Overexpression of celB gene coding for beta-glucosidase from Pyrococcus furiosus using a baculovirus expression vector system in silkworm, Bombyx mori

beta-Glucosidase is a member of the glycosyl hydrolases that specifically catalyze the hydrolysis of terminal nonreducing beta-D-glucose residues from the end of various oligosaccharides with the release of beta-D-glucose. CelB gene, encoding the thermostable beta-glucosidase, was amplified from the Pyrococcus furiosus genome and then cloned into the baculoviral transfer vector under the control of the polyhedrin gene promoter. After co-transfection with the genetically modified parental Bombyx mori nucleopolyhedrovirus (BmNPV), the recombinant virus containing celB gene was used to express beta-glucosidase in silkworm. The recombinant beta-glucosidase was purified to about 81% homogeneity in a single heat-treatment step. The optimal activity of the expressed beta-glucosidase was obtained at pH 5.0 and about 105 degrees C; divalent cations and high ionic strength did not affect the activity remarkably. This suggested that the enzymatic characteristics of recombinant beta-glucosidase were similar to the native counterpart. The expressed beta-glucosidase accounted for more than 10% of silkworm total haemolymph proteins according to the protein quantification and densimeter scanning. The expression level reached 10,199.5 U per ml haemolymph and 19,797.4 U per silkworm larva, and the specific activity of the one-step purified crude enzyme was 885 U per mg. It was demonstrated to be an attractive approach for mass production of thermostable beta-glucosidase using this system.     

Use of fluorescein-di-beta-D-galactopyranoside (FDG) and C12-FDG as substrates for beta-galactosidase detection by flow cytometry in animal, bacterial, and yeast cells.

Fluorescein-di-beta-D-galactopyranoside (FDG) was found to be a useful substrate for beta-galactosidase detection by flow cytometry in gram-negative bacteria, since it entered viable cells and gave a fluorescence emission proportional to the enzymatic activity. C12-FDG, a more lipophilic derivative, gave a very poor signal because of the lack of penetration. On the contrary, C12-FDG was more sensitive than FDG for beta-galactosidase activity determinations in animal cells. In contrast to previous reports, C12-FDG did not enter viable yeast cells, so that the use of the substrate required cell permeabilization. Without this treatment, C12-FDG penetrates only nonviable yeast cells that may occur in populations expressing beta-galactosidase.     

The cellular distribution of some rat-kidney glycosidases

1. Free and total activities of beta-glucosidase, beta-galactosidase, N-acetyl-beta-glucosaminidase and beta-glucuronidase have been determined fluorimetrically in five subcellular fractions of rat kidney. 2. The beta-glucosidase activity appeared in the soluble fraction, beta-glucuronidase had the distribution pattern of a lysosomal enzyme, and both beta-galactosidase and N-acetyl-beta-glucosaminidase had bimodal distributions. 3. Two types of beta-galactosidase activity were found: a sedimentable type, having optimum pH3.7, mol.wt. about 80000 and slow electrophoretic mobility at pH7.0 in starch gel; and a soluble type of much faster mobility, having optimum pH5.5-6.5 and mol.wt. about 40000. 4. Evidence is presented that the beta-glucosidase and the soluble type of beta-galactosidase are the same enzyme. 5. Most of the N-acetyl-beta-glucosaminidase activity was in the lysosome-rich fractions, but a significant proportion occurred in the microsomal fraction in a non-latent form. 6. The use of beta-galactosidase and N-acetyl-beta-glucosaminidase as lysosomal marker enzymes is complicated by the possible presence of multiple forms, but this limitation does not apply to beta-glucuronidase in the rat kidney.     

Use of marker genes in competition studies of Rhizobium

Use of marker genes has several advantages in studying rhizobial competition compared to traditional approaches. Reporter genes such as the ß-glucuronidase gene (gusA) or a thermostable ß-glucosidase gene (celB) allow detection of rhizobial strains in nodules when they are still attached to the root system. Analysis is extremely simple, fast and permits a high data throughput. This detection technique is therefore highly suitable for the study of rhizobial competition and studies using gusA-marked strains of Rhizobium are presented. By making use of gusA and celB, differentially marked strains can be produced and distinguished easily on roots. The availability of two marker genes permits competition studies of two or more than two strains and analysis of dual nodule occupancy. As this methodology does not require sophisticated equipment, a GUS Gene Marking Kit was developed.     

