| Summary: | 碩士 === 國立海洋大學 === 生物科技研究所 === 91 === The yeast Candida rugosa produces five extracellular lipases (LIP-1 ~ LIP-5) which encode 534 amino acid, showing high homology in sequence but partial difference in the substrate specificity. These observation demonstrate that substrate specificity of CRL may be relative to the various CRL sequences. We can using protein engineering to change sequence of LIP-4. In order to study the relationship of enzyme activity with CRL protein sequence.
Using homologous recombination produces thirteen chimeric new lipases NL-A ~ NL-M. Only NL-L show activity in E. coli system. In addition strategy of NL-B and NL-J show activity in Saccharomyces cerevisiae system, that modify these LIPs protein engineering and we to manipulate the LIP-4 gene with glycosylation. As comparison of NL-J, NL-JF does not show enzyme activity and its sequence only has difference of three amino acid.
Thermostability of NL-J is showed the more stable than the other CRL. The difference of NL-J with LIP-4 has twenty-eight amino acid, and they has identity of 95%. The lipase activity of NL-J is more active than LIP-4, showing increase about 1 ~ 4 fold. The lipase activity of NL-J is increased in the substrates of tributyrin (C4:0), tricapryin (C8:0), tristearin (C18:0), triolein (C18:1) and olive oil about 1.7, 2.2, 2.1, 3.3 and 4.2 fold, respectively. The esterase activity of NL-J is less active than LIP-4 but NL-J increases 2 ~ 3 fold than commercial lipase in the esterase activity. Both NL-B and NL-L only have twenty amino acid differently, and they show ninety-five percent identity. However, they are different in the substrate specificity of lipase and esterase.
Error-prone PCR is performed in the LIP-4. A LIP-4 random mutation library was generated by using Error-prone PCR, using tributyrin plate to screen, 11.25 % clones show enzyme activity. This result demonstrates that LIP-4 sequence is critical and mutation in the 88.75 % position of LIP-4 may inactivate the enzyme activity.
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