Inhibition of Monoamine Oxidase by Anithiactins from Streptomyces sp.

Monoamine oxidase (MAO) is found in most cell types and catalyzes the oxidation of monoamines. Three anithiactins (A-C, modified 2-phenylthiazoles) isolated from Streptomyces sp. were tested for inhibitory activity of two isoforms, MAO-A and MAO-B. Anithiactin A was effective and selective for the inhibition of MAO-A, with an IC50 value of 13.0 µM; however, it was not effective for the inhibition of MAO-B. Anithiactins B and C were weaker inhibitors for MAO-A and MAO-B. Anithiactin A was a reversible and competitive inhibitor for MAO-A with a Ki value of 1.84 µM. The hydrophobic methyl substituent in anithiactin A may play an important role in the inhibition of MAO-A. It is suggested that anithiactin A is a selective reversible inhibitor for MAO-A, with moderate potency, and can be considered a new potential lead compound for further development of novel reversible inhibitors for MAO-A.

Characterization of Novel Family IV Esterase and Family I.3 Lipase from an Oil-Polluted Mud Flat Metagenome.

Two genes encoding lipolytic enzymes were isolated from a metagenomic library constructed from oil-polluted mud flats. An esterase gene, est3K, encoded a protein of 299 amino acids (ca. 32,364 Da). Est3K was a family IV esterase with typical motifs, HGGG, and HGF. Although est3K showed high identity to many genes with no information on their enzymatic properties, Est3K showed the highest identity (36 %) to SBLip5.1 from forest soil metagenome when compared to the enzymes with reported properties. A lipase gene, lip3K, encoded a protein of 616 amino acids (ca. 64,408 Da). Lip3K belonged to family I.3 lipase with a C-terminal secretion signal and showed the highest identity (93 %) to the lipase of Pseudomonas sp. MIS38. The presence of several newly identified conserved motifs in Est3K and Lip3K are suggested. Both Est3K and Lip3K exerted their maximal activity at pH 9.0 and 50 °C. The activity of Lip3K was significantly increased by the presence of 30 % methanol. The ability of the enzymes to retain activities in the presence of methanol and the substrates may offer a merit to the biotechnological applications of the enzymes such as transesterification. The activity and the thermostability of Lip3K were increased by Ca(2+). Est3K and Lip3K preferred p-nitrophenyl butyrate (C4) and octanoate (C8), respectively, as the substrate and acted independently on the substrates with no synergistic effect.

Characterization of a GH family 8 ß-1,3-1,4-glucanase with distinctive broad substrate specificity from Paenibacillus sp. X4.

A ß-1,3-1,4 glucanase gene of Paenibacillus sp. X4, bglc8H, was cloned and characterized. BGlc8H was predicted to be a protein of 409 amino acid residues, including a signal peptide of 31 amino acids. The mature enzyme was predicted to have 378 amino acid residues; its [corrected] molecular mass and pI were estimated as 41,561 Da and 7.61, respectively. BGlc8H belongs to glycoside hydrolase family 8 (GH8). Site-directed mutants of Glu95 and Asp156 of BGlc8H showed a near-complete loss of activity, indicating that they are catalytically-active residues. Unlike other GH8 members, BGlc8H had broad substrate specificity and hydrolyzed barley-ß-D-glucan > chitosan > carboxymethyl-cellulose > and lichenan. BGlc8H had a lower ratio of lichenase/barley-ß-D-glucanase activities compared to GH16 enzymes. BGlc8H was optimally active at pH 5 and 50 °C, except for barley-ß-D-glucanase (40 °C) and chitosanase (pH 7) activities. BGlc8H hydrolyzed cello-oligosaccharides (G3-G6) to G3 and G2 but not to G1. Ca(2+) increased the activity and thermostability of BGlc8H for lichenan suggesting its use for the saccharification of cellulosic biomass.

Production of XynX, a large multimodular protein of thermoanaerobacterium sp., by protease-deficient Bacillus subtilis strains [corrected].

XynX of Thermoanaerobacterium sp. [corrected] is a large, multimodular xylanase of 116 kDa. An Escherichia coli transformant carrying the entire xynX produced three active truncated xylanase species of 105, 85, and 64 kDa intracellularly. The Bacillus subtilis WB700 transformant with the xynX, a strain deficient in seven proteases including Vpr, secreted two active truncated xylanase species of 65 and 44 kDa. The B. subtilis WB800 transformant with xynX, a strain deficient in eight proteases including Vpr and WprA, secreted more active enzymes, 8.46 U ml(-1), mostly in the form of 105 and 85 kDa, than the WB700 transformant, 6.93 U ml(-1). This indicates that the additional deletion of wprA enabled the WB800 to secrete XynX in its intact form. B. subtilis WB800 produced more total enzyme activity than E. coli (1,692 ± 274 U vs. 141.9 ± 27.1 U), and, more importantly, secreted almost all the enzyme activity. The results suggest the potential use of B. subtilis WB800 as a host system for the production of large multimodular proteins.

Characterization of xyn10J, a novel family 10 xylanase from a compost metagenomic library.

