A combined approach of sequence-based screening from metagenomic soil DNA and subsequent in silico screening was established to identify novel trehalose synthases (TS, EC 5.4.99.16). Metagenomic DNA was isolated from diverse soil samples and used as template for PCR-based screening targeted against conserved regions of trehalose synthases. This resulted in four metagenomic TS-like fragments with broad sequence diversity (41 - 67% identity to each other). The encoded open reading frames were used as templates for further in silico screening. Two trehalose synthases were discovered using this novel approach and their enzymatic properties were further investigated. The trehalose synthase from Micrococcus terreus MtTS exhibited a broad pH optimum between 6.5 and 7.5 with highest reaction velocity at 35 degrees C and a protruding stability at this temperature (t(1/2) = 50 h). Characteristic of enzymes from thermophilic organisms, the trehalose synthase from Thermobaculum terrenum had a distinct temperature optimum at 50 degrees C, exhibiting also a prominent half time with t(1/2) = 45 h at pH 6.5. Both bioconversions resulted in final trehalose levels of 60%, whereas TtTS produced reduced amounts of the byproduct glucose (10%) compared with MtTS (15%), which is favorable for trehalose production. This combined screening approach intended to circumvent the bottleneck of metagenomic enzyme mining, regarding time and cost of intensive screening procedures for industrial relevant biocatalysts such as trehalose synthases.