Endogenous beta-glucosidase and beta-galactosidase activities from selected probiotic micro-organisms and their role in isoflavone biotransformation in soymilk

AIM: To compare endogenous beta-glucosidases and beta-galactosidases for hydrolysis of the predominant isoflavone glycosides into isoflavone aglycones in order to improve biological activity of soymilk. METHODS AND RESULTS: beta-glucosidase and beta-galactosidase activities of probiotic organisms including Lactobacillus acidophilus ATCC 4461, Lactobacillus casei 2607 and Bifidobacterium animalis ssp. lactis Bb12 in soymilk were evaluated and correlated with the increase in concentration of isoflavone aglycones during fermentation. The concentrations of isoflavone compounds in soymilk were monitored using a Varian model high-performance liquid chromatography (HPLC) with an amperometric electrochemical detector. In all micro-organisms, beta-glucosidase activity was found greater than that of beta-galactosidase. There was an increase in the aglycone concentration with incubation time because of the apparent hydrolytic action on isoflavone glycosides. Aglycone concentration in the soymilk with L. acidophilus 4461, L. casei 2607 and B. animalis ssp. lactis Bb12, increased by 5.37-, 5.52- and 6.10-fold, respectively, after 15 h of fermentation at 37 degrees C. The maximum hydrolytic potential was also observed at 15 h of fermentation for the three micro-organims coinciding with peak activities of the two enzymes. CONCLUSIONS: beta-glucosidase activity was more than 15 times higher than beta-galactosidase activity in soymilk for each of the micro-organisms during fermentation. beta-glucosidase played a greater role in isoflavone glycoside hydrolysis. SIGNIFICANCE AND IMPACT OF THE STUDY: Screening for beta-glucosidase and beta-galactosidase activities among probiotics in soymilk is important for the improvement of biological activity of soymilk and in the selection of micro-organisms for use in the growing industry of functional foods and beverages.     

Electrochemical evaluation of cellular physiological status under stress in Escherichia coli with the rpoS-lacZ reporter gene

We developed an electrochemical detection method for evaluating cellular physiological status based on the stringent response as a means to monitor cell viability. A reporter plasmid was constructed by inserting the beta-galactosidase gene (lacZ) under the control of the rpoS promoter, and then used to transform E. coli cells. Electrochemical responses from the products catalyzed by beta-galactosidase expressed by these E. coli cells were detected using the chronoamperometric technique in a nondestructive manner. Comparisons of response currents between the relA-positive strain and relA-negative strain revealed that increases in these currents were caused by the stringent response due to the stressful alcoholic environment, and thus as a model of stressful cultivating conditions. The current was proportional to the beta-galactosidase activity assayed by a conventional method that required the destruction of cells. The cellular physiological status, which depends on the stringent response as a viability marker, therefore, could then be evaluated online with a current using the rpoS-lacZ reporter gene in the relA-positive strain without pretreatment.     

Determining the evolutionary potential of a gene

In addition to information for current functions, the sequence of a gene includes potential information for the evolution of new functions. The wild-type ebgA (evolved beta-galactosidase) gene of Escherichia coli encodes a virtually inactive beta-galactosidase, but that gene has the potential to evolve sufficient activity to replace the lacZ gene for growth on the beta-galactoside sugars lactose and lactulose. Experimental evidence, which has suggested that the evolutionary potential of Ebg enzyme is limited o two specific amino acid replacements, is limited to examining the consequences of single base- substitutions. Thirteen beta-galactosidases homologous with the Ebg beta-galactosidase are widely dispersed, being found in gram-negative and gram-positive eubacteria and in a eukaryote. A comparison of Ebg beta-galactosidase with those 13 beta-galactosidases shows that Ebg is part of an ancient clade that diverged from the paralogous lacZ beta- galactosidase over 2 billion years ago. Ebg differs from other members of its clade at only 2 of the 15 active-site residues, and the two mutations required for full Ebg beta-galactosidase activity bring Ebg into conformity with the other members of its clade. We conclude that either these are the only acceptable amino acids at those positions, or all of the single-base-substitution replacements that must arise as intermediates on the way to other acceptable amino acids are so deleterious that they constitute a deep selective valley that has not been traversed in over 2 billion years. The evolutionary potential of Ebg is thus limited to those two replacements.      

