Aspergillus niger as an expression system for heterologous expression of ROL and BTL2
Dinçer, Sedef (2010) Aspergillus niger as an expression system for heterologous expression of ROL and BTL2. [Thesis]
Official URL: http://192.168.1.20/record=b1302915 (Table of Contents)
Lipases are esterases that hydrolyze lipids to fatty acids and glycerides. These enzymes have a variety of functions, thus applications, making them attractive targets to be studied. In this study, Bacillus thermocatenulatus lipase 2 (BTL2) and Rhizopus oryzae lipase (ROL) were cloned in pAL85 expression vector under control of constitutive pkiA promoter and trpC terminator. Aspergillus niger 872.11 protoplasts were transformed with these constructs. Transformation was confirmed by PCR from genomic DNA, lipase production was screened on Rhodamine-containing plates and positive strains were selected for shake flask cultures. Samples taken from the shake flask cultures were subjected to further analysis. SDS-PAGE and zymogram assay of BTL2 transformants showed that two transformants were expressing BTL2. Results of activity assay against 4-MU-caprylate and Bradford assay were also consistent with this data. But the results were not reproducible. Computational analysis showed that the enzyme was not suitable for high level expression in A. niger. No bands for ROL transformants could be detected on SDS-PAGE analysis. Activity assay against 4-MU-caprylate did not show a significant activity either. Differences in codon usage preferences between Rhizopus oryzae and Aspergillus niger were investigated in order to suggest an explanation for low efficiency.
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