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Title: Investigation of extracellular esterase activities from thermophilic fungi, and purification and characterization of novel small enzymes
Author: Fan, Xiaolian
ISNI:       0000 0001 3456 8233
Awarding Body: University of Strathclyde
Current Institution: University of Strathclyde
Date of Award: 2001
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The existence of naturally occurring, very small enzymes (<10 kDa, microenzymes) has been established with the findings that some enzyme activities are associated with proteins smaller than 10 kDa. This work investigated the extracellular esterase activities observed in some thermophilic fungi, and purified and characterised three novel thermostable microenzymes with esterase activities. Among the nine thermophilic fungi studied, two esterase-positive organisms, Emericella nidulans and Talaromyces emersonii, exhibited the enzyme activities in less than 10 kDa fraction when grown in either malt extract medium or a synthetic medium. Two small enzymes (E40 and E32) from E. nidulans and one (T40) from T emersonii were identified, and purified to homogeneity by ultrafiltration, gel filtration and reverse-phase HPLC. Nondenaturing gel filtration showed that MWs of E40 and T40 were 1.6 kDa, and E32 was 4.1 kDa, while electrospray and MALDI mass spectrometry indicated monomeric MWs of 510.3,609.3 and 1424.7 Da for E40, T40 and E32 respectively. This was consistent with a trimer structure in solution. Sequence analysis revealed that all the three esterases had the (Gly-Pro-Hyp)n repeating unit, the characteristic of collagen. The esterase activities were associated with small diffusible factor(s). X-ray microanalysis indicated the esterases contained Zn and Al. The esterases exhibited extremely high thermostability and unusual pH stability. They were more active against short chain-length fatty acids than long ones and hydrolysed glycerol esters with 1,3 specificity. An attempt at chemical synthesis of the enzymes showed that the synthetic peptide itself was inactive. Circular dichroism spectra showed that the native esterase possessed more triple-helical conformation than the synthetic peptide.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
Keywords: Bioengineering & biomedical engineering