Molecular cloning and expression of the trichoderma harzianum C4 endo-beta-1,4-Xylanase Gene in Saccharomyces cerevisiae.
An endo-beta-1,4-xylanase (beta-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 on D-xylan. The complementary DNA (cDNA) encoding beta-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 beta- 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 mu-based plasmids, which could render recombinants able to secrete beta-xylanase into the media.
Lee
Jung Min
JM
Skin Biotechnology Center, Graduate School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University,Yongin 446-701, Korea.
Shin
Ji-Won
JW
Nam
Jae-Kook
JK
Choi
Ji-Young
JY
Jeong
Choon-Soo
CS
Han
In-Seob
IS
Nam
Soo-Wan
SW
Choi
Yun-Jaie
YJ
Chung
Dae Kyun
DK
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis / Q000235:genetics; D004271:DNA, Fungal / Q000032:analysis / Q000235:genetics; D043364:Endo-1,4-beta Xylanases / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D007218:Industrial Microbiology; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
D018076:DNA, Complementary; D004271:DNA, Fungal; D011993:Recombinant Fusion Proteins; D043364:Endo-1,4-beta Xylanases