Enhanced production of Trichoderma reesei endoglucanases and use of the new cellulase preparations in producing the stonewashed effect on denim fabric

Abstract

Trichoderma reesei strains were constructed for production of elevated amounts of endoglucanase II (EGII) with or without cellobiohydrolase I (CBHI). The endoglucanase activity produced by the EGII transformants correlated with the copy number of the egl2 expression cassette. One copy of the egl2 expression cassette in which the egl2 was under the cbh1 promoter increased production of endoglucanase activity 2.3-fold, and two copies increased production about 3-fold above that of the parent strain. When the enzyme with elevated EGII content was used, an improved stonewashing effect on denim fabric was achieved. A T. reesei strain producing high amounts of EGI and -II activities without CBHI and -II was constructed by replacing the cbh2 locus with the coding region of the egl2 gene in the EGI-overproducing CBHI-negative strain. Production of endoglucanase activity by the EG-transformant strain was increased fourfold above that of the host strain. The filter paper-degrading activity of the endoglucanase-overproducing strain was lowered to below detection, presumably because of the lack of cellobiohydrolases.

FIG. 1.

Restriction map of the plasmid pALK537. The egl2 cDNA is exactly joined to the cbh1 promoter. A 9.2-kb NotI fragment was isolated from the plasmid for transformation.

FIG. 2.

Restriction map of the plasmid pALK540. An 11.6-kb ClaI-PvuI fragment was isolated from the plasmid for transformation.

FIG. 3.

Southern analysis of transformants ALKO3530 and ALKO3574 in the which cbh1 locus has been replaced with the 9.2-kb NotI fragment from pALK537. (A) Genomic DNA was digested with XhoI. Hybridization was done with a cbh1 probe. Lane 1, VTT-D-79125; lane 2, ALKO3530; lane 3, ALKO3574; lane 4, molecular weight marker λHindIII. (B) Genomic DNA was digested with XhoI. Hybridization was performed with the 9.2-kb NotI fragment used for the transformations. Lanes 1 and 2, molecular weight markers λHindIII and λEcoRI-HindIII; lane 3, ALKO3530; lane 4, ALKO3574; lane 5, VTT-D-79125. (C) Schematic presentation of the organization of the cbh1 chromosomal locus in the host strain and the transformants, showing the XhoI cleavage site.

FIG. 4.

Southern analysis of the transformant ALKO3529 and the host strain VTT-D-79125. (A) Genomic DNA was digested with XhoI. Hybridization was done with a cbh1 probe. Lane 1, VTT-D-79125; lane 2, ALKO3529; lanes 3 and 4, molecular weight markers λHindIII and λEcoRI-HindIII. (B) Genomic DNA was digested with PvuI. Hybridization was done with an egl2 probe. Lane 1, molecular weight marker λHindIII; lane 2, ALKO3529; lane 3, molecular weight marker λHindIII; lane 4, VTT-D-79125.

FIG. 5.

SDS-PAGE of the samples from the culture supernatants of host strain VTT-D-79125 and the EGII transformants ALKO3529, ALKO3530, and ALKO3574. A total of 20 μg of total secreted protein was loaded in each lane. Lane 1, VTT-D-79125; lane 2, ALKO3529; lane 3, ALKO3530; lane 4, ALKO3574; lane 5, 3 μg of purified EGII protein.

FIG. 6.

Southern analysis of the transformant ALKO3528 and the host strain ALKO2698. (A) Genomic DNA was digested with PstI. Hybridization was done with a cbh2 probe. Lane 1, molecular weight marker λHindIII; lane 2, ALKO2698; lane 3, ALKO3528. (B) Genomic DNA was digested with BglII or XbaI-SmaI. Hybridization was done with an egl2 probe. Lane 1, molecular weight marker λHindIII; lane 2, ALKO2698 digested with BglII; lane 3, ALKO3528 digested with BglII; lane 4, ALKO2698 digested with XbaI-SmaI; lane 5, ALKO3528 digested with XbaI-SmaI. (C) Schematic presentation of the organization of the cbh2 locus in the host strain and the ALKO3528 transformant strain showing different cleavage sites and the fragment sizes from the cbh2 locus when probing with the egl2 probe.