Expression in vivo of the lacZ gene, delivered to mouse lungs using synthetic microspheres

A capacity of MF-2 synthetic microspheres to serve as the vehicle for transfer of the marker LacZ gene to mouse lung epithelial cells was studied after a single intranasal administration of the MF-2/gene complex. Two types of plasmids carrying marker gene LacZ were used in the experiments: with cytoplasmic (pCMV-LacZ) and nuclear (pCMV-nlsLacZ) localization of the gene product (beta-galactosidase). As early as 7 days after the complexes MF-2/pCMV-LacZ and MF-2/pCMV-nlsLacZ were administered, specific staining for beta-galactosidase revealed this enzyme activity in the epithelial cells of bronchi, bronchioli, and alveoli. The maximum in vivo of the marker gene in the MF-2/pCMV-LacZ complex was observed at day 14 to 21 after administration and the corresponding gene product was detected during the following two months. The MF-2-mediated gene transfer led to a twofold increase in beta-galactosidase activity relative to the case when the "unbound" pCMV-LacZ plasmid was administered. These results suggest that the synthetic microsphere-mediated transfer of alien genes to the lung of experimental animals is promising. Microspheres may be used in gene therapy for pulmonary affections, in particular cystic fibrosis.     

Evaluation of novel chromogenic substrates for the detection of bacterial beta-glucosidase

AIMS: To evaluate three previously unreported substrates for the detection of beta-glucosidase activity in clinically relevant bacteria and to compare their performance with a range of known substrates in an agar medium. METHODS AND RESULTS: The performance of 11 chromogenic beta-glucosidase substrates was compared using 109 Enterobacteriaceae strains, 40 enterococci and 20 strains of Listeria spp. Three previously unreported beta-glucosides were tested including derivatives of alizarin, 3',4'-dihydroxyflavone and 3-hydroxyflavone. These were compared with esculin and beta-glucoside derivatives of 3,4-cyclohexenoesculetin, 8-hydroxyquinoline and five indoxylics. All substrates yielded coloured precipitates upon hydrolysis in agar. Alizarin-beta-D-glucoside was the most sensitive substrate tested and detected beta-glucosidase activity in 72% of Enterobacteriaceae strains and all enterococci and Listeria spp. The two flavone derivatives showed poor sensitivity with Gram-negative bacteria but excellent sensitivity with enterococci and Listeria spp. CONCLUSIONS: Alizarin-beta-d-glucoside is a highly sensitive substrate for detection of bacterial beta-glucosidase and compares favourably with existing substrates. beta-glucosides of 3',4'-dihydroxyflavone and 3-hydroxyflavone are effective substrates for the detection of beta-glucosidase in enterococci and Listeria spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented allow for informed decisions to be made regarding the optimal choice of beta-glucosidase substrate for detection of pathogenic and/or indicator bacteria.     

Molecular cloning and expression of the novel fungal beta-glucosidase genes from Humicola grisea and Trichoderma reesei

A novel fungal beta-glucosidase gene (bgl4) and its homologue (bgl2) were cloned from the cellulolytic fungi Humicola grisea and Trichoderma reesei, respectively. The deduced amino acid sequences of H. grisea BGL4 and T. reesei BGL2 comprise 476 and 466 amino acids, respectively, and share 73.1% identity. These beta-glucosidases show significant homology to plant beta-glucosidases belonging to the beta-glucosidase A (BGA) family. Both genes were expressed in Aspergillus oryzae, and the recombinant beta-glucosidases were purified. Recombinant H. grisea BGL4 is a thermostable enzyme compared with recombinant T. reesei BGL2. In addition to beta-glucosidase activity, recombinant H. grisea BGL4 showed a significant level of beta-galactosidase activity, while recombinant T. reesei BGL2 showed weak beta-galactosidase activity. Cellulose saccharification by Trichoderma cellulases was improved by the addition of recombinant H. grisea BGL4.     

