Cloning and Expression of β-Glucosidase Genes in Escherichia coli and Saccharomyces cerevisiae Using Shuttle Vector pYES 2.0
ABSTRACT Genes for beta-glucosidase (Bgl) isolated from a genomic library of the cellulolytic bacterium, Cellulomonas biazotea, were cloned in pUC18 in its SacI cloning site and transformed to E. coli. Ten putative recombinants showed blackening zones on esculin plates, yellow zones on pNPG plates, in liquid culture and on native polyacrylamide gel electrophoresis activity gels. They fell into three distinct groups. Three representative E. coli clones carried recombinant plasmids designated pRM54, pRM1 and pRM17. The genes were located on 5.6-, 3.7- and 1.84-kb fragments, respectively. Their location was obtained by deletion analysis which revealed that 5.5, 3.2, and 1.8 kb fragments were essential to code for BglA, BglB, and BglC, respectively, and conferred intracellular production of beta-glucosidase on E. coli. Expression of the bgl genes resulted in overproduction of beta-glucosidase in the three clones. Secretion occurred into the periplasmic fractions. Three inserts carrying bgl genes from the representative recombinant E. coli were isolated with SacI, ligated in the shuttle vector pYES 2.0 in its SacI site and transformed to E. coli and S. cerevisiae. The recombinant plasmids were redesignated pRPG1, pRPG2 and pRPG3 coding for BglA1, BglB1 and BglC1. The cloned genes conferred extracellular production of beta-glucosidase on S. cerevisiae and enabled it to grow on cellobiose and salicin. The gall promoter of shuttle vector pYES 2.0 enabled the organisms to produce twice more beta-glucosidase than that supported by the lacZ-promoter of pUC18 plasmid in E. coli. The cloned gene can be used as a selection marker for introducing recombinant plasmids in wild strains of S. cerevisiae. The enzyme produced by bgl+ yeast and E. coli recombinants resembles that of the donor with respect to temperature and pH requirement for maximum activity. Other enzyme properties of the beta-glucosidases from S. cerevisiae were substantially the same as those from C. biazotea.
- SourceAvailable from: Khadiga Abou-Taleb
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- "2005). The production of cellulases by wild type cells of Bacillus pumilus (Kotchoni and Shonukan 2002), Cellulomonas biazotea (Rajoka 1998) and Trichoderma aureoviride (Zaldivar et al. 2001) in liquid media did not exceed 1.5 U/ml. Production of filter paper cellulase (FPase), endo-β-glucanase and βglucosidase by Cellulomonas biazotea was investigated during growth on different cellulosic substrates (Rajoka and Malik 1997). "
ABSTRACT: Maximum cellulase partial purification was achieved with 80 % (NH4)2SO4 precipitation. The Km and Vmax values were calculated for CMCase, FPase & β-glucosidase. It was found that Km values (0.92±0.28, 3.70, 0.55± 0.17 %) & (0.59±0.24, 3.86±0.39 & 0.65±0.14 %) and Vmax values (4.27± 0.73, 2.50 & 6.34±1.22 U/ ml) & (3.53±0.60, 2.22 & 7.42±1.04 U/ ml) for Bacillus alcalophilus S39 and B. amyloliquefaciens C23, respectively. It was found that the enzymes activity has a broad pH range between 4.8 and 5. The optimum temperature of the enzymes was observed to be around 50 °C, and 97-98 % of the original activity was retained after heat treatment at 80 °C for 15 min. The enzymes activity was stimulated by Ca ++ & Co ++ , weak inhibition of cellulases with EDTA, Cu ++ , Acetone & Methanol, SDS & ethanol was the strongest inhibitor and the enzymes had no effect with Na + , Mn ++ , Tween 80 and Mg ++. Analyses of the enzyme preparation by SDS-PAGE revealed two protein bands showing cellulolytic activity. The molecular weight of these bands was estimated to be around 66.49 and 28.47 KDa for Bacillus geneus.
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- "Product yield (YP/S), specific product yield (YP/X) and product formation rates (QP) of BGL of H. lanuginosa (Table 3) are significantly higher than the values reported for Aspergilus spp., Trichoderma reesei RUT C30, other fungal cultures, different bacteria (37, 45), E. coli and Saccharomyces cerevisiae recombinants harboring heterologous bgl gene (38), Thielavia terrestris and Themoascus crustaceus (41), Thermoascus aurantiacus (25), Humicola spp. (23), Thermomyces lanuginosus (31), other fungi and their DG resistant mutants (8, 21, 45) though substrates used in referred studies were different. "
ABSTRACT: A 2-deoxyglucose-resistant mutant (M7) of Humicola lanuginosa was obtained by exposing conidia to γ-rays and permitting expression in broth containing 0.6% 2-deoxyglucose (DG) and cellobiose (1%) before plating on DG esculin-ferric ammonium citrate agar medium from which colonies showing faster and bigger blackening zones were selected. Kinetic parameters for enhanced ß-glucosidase (BGL) synthesis by M7 were achieved when corncobs acted as the carbon source. The combination between corncobs and corn steep liquor was the best to support higher values of all product formation kinetic parameters. Effect of temperature on the kinetic and thermodynamic attributes of BGL production equilibrium in the wild organism and M7 was studied using batch process at eight different temperatures in shake-flask studies. The best performance was found at 45°C and 20 g L(-1) corncobs in 64 h. Both growth and product formation (17.93 U mL(-1)) were remarkably high at 45°C and both were coupled under optimum working conditions. Product yield of BGL from the mutant M7 (1556.5 U g(-1) dry corncobs) was significantly higher than the values reported on all fungal and bacterial systems. Mutation had thermo-stabilization influence on the organism and mutant required lower activation energy for growth and lower magnitudes of enthalpy and entropy for product formation than those demanded by the wild organism, other mesophilic and thermo-tolerant organisms. In the inactivation phase, the organisms needed lower values of activation energy, enthalpy and entropy for product formation equilibrium, confirming thermophilic nature of metabolic network possessed by the mutant organism.Brazilian Journal of Microbiology 10/2008; 39(4):724-33. DOI:10.1590/S1517-838220080004000024 · 0.59 Impact Factor
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- "Cellulomonas biazotea and Saccharomyces cerevisiae were recovered by centrifugation (10 000 g, 15 min, 10 • C). Cell-free extracts and cell extracts were obtained as described earlier (Rajoka et al. 1998). "
ABSTRACT: The maximum productivity of -glucosidase by Saccharomyces cerevisiaerecombinants under the control of GALI promoter was 100IUl–1h–1. The highest productivity of -glucosidase by a S.cerevisiae recombinant was 16-fold more than that supported by Cellulomonas biazotea. The recombinants also co-produced ethanol from cellobiose: maximum product yield and productivity were 0.5 and 1.1g ethanolg–1 cellobiose and g ethanoll–1h–1, respectively.Biotechnology Letters 05/2003; 25(12):945-948. DOI:10.1023/A:1024042401544 · 1.59 Impact Factor