BackgroundAlkaline phosphatase (AP) catalyzes the hydrolytic cleavage of phosphate monoesters under alkaline conditions and plays important roles in microbial ecology and molecular biology applications. Here, we report on the first isolation and biochemical characterization of a thermolabile AP from a metagenome.ResultsThe gene encoding a novel AP was isolated from a metagenomic library constructed with ocean-tidal flat sediments from the west coast of Korea. The metagenome-derived AP (mAP) gene composed of 1,824 nucleotides encodes a polypeptide with a calculated molecular mass of 64 kDa. The deduced amino acid sequence of mAP showed a high degree of similarity to other members of the AP family. Phylogenetic analysis revealed that the mAP is shown to be a member of a recently identified family of PhoX that is distinct from the well-studied classical PhoA family. When the open reading frame encoding mAP was cloned and expressed in recombinant Escherichia coli, the mature mAP was secreted to the periplasm and lacks an 81-amino-acid N-terminal Tat signal peptide. Mature mAP was purified to homogeneity as a monomeric enzyme with a molecular mass of 56 kDa. The purified mAP displayed typical features of a psychrophilic enzyme: high catalytic activity at low temperature and a remarkable thermal instability. The optimal temperature for the enzymatic activity of mAP was 37°C and complete thermal inactivation of the enzyme was observed at 65°C within 15 min. mAP was activated by Ca2+ and exhibited maximal activity at pH 9.0. Except for phytic acid and glucose 1-phosphate, mAP showed phosphatase activity against various phosphorylated substrates indicating that it had low substrate specificity. In addition, the mAP was able to remove terminal phosphates from cohesive and blunt ends of linearized plasmid DNA, exhibiting comparable efficiency to commercially available APs that have been used in molecular biology.ConclusionsThe presented mAP enzyme is the first thermolabile AP found in cold-adapted marine metagenomes and may be useful for efficient dephosphorylation of linearized DNA.

Cyclodipeptides (CDP) represent a diverse family of small, highly stable, cyclic peptides that are produced as secondary functional metabolites or side products of protein metabolism by bacteria, fungi, and animals. They are widespread in nature, and exhibit a broad variety of biological and pharmacological activities. CDP synthases (CDPSs) and non-ribosomal peptide synthetases (NRPSs) catalyze the biosynthesis of the CDP core structure, which is further modified by tailoring enzymes often associated with CDP biosynthetic gene clusters. In this review, we provide a comprehensive summary of CDP biosynthetic pathways and modifying enzymes. We also discuss the biological properties of some known CDPs and their possible applications in metabolic engineering.

Large-scale screening of enzyme libraries is essential for the development of cost-effective biological processes, which will be indispensable for the production of sustainable biobased chemicals. Here, we introduce a genetic circuit termed the Genetic Enzyme Screening System that is highly useful for high-throughput enzyme screening from diverse microbial metagenomes. The circuit consists of two AND logics. The first AND logic, the two inputs of which are the target enzyme and its substrate, is responsible for the accumulation of a phenol compound in cell. Then, the phenol compound and its inducible transcription factor, whose activation turns on the expression of a reporter gene, interact in the other logic gate. We confirmed that an individual cell harboring this genetic circuit can present approximately a 100-fold higher cellular fluorescence than the negative control and can be easily quantified by flow cytometry depending on the amounts of phenolic derivatives. The high sensitivity of the genetic circuit enables the rapid discovery of novel enzymes from metagenomic libraries, even for genes that show marginal activities in a host system. The crucial feature of this approach is that this single system can be used to screen a variety of enzymes that produce a phenol compound from respective synthetic phenyl-substrates, including cellulase, lipase, alkaline phosphatase, tyrosine phenol-lyase, and methyl parathion hydrolase. Consequently, the highly sensitive and quantitative nature of this genetic circuit along with flow cytometry techniques could provide a widely applicable toolkit for discovering and engineering novel enzymes at a single cell level.

Nitric oxide (NO) which is produced by inducible nitric oxide synthase (iNOS) is a pro-inflammatory radical and an important biological mediator. 1) prostaglandin E 2 (PGE 2 ), released by arachidonic acid metabolites, is also an important mediator of acute and chronic inflammation, and it is associated with the enzyme, cyclooxygenase-2 (COX-2).2) In addition to these oxygen and arachidonic acid metabolites, tumor necrosis factor-a (TNF-a) acting as a cytokine or inflammatory mediator plays a major role in various inflammatory diseases, including septic shock and rheumatoid arthritis. 3,4) Nuclear factor-kB (NF-kB) appears to play a primary role in the transcriptional regulation of these iNOS, COX-2 and TNF-a gene expressions.5) NF-kB exists as a latent form in the cytoplasm of unstimulated cells and is bound to the inhibitory protein, IkB. 6) Phosphorylation of IkB leads to its degradation and the subsequent translocation of NF-kB to the nucleus where it activates transcriptions of target genes. 7,8) It is noteworthy that the use of medicinal plants or their crude extracts in the prevention and/or treatment of several chronic diseases has been traditionally practiced in different societies worldwide. Moreover, extracts of Ginkgo biloba LINNE (Ginkoaceae) have been used for centuries in traditional Chinese medicine. Today, a standardized extract (EGb 761) is prepared from Ginko biloba leaves and is prescribed commonly in Europe for the treatment of memory disorders, obstructive arteriosclerosis, Alzheimer's disease, ischaemic heart disease, cerebral infarction, aging, and age-related macular degeneration.9-11) Several modes of action of EGb 761 have been described: (1) its effects on the blood circulation, such as its vasoregulatory activity and rheological effects on blood (decreased viscosity, anti-platelet activating factor activity) 11) ; (2) its effects on metabolism changes, such as on neuron metabolism (increased tolerance to anoxia) 9,12,13) ; (3) its inhibition of cell membrane damage caused by free radicals 11,[13][14][15] ; and (4) its gene-regulatory effects, which suggest that it has anticancer activity. 16,17) EGb 761 contains two groups as its main active constituents: 24% flavonol glycoside (ginkgo-flavonol glycosides; quercetin, kaempferol, isorhamnetin) and 6% terpene (bilobalide and ginkgolides A, B, C).18) In the present study, we prepared a standardized Ginkgo biloba extract (GBB), i.e., an extract of Ginkgo biloba leaves standardized in terms of its total terpenes (12Ϯ3%), biflavonoid (4.5Ϯ1.5%), flavonol glycoside (Ͻ8%), and proanthocyanidine (under detection limit) content. The difference between GBB and EGb 761 is that it contains a higher level of terpene, a lower level of flavonol glycoside, and no detectable proanthocyanidine.In the present study, we compared the effects of these Ginkgo biloba extracts (GBB and EGb 761) on lipopolysaccharide (LPS)-induced NO and PGE 2 release. To further explore the possible mechanisms of these inhibitions by GBB, we investigated the expression levels...