The use of lacZ gene fusions in the studies of mammalian development: developmental regulation of mammalian homeobox genes in the CNS

1. Mammalian cells can produce bacterial beta-galactosidase when they carry the lacZ gene under the control of mammalian regulatory elements. The single cell resolution of the beta-galactosidase histochemical detection method makes this molecule an excellent marker in studies of development at the cellular and molecular level. Different lacZ fusion genes can be engineered to study the histological diversification of cell lineages, the developmental regulation of isolated genes, or to recognize and clone genes with new expression profile. 2. Transgenic mice carrying lacZ gene fusions provide information on the cell type, developmental stage and spatial specificity of cis-acting regulatory regions linked to a mammalian homeobox gene. We describe our strategy for designing the gene fusions. 3. The scord region of the Hox 1.3 gene is sufficient to determine spatially restricted expression of a heterologous protein in the midgestational spinal cord. We propose to use this region to alter the expression pattern of other homeobox gene products. Developmental alterations due to a variant expression pattern would point to the function of the misexpressed gene.     

Enzymatic properties and intracellular localization of the novel Trichoderma reesei beta-glucosidase BGLII (cel1A)

This paper describes the characterization of an intracellular beta-glucosidase enzyme BGLII (Cel1a) and its gene (bgl2) from the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). The expression pattern of bgl2 is similar to that of other cellulase genes known from this fungus, and the gene would appear to be under the control of carbon catabolite repression mediated by the cre1 gene. The BGLII protein was produced in Escherichia coli, and its enzymatic properties were analyzed. It was shown to be a specific beta-glucosidase, having no beta-galactosidase side activity. It hydrolyzed both cellotriose and cellotetraose. BGLII exhibited transglycosylation activity, producing mainly cellotriose from cellobiose and sophorose and cellobiose from glucose. Antibodies raised against BGLII showed the presence of the enzyme in T. reesei cell lysates but not in the culture supernatant. Activity measurements and Western blot analysis of T. reesei strains expressing bgl2 from a constitutive promoter further confirmed the intracellular localization of this beta-glucosidase.     

Expression in Escherichia coli of the human leukocyte interferon alpha2 gene fused with N-terminal fragment of beta-galactosidase

Genes for leucocyte interferon and alpha-donor of galactosidase were fused by deletion mutagenesis or by site-directed mutagenesis. In both cases the fused protein was expressed. The protein having an antiviral activity of leucocyte interferon was easily detected in bacteria and solutions by the reaction of beta-galactosidase alpha-complementation and retained the antigenic determinants of interferon and beta-galactosidase. The use of fused proteins for optimization of gene expression and for the analysis of interferon structure-function relationship is discussed.     

Evaluation of novel fluorogenic substrates for the detection of glycosidases in Escherichia coli and enterococci

