Expression Of Β-glucosidase Research Articles

Differential expression of β-glucosidase in tomato — stress stimuli systems

In the present work the responses of β-glucosidase in leaves of tomato plants subjected to various stress factors of both pathogenic (fungi, bacteria, viruses) and abiotic origin (heat shock) were studied. Biochemical and cytochemical methods were applied. It was established that an increase of β-glucosidase activity is induced uniquely by fungal pathogens. The cytochemical tests confirm the finding. Hence, the conclusion can be drawn that β-glucosidase response is a specific character of fungal pathogenesis in tomato; probably, the enzyme is involved in plant — fungi recognition. The data are in accordance with our previous results on tobacco and wheat — stress stimuli systems.

Cloning and expression of β-glucosidase from Flavobacterium meningosepticum: a new member of family B β-glucosidase

A β-glucosidase gene from Flavobacterium meningosepticum has been cloned and expressed in Escherichia coli. The entire nucleotide sequence was determined and analyzed. An open-reading frame of 2,178 bp encoding a polypeptide of 726 amino acids with a calculated M r of 79,952 was derived from the sequence. The coding region is flanked by a putative promoter and transcription terminator sequences. The nucleotide sequence accession number of the complete gene sequence in Genbank is AF015915. Based on the comparison of amino acid sequences, this enzyme is classified as a family B β-glucosidase and is highly homologous with sequences from Clostridium thermocellum, Kluyvermyces fragilis, and Agrobacterium tumefaciens. The cloned enzyme was purified to near homogeneity by ammonium sulfate fractionation (35 ∼ 75%) and chromatography on a cation-exchanged column at pH 6.9. This expressed β-glucosidase has optimum activity at pH 4.2 ∼ 5.0 and 50°C and is stable in the pH range of 5.0 ∼ 8.1 at 25°C. It showed a high specificity on the glycone portion of aryl-β- d-glycosides. All of these characteristics are highly consistent with those of the native β-glucosidase.

Utilization of glucose and cellobiose byCandida molischiana

Some of the factors that influence the biosynthesis of the Candida molischiana β-glucosidase were investigated. The yeast produced maximal enzyme activity when grown at 28 °C in a carbohydrate-containing complex medium (YM) in which the initial pH was adjusted to 6.0. The enzyme appeared to be produced constitutively, as activity was detected when either ethanol, glycerol, xylose, glucitol, mannitol, maltose, trehalose, cellobiose, cellodextrins, or soluble starch was used as the carbohydrate source. The presence of either glucose, mannose, or fructose (> 25 g/L) repressed β-glucosidase expression; however, C. molischiana did produce β-glucosidase when the initial glucose concentration was <25 g/L. When the yeast was grown in YM medium containing glucose plus cellobiose, diauxic utilization of the carbon sources was observed, and β-glucosidase activity was not detected until the glucose was depleted from the medium.Key words: β-glucosidase, glucose repression, fermentation, yeast.

Properties of theβ-glucosidase fromCellulomonas flavigena and fromEscherichia coli harboring the recombinant plasmid pJS3

Plasmid-coded β-glucosidase produced byEscherichia coli was characterized and compared to the enzyme produced byCellulomonas flavigena. Cell-free extracts, non-denaturing PAGE and 5-bromo-4-chloro-3-indolyl-β-d-glucopyranoside (X-glu) as substrate were used to compare both enzymes. The β-glucosidase was assayed for cellobiose andp-nitrophenyl-glucopyranoside (PNPG). Cellobiose hydrolysis was performed at 50°C for the enzyme fromC. flavigena and at 37°C for that fromE. coli pJS3, both with an optimal pH of 6.5. For PNPG hydrolysis, the optimal conditions were pH 5.5 and 37°C for both cell extracts. Most of the β-glucosidase activity was intracellular. When cultures ofC. flavigena were grown with cellobiose or carboxymethylcellulose (CMC) as inducers, the expression of β-glucosidase was increased considerably.E. coli pJS3 produces a cellobiase which hydrolyzes cellobiose and PNPG. TheKm values for cellobiose and PNPG indicated that the β-glucosidase activity ofC. flavigena had a higher affinity for cellobiose as substrate, whereas the β-glucosidase fromE. coli pJS3 showed higher affinity for PNPG.

