THESIS
1998
xiii, 80 leaves : ill., col. photos ; 30 cm
Abstract
A 4.7-kb DNA insert encoding a cellobiase was cloned from Cellulomonas biazotea in Escherichia coli using an excretion vector, pM, to form a recombinant construct, pBZ 4.7. It is the first demonstration of an external production of a recombinant cellobiase of which part of the activity was detected in the periplasm and some could even be in the culture supematant. The cellobiase (Cba) was shown to be able to act synergistically in the hydrolysis of a packing material with an endoglucanase and an exoglucanase of Cellulomonas fimi produced as recombinant enzymes in E. coli. The purification of the Cba from the supematant of the induced JM101(pBZ 4.7) was achieved by simply two purification steps: a gel-filtration chromatography followed by an anion-exchange chromatography. A mature Cba as...[
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A 4.7-kb DNA insert encoding a cellobiase was cloned from Cellulomonas biazotea in Escherichia coli using an excretion vector, pM, to form a recombinant construct, pBZ 4.7. It is the first demonstration of an external production of a recombinant cellobiase of which part of the activity was detected in the periplasm and some could even be in the culture supematant. The cellobiase (Cba) was shown to be able to act synergistically in the hydrolysis of a packing material with an endoglucanase and an exoglucanase of Cellulomonas fimi produced as recombinant enzymes in E. coli. The purification of the Cba from the supematant of the induced JM101(pBZ 4.7) was achieved by simply two purification steps: a gel-filtration chromatography followed by an anion-exchange chromatography. A mature Cba as analyzed by SDS-PAGE had an apparent molecular weight of about 86 kDa. The information suggested that the coding sequence for Cba, designated the cba gene, had a size of about 2.6 kb. The purified Cba are used to immunized a rabbit, and an anti-Cba antibody of a titre of 50,000K was obtained for future work on the analysis and purification of Cba. DNA sequencing of the cba insert revealed that the coding sequence for Cba contained a single open reading frame of 2,484 bp. The cba gene has a high G+C content of 76.4 % and is flanked by a putative ribosomal-binding site and potential transcriptional termination signals upstream and downstream from its coding sequence, respectively. Comparison of the deduced Cba sequence with other β-glucosidases revealed a potential active site at Asp
228.
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