AIMS: Enzyme substrates based on 4-methylumbelliferone are widely used for the detection of Escherichia coli and enterococci in water, by detection of beta-glucuronidase and beta-glucosidase activity respectively. This study aimed to synthesize and evaluate novel umbelliferone-based substrates with improved sensitivity for these two enzymes. METHODS AND RESULTS: A novel beta-glucuronide derivative based on 6-chloro-4-methylumbelliferone (CMUG) was synthesized and compared with 4-methylumbelliferyl-beta-D-glucuronide (MUG) using 42 strains of E. coli in a modified membrane lauryl sulfate broth. Over 7 h of incubation, the fluorescence generated from the hydrolysis of CMUG by E. coli was over twice that from MUG, and all of the 38 glucuronidase-positive strains generated a higher fluorescence with CMUG compared with MUG. Neither substrate caused inhibition of bacterial growth in any of the tested strains. Four beta-glucosidase substrates were also synthesized and evaluated in comparison with 4-methylumbelliferyl-beta-D-glucoside (MU-GLU) using 42 strains of enterococci in glucose azide broth. The four substrates comprised beta-glucoside derivatives of umbelliferone-3-carboxylic acid and its methyl, ethyl and benzyl esters. Glucosides of the methyl, ethyl and benzyl esters of umbelliferone-3-carboxylic acid, were found to be superior to MU-GLU for the detection of enterococci, especially after 18 h of incubation, while umbelliferone-3-carboxylic acid-beta-D-glucoside was inferior. However, the variability in detectable beta-glucosidase activity among the different strains of enterococci in short-term assays using the three carboxylate esters (7 h incubation) may compromise their use for rapid detection and enumeration of these faecal indicator bacteria. CONCLUSIONS: The beta-glucuronidase substrate CMUG appears to be a more promising detection system than the various beta-glucosidase substrates tested. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel substrate CMUG showed enhanced sensitivity for the detection of beta-glucuronidase-producing bacteria such as E. coli, with a clear potential for application in rapid assays for the detection of this indicator organism in natural water and other environmental samples.     

Distribution of beta-glucosidase and beta-glucuronidase activity and of beta-glucuronidase gene gus in human colonic bacteria

beta-Glycosidase activities present in the human colonic microbiota act on glycosidic plant secondary compounds and xenobiotics entering the colon, with potential health implications for the human host. Information on beta-glycosidases is currently limited to relatively few species of bacteria from the human colonic ecosystem. We therefore screened 40 different bacterial strains that are representative of dominant bacterial groups from human faeces for beta-glucosidase and beta-glucuronidase activity. More than half of the low G+C% Gram-positive firmicutes harboured beta-glucosidase activity, while beta-glucuronidase activity was only found in some firmicutes within clostridial clusters XIVa and IV. Most of the Bifidobacterium spp. and Bacteroides thetaiotaomicron carried beta-glucosidase activity. A beta-glucuronidase gene belonging to family 2 glycosyl hydrolases was detected in 10 of the 40 isolates based on degenerate PCR. These included all nine isolates that gave positive assays for beta-glucuronidase activity, suggesting that the degenerate PCR could provide a useful assay for the capacity to produce beta-glucuronidase in the gut community. beta-Glucuronidase activity was induced by growth on d-glucuronic acid, or by addition of 4-nitrophenol-glucuronide, in Roseburia hominis A2-183, while beta-glucosidase activity was induced by 4-nitrophenol-glucopyranoside. Inducibility varied between strains.     

Expression in Trichoderma reesei and characterisation of a thermostable family 3 beta-glucosidase from the moderately thermophilic fungus Talaromyces emersonii

The gene encoding a thermostable beta-glucosidase (cel3a) was isolated from the thermophilic fungus Talalaromyces emersonii by degenerate PCR and expressed in the filamentous fungus Trichoderma reesei. The cel3a gene encodes an 857 amino acid long protein with a calculated molecular weight of 90.59 kDa. Tal. emersonii beta-glucosidase falls into glycosyl hydrolase family 3, showing approximately 56 and 67% identity with Cel3b (GenBank ) from T. reesei, and a beta-glucosidase from Aspergillus Niger (GenBank ), respectively. The heterologously expressed enzyme, Cel3a, was a dimer equal to 130 kDa subunits with 17 potential N-glycosylation sites and a previously unreported beta-glucosidase activity produced extracellularly by Tal. emersonii. Cel3a was thermostable with an optimum temperature of 71.5 degrees C and half life of 62 min at 65 degrees C and was a specific beta-glucosidase with no beta-galactosidase side activity. Cel3a had a high specific activity against p-nitrophenyl-beta-D-glucopyranoside (Vmax, 512 IU/mg) and was competitively inhibited by glucose (k(i), 0.254 mM). Cel3a was also active against natural cellooligosacharides with glucose being the product of hydrolysis. It displayed transferase activity producing mainly cellobiose from glucose and cellotetrose from cellobiose.