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Heterologous Expression And Modification Of Laccase From Panus Rudis

Posted on:2006-01-06Degree:DoctorType:Dissertation
Country:ChinaCandidate:J Q YangFull Text:PDF
GTID:1100360155957493Subject:Microbiology
Abstract/Summary:PDF Full Text Request
Laccase is a multicopper-containing enzyme and has many potential application in industry. But they are generally difficulty to expression in nonfungal system in an active form.because a series of post-translational modifications such as glycosylation exist in synthesis and secretion. At the same time laccases have some limited characters such as low reduction potential and less-thermostable. All of this block the widely application in industry.A new laccase produceded by the white rot fungus Panus rudis has been purified and chanracterized enzymologically. The cDNA encoding the laccase was cloned and sequenced. Panus rudis laccase shows good thermostability and is valuable for further studies on laccase catalytic mechanisms and industry applications.In this study, a modified gene 1ccT obtained by adding 15 nucleotides which high usage in yeast to 3' terminal of the cDNA encoding the laccase from Panus rudia using PCR method. Then the gene of 1cc and lccT were cloned into the expression vector pPICZaA and transformed into the X-33, respectively. The cultivition temperature and the concentation of copper ions in growth medium are two important factors influenced the activity of laccase. In order to obtain higher activity of recombinant laccase, the parameter of the two factor were optimise .The result showed the copper concentration of 0.3mM and the cultivition temperature at 20℃ produced the highest level laccase. Comparison of the activity level of transformant (pPICZαA/lcc-X-33) and (pPICZαA/lccT-X-33) expression in this condition, the highest activity (value reached 192U L~-1) of transformant (pPICZaA/lccT-X-33) was 4.5-fold higher than that(value reached 42.9U L~-1) of (pPICZaA/lcc-X-33).It suggested that the C-terminal processing of laccase may play a important role in activition of laccase.The recombinant LCCT was purified to electrophoretic homogeneity by sequential steps of gel filtration and ion-exchange charomatography. The special activity of the purified protein at pH 4.5 was 3.68U mg~-1, the apparent molecular mass was approximately 61.6 kDa determined by SDS-PAGE. With ABTS as the substrate, the enzyme optimal pH is 2.6, the optimal temperature is 45℃, the K_m is 0.16mM and V_max is 20.04 μM min~-1 .The LCCT wasrelatively stable after lh at 50°C. Over the same period of time residual activity decrease to approximately 71% of the original value when incubated at 50°C. The residual activity decreaseed to 50% of the original value when incubated at 60 °C for 8min .To improvement of the activity of laccase, the gene lccT was modified using site-directed mutation and error-prone PCR separately.With the site-directed mutation, the Asp took place of the Glu in position 459 belonged to a pentapeptide segment located near the type-I Cu site in LCCT. The mutant LCCTM459 was purified like above steps. The special activity of the purified protein at pH 4.5 was 8.54U mg"', the apparent molecular mass was approximately 61.5 kDa determined by SDS-PAGE. With ABTS as the substrate, the enzyme optimal pH is 3.0, the optimal temperature is 45°C, the Km is 0.16mM and Vmax is 20.04 uM min"1 .The LCCTM459 was relatively stable after lh below 50°C. Over the same period of time residual activity decrease to approximately 43% of the original value when incubated at 50 °C. The residual activity decreaseed to 50% of the original value when incubated at 60 °C for8minUsing error-prone PCR random mutagenesis, the mutated gene lccT were expressed in pichia pastoris and the variants were screened over the activity using a plate methed. A higher activity mutant was isolated.and the enzyme (LCCTE) were isolated and characterized. The LCCTE had three amino acid substitution (Asnl93Ser, Asn205Asp, Ala304Thr) Its special activity of the purified protein at pH 4.5 was 16.17U mg'1, the apparent molecular mass was approximately 59.8kDa determined by SDS-PAGE. With ABTS as the substrate, the enzyme optimal pH is3.6, the optimal temperature is 55°C, the Km is 0.15mM and Vmax is 74.07 uM min'1 .The LCCTE was relatively stable after lh below 50°C. Over the same period of time residual activity decrease to approximately 70% of the original value when incubated at 50°C.The residual activity decreaseed to 50% of the original value when incubated at 60°C for15min.Compareation with the LCCT and LCCTM459, LCCTE had the highest special activity which is 2.3-fold and 4.4-fold than that of LCCTM459 and LCCT. Tts optimal temperature improved ten degree and its optimal pH shifted 1 unit higer. But its...
Keywords/Search Tags:laccase, modification, site-directed mutation, error-prone PCR, pichia pastoris, Escherichia coli
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