Cloning and expression of ß-glucosidase genes in Escherichia coli and Saccharomyces cerevisiae using shuttle vector pYES2.0

National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan.
Folia Microbiologica (Impact Factor: 1.15). 02/1998; 43(2):129-35. DOI: 10.1007/BF02816497
Source: PubMed

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.

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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). "
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    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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    • "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). "
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    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 · 1.74 Impact Factor
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    • "For β-glucosidase, assays were performed towards p-nitrophenyl β-D-glucopyranoside (pNPG) as described earlier (Rajoka et al. 1998b). Enzyme activity was expressed as IU ml −1 min −1 or kinetic parameters for product formation were determined. "
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    ABSTRACT: Maximum productivities of -glucosidase by E. coli recombinants, under the control of either lacZ or GALI promoters, were 33 10 and 100 5 IU l–1 h–1, respectively.GAL1 promoter of pYES2 enabled the E. colirecombinants to produce 3.1- and 15.1-fold more -glucosidase than that supported by lacZ promoter of pUC18 in E. coli recombinants and donor, respectively.
    Biotechnology Letters 10/2002; 24(21):1803-1806. DOI:10.1023/A:1020677725691 · 1.74 Impact Factor
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