The recombinant Osh69 protein over-expressed in Escherichia coli has displayed optimal alpha-galactosidase activity at pH 8.0. The enzyme showed good hydrolytic activities towards alpha-1,6-galactosyl oligosaccharides and galactolipid digalactosyl diacylglycerol. Immunoelectron microscopic analysis demonstrates that Osh69 is specifically localized in the chloroplasts of senescing leaves. These findings strongly suggest an important role for Osh69 in the degradation of chloroplast galactolipids during leaf senescence.

Amino Acid Sequence↗
PubMed2006

Isolation of the promoter of a cotton beta-galactosidase gene (GhGal1) and its expression in transgenic tobacco plants.

Beta-galactosidases (EC 3.2.1.23) constitute a widespread family of glycosyl hydrolases in plants and are thought to be involved in metabolism of cell wall polysaccharides. A cDNA of the cotton (Gossypium hirsutum) beta-galactosidase gene, designated GhGal1, has previously been identified and its transcripts are highly abundant at the elongation stage of the cotton fiber. To examine the temporal and spatial control of GhGal1 expression, a transcriptional fusion of the GhGal1 promoter region (1770 bp) with the beta-glucuronidase (GUS) reporter gene was introduced into tobacco plants by the Agrobacterium infection method. The resulting transgenic plants showed higher GUS activity of fruit in the transgenic plants than that in the negative and positive controls. Histochemical localization of GUS activity demonstrated that the expression of the GUS gene could be found in the meristem zones of roots, cotyledons, vascular tissues, fruit and trichomes in transgenic tobacco plants. Additionally, sequence analysis of the regulatory region also revealed several conserved motifs among which some were shared with previously reported fruit/seed-specific elements and the others were related with trichome expression. These results indicated the temporal and spatial expression characterization of the GhGal1 promoter in transgenic tobacco plants and provided an important insight into the roles of GhGal1 in cotton fiber development.

Base Sequence↗
PubMed1991

Flow injection analysis of lactose using covalently immobilized beta-galactosidase, mutarotase, and glucose oxidase/peroxidase on a 2-fluoro-1-methylpyridinium salt-activated Fractogel support.

Milk samples were analyzed for their lactose content using flow injection analysis and incorporating immobilized beta-galactosidase or beta-galactosidase/mutarotase and glucose oxidase/peroxidase bioreactors. These enzymes were immobilized, under mild conditions, on to a 2-fluoro-1-methylpyridinium salt-activated Fractogel support. The use of a phosphate buffer (0.15 M) was found to facilitate the rapid mutarotation of alpha-D-glucose and hence could obviate the need for the more expensive mutarotase. The chromogenic agents of choice for monitoring the reaction were 3-methyl-2-benzothiazolinone hydrazone and 3-dimethylaminobenzoic acid. Linearity was observed over the concentration range 16-160 micrograms/ml using lactose standards (r = 0.996). Between 30 and 40 milk samples/h can be analyzed. Comparisons are made with existing HPLC and alkaline methylamine methods for a range of milk matrices. The FIA method consistently gives the lowest standard deviations and coefficient of variation for the various milk matrices analyzed.

Animals↗
PubMed1975

Purification of G-M-1-ganglioside and ceramide lactoside beta-galactosidase from rabbit brain.

The major beta-galactosidase of rabbit brain has been purified over 400-fold. The enzyme converts G-M-1-ganglioside; Gal beta-1 yields 3 GalNAc beta-1 yields 4 (NANalpha-2 yields 3) Gal beta-1 yields 4 Glc yields ceramide (G-M-1) into Tay Sachs ganglioside GalNAc beta-1 yields 4 (NANalpha-2 yields 3) Gal beta-1 yields 4 Glc yields ceramide (G-M-2-ganglioside) and ceramide lactoside, Gal beta-1 yields 4 Glc yields ceramide (Gal-Glc-Cer) into glucocerebroside, Glc yields ceramide (Glc-Cer). The enzyme also hydrolyzes the synthetic substrates NPh-Gal and MeUmb-Gal. It is eluted as a single peak from Sephadex G-200 columns when natural and synthetic substrates were used and has an isoelectric point of 6.3. We were unable to resolve activity towards G-M-1-ganglioside and Gal-Glc-Cer by polyacrylamide electrophoresis in two buffer systems. With G-M-1 the pH optimum was 4.3 in acetate buffer and the K-m value 78 mu-M while with Gal-Glc-Cer, a pH optimum of 4.5 and a K-m of 17 mu-M were found. Hydrolysis of both natural and synthetic substrates was inhibited by gamma-D-galactonolactone, D-galactose and lactose. The data strongly suggest that a single beta-galactosidase hydrolyzes all the substrates tested.

Animals↗