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Dissertation zugänglich unter
Metagenome derived carbohydrate active enzymes for directed modification of polyphenols
Kohlenhydrat-aktive Enzyme aus Metagenomen zur gerichteten Modifizierung von Polyphenolen
(2013) Journal of Biotechnology 191:38-45, Applied and Environmental Microbiology 79:4551–4563
Dokument 1.pdf (3.966 KB)
Glycosyltransferasen , Glykosidasen , Metagenom , Enzym , Glykosylierung , Biotransformation , Biokonversion , Biokatalyse , Bakterien , Mikroorganism
Freie Schlagwörter (Deutsch):
Kohlenhydrat-aktive Enzyme , alpha-L-Rhamnosidasen , funktionelle Metagenomik
Streit, Wolfgang (Prof. Dr.)
Tag der mündlichen Prüfung:
Kurzfassung auf Englisch:
The functional detection of novel enzymes other than hydrolases from metagenomes is limited since only a very few reliable screening procedures are available that allow the rapid screening of large clone libraries. For the discovery of flavonoid-modifying enzymes in genome and metagenome clone libraries, we have developed a new screening system based on high-performance thin-layer chromatography (HPTLC). This metagenome extract thin-layer chromatography analysis (META) allows the rapid detection of glycosyltransferase (GT) and also other flavonoid-modifying activities. The developed screening method is highly sensitive, and an amount of 4 ng of modified flavonoid molecules can be detected. This novel technology was validated against a control library of 1,920 fosmid clones generated from a single Bacillus cereus isolate and then used to analyze more than 38,000 clones derived from two different metagenomic preparations. Thereby we identified two novel UDP glycosyltransferase (UGT) genes. The metagenome-derived gtfC gene encoded a 52-kDa protein, and the deduced amino acid sequence was weakly similar to sequences of putative UGTs from Fibrisoma and Dyadobacter. GtfC mediated the transfer of different hexose moieties and exhibited high activities on flavones, flavonols, flavanones, and stilbenes and also accepted isoflavones and chalcones. From the control library we identified a novel macroside glycosyltransferase (MGT) with a calculated molecular mass of 46 kDa. The deduced amino acid sequence was highly similar to sequences of MGTs from Bacillus thuringiensis. Recombinant MgtB transferred the sugar residue from UDP-glucose effectively to flavones, flavonols, isoflavones, and flavanones. Moreover, MgtB exhibited high activity on larger flavonoid molecules such as tiliroside.
Further, a novel alpha-L-rhamnosidase was identified. A combined sequence- and function-based analysis of a metagenomic library DNA derived from elephant feces led to the identification of a novel bacterial α-L-rhamnosidase belonging to glycoside hydrolase family 78 (GH78). The gene was designated rhaB (4,095 bp) and encoded for a putative protein of 1,364 amino acids. The C-terminal part of the enzyme revealed an amino acid (AA) sequence identity of 58% to a predicted bacterial α-L-rhamnosidase from Bacteroides nordii. Interestingly, the N-terminal region of the deduced enzyme RhaB contained a GDSL-like lipase motif and an acetyl-xylan esterase (DAP2) motif. While heterologous expression of the complete rhaB failed, subcloning of the gene identified the most active open reading frame (ORF) to be of 3,081 bp, which we designated rhaB1. The enzyme RhaB1 was overexpressed in E. coli BL21 (DE3) and was purified to an amount of 75 mg per liter of culture medium. In accordance to the intestinal origin, RhaB1 showed a preference for mesophilic conditions with an optimum activity at a temperature of 40 °C and at pH 6.5, respectively. The recombinant protein had a Km value of 0.79 mM and a specific activity vmax of 18.4 U for pNP-α-L-rhamnose, a calculated Km of 6.36 mM and Vmax of 2.9*10-3 U for naringin, and a Km of 6.75 mM and specific activity Vmax of 8.63*10-2 U for rutin, respectively. Phylogenetic analysis and amino acid domain architecture comparison revealed that RhaB1 belongs to a new subclass of bacterial B type α-L-rhamnosidases of GH 78. To our knowledge RhaB1 is the first biochemically-characterized enzyme of this subclass.