Cloning of cellulase gene(s) of trichoderma reesei in sacchromyces cerevisiae
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Date
2007
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Publisher
CCSHAU
Abstract
A total of seven Trichoderma reesei cultures were purified by
reculturing on potato dextrose agar medium. Cellulase production by T.
reesei cultures varied from 0.73 IU/ml to a maximum of 3.11 IU/ml in
cellulose medium. Among all the strains evaluated, T.reesei culture 5A
showed the maximum cellulase activity (3.11 IU). Cellulase activity of T.
reesei 5A was monitored at different interval of time (12-96 hrs.) and
maximum activity was observed after 96 hrs. of growth. Genomic DNA
of T. reesei 5A was isolated and checked for its quality by agarose gel
VI
electrophoresis and by UV spectrophotometer. The quantity of DNA was
found to range between 195 μg/ml to 2607.5 μg/ml. Different
parameters i. e. growth period of 36 hrs., CTAB concentration 1.5% and
incubation period of 90 min., were found to be optimum during DNA
isolation. Partial digestion of genomic DNA was done with Sau 3A and
the digested DNA was ligated to Bam H1 treated Yeast-E. coli shuttle
vector YEpFLAG-1. The construct was used for transformation of E.
coli. Recombinant clones of E. coli were screened on carboxy methyl
cellulose and ampicillin containing Reese medium. The recombinant
plasmids YEpFLAG-1-cel-exo and YEpFLAG-1-cel-endo were isolated
from E. coli recombinant clones and checked for amplification of
cellulase gene by PCR using the standard conditions. PCR amplification
was observed for exoglucanase and endoglucanase gene. Protoplast of
yeast Saccharomyces cerevisiae were transformed with the recombinant
plasmids YEpFLAG-1-cel-exo and YEpFLAG-1-cel-endo isolated from
E.coli. The recombinant clones of yeast were screened on YEP medium
containing 1% phosphocellulose and ampicillin. Expression of cellulase
gene(s) in S. cerevisiae transformants was checked by monitoring the
cellulase activity and ethanol production. CMCase activity was
observed in all the three yeast transformants as 0.80 IU/ml, 0.50
IU/ml and 0.70 IU/ml respectively. No FPase activity could be detected
in any of the yeast transformants. Different concentrations of
phosphocellulose (2% and 5%) were used for ethanol production by the
yeast transformants. Yeast transformant-6 produced maximum 1.5%
alcohol when inoculated in 5% phosphocellulose containing YEP
medium. Yeast transformant-2 and transformant-4 also produced
ethanol in rice straw medium.
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Keywords
Trichoderma reesei, E. coli, Saccharomyces cerevisiae, Cellulase, Cloning