Kentaro Miyazaki

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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 ~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 share identity(More)
The conventional method for cloning a DNA fragment is to insert it into a vector and ligate it. Although this method is commonly used, it is labor intensive because the ratio and concentrations of the DNA insert and the vector need optimizing. Even then, the resultant library is often plagued with unwanted plasmids that have no inserts or multiple inserts.(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 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)
β-glucosidases (BGLs) hydrolyze cello-oligosaccharides to glucose and play a crucial role in the enzymatic saccharification of cellulosic biomass. Despite their significance for the production of glucose, most identified BGLs are commonly inhibited by low (∼mM) concentrations of glucose. Therefore, BGLs that are insensitive to glucose inhibition have great(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)
Random point mutagenesis does not access a large fraction of protein sequence space corresponding to primarily nonconservative amino acid substitutions. The cost of this limitation during directed evolution is unknown. Random point mutagenesis over the entire gene encoding the psychrophilic protease subtilisin S41 identified a pair of residues (Lys211 and(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)