Protein engineering and covalent modification of trichoderma reesei cellulases in pichia pastoris for textile biofinishing
Bayram Akçapınar, Günseli (2011) Protein engineering and covalent modification of trichoderma reesei cellulases in pichia pastoris for textile biofinishing. [Thesis]
Official URL: http://192.168.1.20/record=b1378267 (Table of Contents)
Cellulase enzymes have been extensively used for the biopolishing of cellulosic fabrics but they are inefficient to prevent pilling in viscose fabrics. Moreover, their application causes a loss in the fabric strength due to the aggressive action of the enzymes. One solution to this problem is the design and production of enzymes with increased molecular weights so that aggressive action of the cellulases would be limited to the fabric surface. In the framework of this study, cellulases and cellulase formulations that can ameliorate the problem of pilling and prevent loss of tensile strength in viscose fabrics were designed and produced . For this purpose, both protein engineering and chemical modification methods were used seperately and in combination to obtain cellulases with desired properties. Trichoderma reesei Endoglucanase I (EGI), Endoglucanase III (EGIII), Cellobiohydrolase I (CBHI) enzymes were successfully cloned and expressed in Pichia pastoris under the control of AOX1 promoter to mg/L quantities. A loop mutant of EGI, (EGI_L5) was prepared by introduction of a ten aminoacid long loop by molecular modelling and site directed mutagenesis for the creation of hotspots for directed crosslinking of the enzyme. The mutant enzyme was crosslinked using crosslinked enzyme aggregate (CLEA) technology. The effect of codon optimization on EGI production was analyzed. A mutant of EGI was prepared by inserting a second catalytic domain to EGI and thereby forming a bicatalytic mutant of EGI (EGI_BC) with increased molecular weight. All of the recombinant enzymes were produced in a laboratory scale fermenter and characterized. A commercial cellulase preparation was crosslinked using CLEA technology and fractionated according to the particle size. The effects of native, engineered and chemically modified cellulases on viscose fabrics were evaluated. It was found that commercial cellulase preparation crosslinked using CLEA technology, recombinant EGI and EGI_L5 produced in P. pastoris improved the pilling values of viscose fabrics by 20 % without much loss in the strength of the fabrics.
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