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A copper-inducible laccase activity was detected in Thermus thermophilus HB27. The enzyme was partially purified and separated by SDS-PAGE. After staining, a gel slice containing a approximately 53-kDa protein was excised and treated with trypsin, and the in-gel digests were analyzed by mass spectrometry. By mass fingerprinting, the peptides were found to(More)
MEGAWHOP allows for the cloning of DNA fragments into a vector and is used for conventional restriction digestion/ligation-based procedures. In MEGAWHOP, the DNA fragment to be cloned is used as a set of complementary primers that replace a homologous region in a template vector through whole-plasmid PCR. After synthesis of a nicked circular plasmid, the(More)
Metagenomics has emerged as an alternative approach to conventional microbial screening that allows exhaustive screening of microbial genomes in their natural environments. Despite the potential usefulness of this approach, functional analysis of the metagenome is often problematic because of insufficient and biased expression of the cloned genes in(More)
A reporter assay-based screening method for enzymes, which we named product-induced gene expression (PIGEX), was developed and used to screen a metagenomic library for amidases. A benzoate-responsive transcriptional activator, BenR, was placed upstream of the gene encoding green fluorescent protein and used as a sensor. Escherichia coli sensor cells(More)
A metagenomic approach was taken to retrieve catabolic operons for aromatic compounds from activated sludge used to treat coke plant wastewater. Metagenomic DNA extracted from the sludge was cloned into fosmids and the resulting Escherichia coli library was screened for extradiol dioxygenases (EDOs) using catechol as a substrate, yielding 91 EDO-positive(More)
The β-glucosidase encoded by the td2f2 gene was isolated from a compost microbial metagenomic library by functional screening. The protein was identified to be a member of the glycoside hydrolase family 1 and was overexpressed in Escherichia coli, purified, and biochemically characterized. The recombinant β-glucosidase, Td2F2, exhibited enzymatic activity(More)
Oligoxyloglucan reducing end-specific cellobiohydrolase (OXG-RCBH) is a unique exo-beta-1,4-glucanase that belongs to glycoside hydrolase family 74. The enzyme recognizes the reducing end of xyloglucan oligosaccharides and releases two glucosyl residue segments from the reducing end of the main chain. Previously, we reported that OXG-RCBH consists of two(More)
A metagenomic library of activated sludge was screened for bleomycin resistance genes. Two genes were identified that differed greatly from each other, from the genes of bleomycin-producing actinomycetes, and from those of clinical isolates. Therefore, the nonclinical environment is a rich reservoir of new resistance elements, and metagenomics can be used(More)
Geotrichum sp. M128 possesses two xyloglucan-specific glycoside hydrolases belonging to family 74, xyloglucan-specific endo-beta-1,4-glucanase (XEG) and oligoxyloglucan reducing-end-specific cellobiohydrolase (OXG-RCBH). Despite their similar amino acid sequences (48% identity), their modes of action and substrate specificities are distinct. XEG catalyzes(More)