?-Glucosidase genes of naturally occurring and cellulolytic Streptomyces species: characterization of two such genes in Streptomyces lividans

To learn more about the mechanisms by which Streptomyces converts lignocellulosic biomass, we have isolated a number of cellulolytic Streptomyces strains from Northern Sweden. Genes encoding intracellular β-glucosidase (cellobiase) of Streptomyces UM-2 and S. UM-d, have been cloned into Streptomyces lividans. This was accomplished by using the chromogenic compound X-glu (5-bromo-4-chloro-3-indoly β-d-glucopyranoside) for the detection of cloned β-glucosidase. The β-glucosidase genes of S. UM-2 and S. UM-d were isolated on 7.5 kb and 18 kb BamHI fragments, respectively. The S. UM-d gene was further subcloned to a 7.5 kb fragment. Each of the two BamHI fragments encompassing the β-glucosidase genes hybridized to fragments of the corresponding size in chromosomal DNA from the respective strain used for the cloning. The cloned genes expressed β-glucosidase activities in S. lividans with levels depending on the carbon source. Regulation of the expression of β-glucosidase was exerted at the transcriptional level, since β-glucosidase mRNA was expressed at higher levels with cellobiose, as compared to Avicel or glucose as carbon sources.

Regulation of β-1, 4-Glucosidase Expression by Candida wickerhamii

Candida wickerhamii NRRL Y-2563 expressed beta-glucosidase activity (3 to 8 U/ml) constitutively when grown aerobically in complex medium containing either glycerol, succinate, xylose, galactose, or cellobiose as the carbon source. The addition of a high concentration of glucose (>75 g/liter) repressed beta-glucosidase expression (<0.3 U/ml); however, this yeast did produce beta-glucosidase when the initial glucose concentration was </=50 g/liter. When grown aerobically in medium containing glucose plus the above-listed carbon sources, diauxic utilization of the carbon source was observed and the expression of beta-glucosidase was glucose repressed. Surprisingly, glucose repression did not occur when the cells were grown anaerobically. When grown anaerobically in medium containing 100 g of glucose per liter, C. wickerhamii produced 6 to 9 U of enzyme per ml and did not demonstrate diauxic utilization of glucose-cellobiose mixtures. To our knowledge, this is the first report of apparent derepression of a glucose-repressed enzyme by anaerobiosis.

The role and expression of β-glucosidase during spore germination of mutant and wild-type Dictyostelium discoideum

Dormant spores of Dictyostelium discoideum are contained in a viscous matrix of extracellular material at the top of sorocarps. Analysis of crude extracts of the sorus demonstrates that there is a higher specific activity of β-glucosidase in the matrix than in the dormant spores. Polyacrylamide gel electrophoresis of the matrix material reveals the presence of β-glucosidase 2 with trace amounts of β-glucosidase 1. In the dormant spores only β-glucosidase 2 is detected. Upon heat activation of the spores, β-glucosidase 2 activity decreases to approximately 25% of the activity found in the preheated spores. The enzyme appears to have a surface location in dormant spores, since trypsin treatment will destroy all of the β-glucosidase 2 activity. The normal germination of dormant spores after trypsin treatment suggests that β-glucosidase 2 activity is not required for activation, swelling, or emergence processes.During the emergence of myxamoebae, β-glucosidase 2 is replaced by a newly synthesized enzyme, β-glucosidase 1. The formation of this new enzyme requires both RNA and protein syntheses. The complete germination of a β-glucosidase 1 minus mutant, however, casts doubt on the necessity for β-glucosidase1 activity during emergence. This is further supported by the normal germination of strain SG1 which contains four- to five-fold higher β-glucosidase activity than that found in wild-type amoebae at the end of the germination period. In addition, strain SG1 may be activated with a number of techniques which results in a separation of the time of β-glucosidase synthesis from the time of myxamoebae emergence. Furthermore, spores (which have been blocked from emerging by cycloheximide treatment) will slowly release spheroplasts when treated with only pronase and D. discoideum cellulase. However, addition of D. discoideum β-glucosidase 1 to the cellulose–pronase-treated spores does accelerate the rate of spheroplast formation. Thus, β-glucosidase 1 activity may only play a minor and dispensable role in D. discoideum spore germination.