Metagenomanalysen von zwei Habitaten mit (hemi-)cellulolytischen mikrobiellen Gemeinschaften

Abstract

The analysis of metagenomes not only can give an insight into the microbial diversity of a habitat, it can also reveal new biocatalysts by screening of metagenomic DNA-libraries. In the present dissertation, the bacterial diversity of the digestive tract of the beaver (Castor fiber albicus) was analyzed. Furthermore, a metagenomic fosmid library derived from soil samples of the Avachinsky crater (Siberia) was screened for glycoside hydrolase genes. The phylogenetic composition of the bacterial community in the appendix and large intestine of the beaver was accessed by analyzing the sequence data of amplified partial 16S rRNA genes which were sequenced using the Sanger method and pyrosequencing. By the construction of phylogenetic trees and the evaluation of the 454-pyrosequencing data set the phyla Proteobacteria, Verrucomicrobia, Firmicutes, Bacteroidetes, Fusobacteria, Cyanobacteria and Actinobacteria were identified. The predominant phylum in the appendix was the Firmicutes with over 60 % of all 16S rRNA sequences, whereas in the large intestine the Verrucomicrobia (43.5 %) and Firmicutes (37.4 %) prevailed. The metagenomic fosmid library derived from soil samples of the Avachinsky crater harbored approximately 182 Mbp DNA within 5200 E.coli fosmid library clones. The function-based screening revealed eleven clones with hydrolytic activity which contained six different (hemi-)cellulase open reading frames (ORFs). Two of these ORFs were heterologously expressed in E.coli BL21. The purified enzymes, a xylanase (Xyn1015) and a lichenase (Bga48), were biochemically characterized. The maximum hydrolytic activity of Xyn1015 was found to be at 95 °C, pH 7, and a NaCl concentration of 1 M. The enzyme showed long-term stability against thermal inactivation with a half life of 24 h at 95 °C. With beechwood xylan as a substrate a Km of 2 mg/ml and Vmax of 400 U/mg was determined (at 95 °C, pH7, and 1 M NaCl). Bga48 showed the highest hydrolytic activity at 90 °C and pH 6. The enzyme revealed a high stability against heat inactivation. After five hours at 75 °C a loss of only approximately 40 % of the enzyme`s activity was detected. Bga48 displayed a Km of 0.24 mg/ml and a Vmax of 200 U/mg (barley ß-glucan, 90 °C, pH 6).