Regulation of Substrate Selectivity of a Lipase from Thermomyces lanuginosus by C-terminus Modification
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Abstract:
In this study, the C-terminus (I241-P256) of Thermomyces lanuginosus lipase (TLL) was replaced by the corresponding region of Aspergillus oryzae lipase (Y246-H266) through site-directed substitution, and then the mutant of TLL, named TLL_CC, was successfully expressed in P. pastoris strain X-33. The hydrolysis experiments of emulsified olive oil indicated that the substitution for C-terminus of TLL led to a reduction of 15 ℃ in optimum reaction temperature but didn’t change the pH-depedent profile of the activity, implying that long C-terminus may increase the overall flexibility of enzymes and reduce the optimum reaction temperature. Hydrolysis experiments of triolein showed that the TAG substrate remained 28.31% and 48.12% in reaction systems of TLL lipase and mutant TLL_CC, respectively. On the other hand, esterification reactions showed that the yield of TAG produced by TLL_CC was 56.67% of TLL, while the same amount of DAG was obtained. The results indicated that the substitution of C-terminus of TLL distinctly decreased its ability to hydrolyze or produce TAG, but did not affect its ability to synthesize DAG.. Thus, the substitution of amino acids on C-terminus may play important role in regulating substrate selectivity of TLL.
