Molecular Cloning and Expression of the Trichoderma harzianum C4 Endo-β-1,4-Xylanase Gene in Saccharomyces cerevisiae
2009
Lee, J.M., Kyung Hee University, Yongin, Republic of Korea | Shin, J.W., Kyung Hee University, Yongin, Republic of Korea | Nam, J.K., Kyung Hee University, Yongin, Republic of Korea | Choi, J.Y., University of Ulsan, Ulsan, Republic of Korea | Jeong, C.S., University of Ulsan, Ulsan, Republic of Korea | Han, I.S., University of Ulsan, Ulsan, Republic of Korea | Nam, S.W., Dong-Eui University, Busan, Republic of Korea | Choi, Y.J., Seoul National University, Seoul, Republic of Korea | Chung, D.K., Kyung Hee University, Yongin, Republic of Korea
An endo-β-1,4-xylanase (β-xylanase) from Trichoderma harzianum C4 was purified without cellulase activity by sequential chromatographies. The specific activity of the purified enzyme preparation was 430 units/mg protein on D-Xylan. The complementary DNA (cDNA) encoding β-xylanase (xynⅡ) was amplified by PCR and isolated from cDNA PCR libraries constructed from T. harzianum C4. The nucleotide sequence of the cDNA fragment contained an open reading frame of 663 bp that encodes 221 amino acids, of which the mature protein is homologous to several β-xylanases Ⅱ. An intron of 63 bp was identified in the genomic DNA sequence of xynⅡ. This gene was expressed in Saccharomyces cerevisiae strains under the control of adh1 (alcohol dehydrogenase Ⅰ) and pgk1 (phosphoglycerate kinase Ⅰ) promoters in 2 μ-based plasmids, which could render recombinants able to secrete β-xylanase into the media.
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