Heterologous expression and biophysical characterization of non-catalytic proteins secreted by lignocellulose-degrading fungi

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Title: Heterologous expression and biophysical characterization of non-catalytic proteins secreted by lignocellulose-degrading fungi
Author: Leamon, A K M Al Amin
Contributor: University of Helsinki, Faculty of Agriculture and Forestry, Department of Food and Environmental Sciences
Publisher: Helsingin yliopisto
Date: 2019
Language: eng
URI: http://urn.fi/URN:NBN:fi:hulib-201910153664
Thesis level: master's thesis
Discipline: Biotekniikka (EYT)
Biotechnology (EYT)
Bioteknik (EYT)
Abstract: Cellulose is a major component of plant biomass that can be used to produce bio-derived materials, chemicals, and fuels. Whereas the conversion of cellulose to chemicals and fuels is limited by the surface functionality of cellulose fibers due to their highly compact structure. A group of non-catalytic proteins including expansins, loosenins and cerato-platanins are known to alter cellulose fiber structures, presumably by breaking hydrogen bonds between cellulose microfibrils. This makes them interesting targets for the chemical and biophysical modification of plant polysaccharides. In this work, seven non-catalytic target genes were selected from the transcriptome of Phanerochaete carnosa. Pichia pastoris was transformed with expression constructs harboring the respective genes under control of the AOX1 promoter. Protein expression was analyzed by colony blot assay and small-scale expression in liquid media. Two cerato-platanins (CP1, CP4) and 3 loosenin-like proteins (LOOL7, LOOL9 and LOOL12) were successfully produced in large-scale. The identity of CP1, LOOL7, LOOL9 and LOOL12 was confirmed by MALDI-TOF MS analysis. The proteins showed no hydrolytic activity when tested on carboxymethyl cellulose, xylan or glucomannan. Defibrillation assay results of CP1, CP4, LOOL7 and LOOL9 suggested that the non-catalytic proteins lack specific weakening effect on cellulose filter paper. Considerably high protein blank absorbance of LOOL7 indicates possibility for self-assembling abilities of the protein. Incubation of filter paper with CP1 seemed to improve the hydrolytic action of cellulases on Avicel and filter paper, as shown by complementation assays. The effect of LOOLs on enzymatic cellulose hydrolysis varied over the time course of different experiments. Most notably, LOOL7 and LOOL12 were able to increase the reducing sugar release from Avicel and filter paper by cellulase at different time points. The maximum increase of cellulose conversion achieved by LOOL12 was 30% after 10 min when non-catalytic proteins and Cellic CTec2 were added simultaneously. LOOL7 showed highest conversion improvement of 21% after 6 h pre-incubation assay with filter paper. Upon prolonged pre-incubation (72 h), significant improvement of filter paper hydrolysis was noticed for CP4 and CP1, while LOOLs unable to enhance the hydrolytic activity of the tested cellulase mixture. For the first time, a comparative study of cerato-platanins and loosenin-like proteins was carried out and the findings presented in this thesis suggest that especially CP1 can be a promising accessory protein for efficient bioconversion of cellulose. Nevertheless, a deeper understanding of the structure and function of these non-catalytic proteins will be helpful in determining their potential biotechnological applications.
Subject: cellulose
non-catalytic protein
weakening effect
hydrolytic activity

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