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Title:	Functional studies of fungal cutinases and protein engineering of Thielavia terrestris cutinase: insight into structural, catalytic and thermal stability
Authors:	Khadijah Ahmed Y Altammar (P59695)
Supervisor:	Farah Diba Abu Bakar, Dr.
Keywords:	Fungal cutinases Thielavia terrestris Cutin hydrolase Protein engineering Dissertations, Academic -- Malaysia
Issue Date:	2-May-2016
Description:	Cutin hydrolase is an enzyme that belongs to the serine esterase super-family and is regarded as an evolutionary bridge between esterases and lipases. Its hydrolytic activity on a variety of water-soluble esters and emulsified triglycerides makes this enzyme a potential catalyst in a variety of industries. Most enzymes however, are relatively unstable at extreme temperatures and pH, whilst their efficiency is restricted due to limitations of the substrate recognition pocket. Thus, continued screening of new genes from nature is an excellent approach to discover enzymes such as cutinases with novel potential properties. Protein engineering is another approach to enhance the properties of existing enzymes. In this study, genome mining of the mesophile Aspergillus niger and Trichoderma virens alongside the thermophile Thielavia terrestris was carried out. A total of five cDNA sequences encoding cutinases were cloned and expressed in Pichia pastoris. Of the five cDNA sequences, three were obtained from A. niger (Ancut1, Ancut2, Ancut3), one from T. virens (Tvcut) and one from T. terrestris (Ttcut). Extracellular expression of AnCUT1, AnCUT2, TvCUT and TtCUT showed that these recombinant enzymes are approximately 30, 42, 27 and 30 kDa, respectively; whilst AnCUT3 was represented by proteins of about 24 and 50 kDa. The recombinant TtCUT displayed an optimum activity between 25°C and 40°C and retained more than 50% of its initial activity for up to 60 min at temperatures from 60 to 80°C. The mesophilic cutinases showed an optimum temperature between 25°C and 40°C and retained less than 40% of its relative activity at the same condition. The thermo-tolerant cutinase showed a maximum activity between pH 7.0 and pH 8.0 whereas the mesophilic cutinases displayed an optimum activity at acidic pHs (pH 5.0-6.0). AnCUT1, AnCUT2, AnCUT3 and TtCUT prefer short carbon chain length substrates (C4, C5), whereas TvCUT has a preference towards the medium chain substrate (C10). Enzymatic degradation of polycaprolactone (PCL) synthetic polymer showed that the degradation rate of PCL by T. terrestris cutinase is the highest. Therefore, TtCUT was chosen for mutagenesis to enhance its stability and enzymatic performance. The disruption of the native short-ranged disulfide bond (C54R) resulted in a misfolded protein that accumulated in the cytoplasm as inclusion bodies. The catalytic properties of TtCUT variants were similar to those of the wild type (WT). Cutinase H157K exhibited slightly higher induced unfolding temperature and specific activity towards high molecular weight substrate (PCL) compared to the cutinase WT. The disulfide bridge variant A58C/V71C had improved unfolding temperature when compared to WT. However, this enzyme variant exhibited less enzymatic activity towards PCL. The variants designed for prevention of deamidation (N162D) did not show increased thermostability over the WT enzyme. This work illustrated the differences in structure and function between cutinases of different fungal origins. It was also shown that protein engineering techniques can improve properties of TtCUT.,Certification of Master's/Doctoral Thesis" is not available
Pages:	223
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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