PURIFICATION, CHARACTERISATION AND CLONING OF -1,3 GLUCANASE GENE FROM Trichoderma harzinum
Abstract
Dual culture technique was used to evaluate the effect of three species of Trichoderma that showed a potential control of Fusarium oxysporium. Trichoderma harzianum showed maximum growth inhibition (79.3%) followed by Trichoderma viridi (68.5%) and Trichoderma longibrachiatum (44.3%). β-1,3-glucanases was purified from Trichoderma harzianum to homogeneity by ion exchange chromatography on DEAE-Sephacel and gel filtration on Sephadex G100. A typical procedure provided 20-fold purification with 11.9% yield. The apparent molecular mass was 30 kD and it was active on a broad pH range, however the maximal activity was detected at pH 7.5. The optimum temperature of the β-1,3-glucanase was 55C. Polymerase chain reaction (PCR) was used to amplify a fragment about 600 bp from β-1,3 gluanase gene using specific glu forward and reverse primers. The eluted DNA was ligated into pGEM-T-Easy vector and transformed into competent E. coli JM109. White transformed colony, named T1glu, containing recombinant plasmid was validated by PCR using both glu forward and reverse and M13 forward and reverse primers to confirm the presence of β 1,3 glucanase gene insert in right orientation whereas, the fragment amplified with glu forward and glu reverse primers was 600 bp. Partial sequence of the amplified DNA fragment showed 97% sequence homology with the other published sequences.References
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