Cloning, expression and characterization of novel ....

Cloning, expression and characterization of novel ....

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Author: Nele Königsfeld
Length: 122 page(s)
Written: Sep 2013
Sales Rank: - XinXii Sales Rank
Views: 1238

Category: Sciences & Research » Natural Sciences  |  Work: Dissertation
Keywords: enzymes, microorganisms, microbial consortia, breakdown of plant

Dissertation Technische Mikrobiologie

Cloning, expression and characterization of novel thermostable plant cell wall degrading enzymes

A great variety of microorganisms living closely together in so called microbial consortia are required for the effective breakdown of plant derived matter. Instead of trying to cultivate these microorganisms individually in special media, this study dealt with a metagenomic approach in order to isolate new enzymes. Two novel enzymes were identified from two different enrichment cultures.
In the first part of this study, compost samples were enriched on apple pectin at 70 °C followed by the construction of metagenomic libraries. Activity based screening led to the discovery of a novel pectate lyase gene, designated pekT. The 1308 bp gene was expressed in Escherichia coli and the recombinant enzyme, designated PekT, was purified and further characterized. It was found to have maximal activity at 70 °C under slightly acidic conditions, different from most pectate lyases described so far. The enzyme exhibited a strong dependence on Ca2+ as cofactor which could not be replaced by other divalent cations. The addition of 5 mM ethylenediaminetetraacetic acid (EDTA) resulted in complete inhibition of PekT. The enzyme preferred polygalacturonic acid (PGA) and citrus pectin as substrates (100% and 70% relative activity respectively), whereas apple pectin was degraded to a much lesser extent (2%). Determined from Michaelis-Menten plot, the Km value for PGA was 1.6 mg/ml with the Vmax value of 131 μmol/min*mg and a kcat of 108 substrate molecules per second.
In the second part, a mixed culture from a volcanic hot spring (Azores) was enriched on minimal media containing rye straw at 70 °C. A metagenomic library was constructed and screened for enzymatic activities. A xylanase gene coding for a 338 amino acid peptide was identified, cloned and expressed in E. coli. The novel xylanase, designated XylT, belongs to the glycosyl hydrolase family 10. The recombinant enzyme exhibited its maximum activity at 80 °C and pH 7.0. It was quite stable in the presence of various inhibitors and was not dependent on an ion as cofactor. The preferred substrate of XylT was beechwood xylan with a Km value of 0.7 mg/ml and a Vmax value of 5331 μmol/min*mg. The turnover number of the xylanase was 3469 substrate molecules per second. It degraded oat spelt xylan and birchwood xylan to a lesser extent (70% and 47% respectively) and it showed no activity towards cellulose. XylT exhibited a high thermostability with a half life of two hours at 70 °C.
Investigating the influence of pressure on the thermal stability of XylT, the enzyme was incubated for one hour at 80 °C under varying pressures. Under atmospheric pressure no residual xylanase activity was found after one hour incubation. However, when 5 MPa was applied, already 50% residual activity could be measured and the application of 18.5 MPa stabilized XylT nearly completely towards thermal deactivation.

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