A gene encoding an extracellular xylanase was cloned from a compost metagenomic library. The xylanase gene, xyn10J, was 1,137 bp in length and was predicted to encode a protein of 378 amino acid residues with a putative signal peptide of 27 amino acid residues. The molecular mass of the mature Xyn10J was calculated to be 39,882 Da with a pI of 6.09. Xyn10J had a motif GVKVHFTEMDI characteristic of most members of glycosyl hydrolase family 10. The amino acid sequence of Xyn10J showed 60.0% identity to that of XynH, a xylanase from an uncultured soil bacterium and 55% identity to XylC of Cellvibrio mixtus. Site-directed mutagenesis of the expected active site based on the sequence analysis indicated that an aspartic acid residue (Asp207), in addition to the identified catalytic residues Glu165 and Glu270, plays a crucial role for the catalytic activity. The purified Xyn10J had a mass of about 40 kDa and was optimally active at pH 7.0 and 40 °C. Xyn10J hydrolyzed beechwood xylan > birchwood xylan > oat spelt xylan > arabinoxylan. Xyn10J hydrolyzed xylotetraose and xylohexaose exclusively to xylobiose, xylopentaose, and xylotriose mainly to xylobiose with transglycosylation activity. The saccharification of reed (Phragmites communis) powder by commercial enzymes was significantly increased by the addition of a small amount of Xyn10J to the commercial preparation. Xyn10J is the first xylanase screened directly from a compost metagenomic library, and the enzyme has the potential to be used in the conversion of biomass to fermentable sugars for biofuel production.

Chitinibacter suncheonensis sp. nov., a chitinolytic bacterium from a mud flat in Suncheon Bay.

A chitinolytic bacterium, designated strain SK16(T), was isolated from a mud flat in Suncheon Bay, Republic of Korea. Strain SK16(T) is Gram-negative, strictly aerobic, motile by a polar flagellum, and short rod-shaped. Phylogenetic analyses based on 16S rRNA gene sequences showed that the strain belonged to the genus Chitinibacter and was most closely related to Chitinibacter tainanensis S1(T) (98.2% similarity). DNA-DNA hybridization analyses showed a low association value of 20.45±4.08% between them. The major cellular fatty acids, the G+C content of the genomic DNA, and the predominant quinone of the strain were summed feature 3 (iso-C(15:0) 2-OH and/or C(16:1) ω7c; 50.5%) and C(12:0) (12.5%), 52.26 mol%, and Q-8, respectively. Based on the phylogenetic, chemotaxonomic, and phenotypic properties, strain SK16(T) represents a novel species of the genus Chitinibacter, for which the name Chitinibacter suncheonensis sp. nov. is proposed. The type strain is SK16(T) (=KCTC 23839(T) =DSM 25421(T)).

Molecular cloning and characterization of a novel family VIII alkaline esterase from a compost metagenomic library.

A metagenomic library was constructed from completely fermented compost using a fosmid vector. From a total of 23,400 clones, 19 esterase-positive clones were selected on LB plates containing 1% glyceryl tributyrate as the substrate. The esterase gene of an esterase-positive clone, est2K, was on an ORF of 1299 bp and encoded a protein of 432 amino acids. Est2K had a SMTK motif and was a family VIII esterase. Unlike most family VIII esterases, Est2K had a signal peptide of 27 amino acids. The molecular mass and pI of the mature Est2K was calculated to be 44,668 Da and 4.48, respectively. The amino acid sequence of Est2K showed 72% identity with that of EstC, an esterase of an uncultured bacterium from leachate. The purified Est2K was optimally active at pH 10.0 and 50 degrees C. Est2K was stable in the presence of 30% methanol and exhibited a 2.4-fold higher activity in the presence of 5% methanol than in the presence of 1% isopropanol. Est2K preferred short to medium length p-nitrophenyl esters, especially p-nitrophenyl butyrate, as the substrate. Est2K did not hydrolyze beta-lactam antibiotics ampicillin and nitrocefin, even though Est2K showed the highest similarity to EstC.

The role of carbohydrate-binding module (CBM) repeat of a multimodular xylanase (XynX) from Clostridium thermocellum in cellulose and xylan binding.

A non-cellulosomal xylanase from Clostridium thermocellum, XynX, consists of a family-22 carbohydratebinding module (CBM22), a family-10 glycoside hydrolase (GH10) catalytic module, two family-9 carbohydrate-binding modules (CBM9-I and CBM9-II), and an S-layer homology (SLH) module. E. coli BL21(DE3) (pKM29), a transformant carrying xynX', produced several truncated forms of the enzyme. Among them, three major active species were purified by SDS-PAGE, activity staining, gel-slicing, and diffusion from the gel. The truncated xylanases were different from each other only in their C-terminal regions. In addition to the CBM22 and GH10 catalytic modules, XynX(1) had the CBM9-I and most of the CBM9-II, XynX(2) had the CBM9-I and about 40% of the CBM9-II, and XynX(3) had about 75% of the CBM9-I. The truncated xylanases showed higher binding capacities toward Avicel than those toward insoluble xylan. XynX(1) showed a higher affinity toward Avicel (70.5%) than XynX(2) (46.0%) and XynX(3) (42.1%); however, there were no significant differences in the affinities toward insoluble xylan. It is suggested that the CBM9 repeat, especially CBM9-II, of XynX plays a role in xylan degradation in nature by strengthening cellulose binding rather than by enhancing xylan binding.

In vivo gene therapy of type I diabetic mellitus using a cationic emulsion containing an Epstein Barr Virus (EBV) based plasmid vector.

A cationic emulsion containing an insulin expression plasmid was prepared for the treatment of type 1 diabetic mellitus (DM) in vivo. A rat proinsulin-1 gene was inserted to EBV-based plasmid vectors containing CAG promoter. Cationic emulsion composed of DOTAP and squalene was complexed with the plasmid DNA. An intravenous injection of cationic emulsion containing proinsulin gene decreased blood glucose levels for 7 days within normal range. The cationic emulsion exerted more profound effect on blood glucose levels compared to naked DNA. RT-PCR results confirmed that the proinsulin was expressed in several organs containing liver, lung, spleen, and kidney. The refractory response was invoked by multiple injections of naked DNA or cationic emulsion/DNA complex, which was later proven to be an immune response against expressed proinsulin. Therefore, the cationic emulsion showed a promising result as a novel insulin gene therapy vehicle by decreasing blood glucose level for a month.