Improvement of enzyme activity of ß-1,3-1,4-glucanase from Paenibacillus sp. X4 by error-prone PCR and structural insights of mutated residues.

ß-1,3-1,4-Glucanase (BGlc8H) from Paenibacillus sp. X4 was mutated by error-prone PCR or truncated using termination primers to improve its enzyme properties. The crystal structure of BGlc8H was determined at a resolution of 1.8 Å to study the possible roles of mutated residues and truncated regions of the enzyme. In mutation experiments, three clones of EP 2-6, 2-10, and 5-28 were finally selected that exhibited higher specific activities than the wild type when measured using their crude extracts. Enzyme variants of BG2-6, BG2-10, and BG5-28 were mutated at two, two, and six amino acid residues, respectively. These enzymes were purified homogeneously by Hi-Trap Q and CHT-II chromatography. Specific activity of BG5-28 was 2.11-fold higher than that of wild-type BGwt, whereas those of BG2-6 and BG2-10 were 0.93- and 1.19-fold that of the wild type, respectively. The optimum pH values and temperatures of the variants were nearly the same as those of BGwt (pH 5.0 and 40 °C, respectively). However, the half-life of the enzyme activity and catalytic efficiency (k cat/K m) of BG5-28 were 1.92- and 2.12-fold greater than those of BGwt at 40 °C, respectively. The catalytic efficiency of BG5-28 increased to 3.09-fold that of BGwt at 60 °C. These increases in the thermostability and catalytic efficiency of BG5-28 might be useful for the hydrolysis of ß-glucans to produce fermentable sugars. Of the six mutated residues of BG5-28, five residues were present in mature BGlc8H protein, and two of them were located in the core scaffold of BGlc8H and the remaining three residues were in the substrate-binding pocket forming loop regions. In truncation experiments, three forms of C-terminal truncated BGlc8H were made, which comprised 360, 286, and 215 amino acid residues instead of the 409 residues of the wild type. No enzyme activity was observed for these truncated enzymes, suggesting the complete scaffold of the α6/α6-double-barrel structure is essential for enzyme activity.

Characterization of a Novel Alkaline Family VIII Esterase with S-Enantiomer Preference from a Compost Metagenomic Library.

A novel esterase gene, est7K, was isolated from a compost metagenomic library. The gene encoded a protein of 411 amino acids and the molecular mass of the Est7K was estimated to be 44,969 Da with no signal peptide. Est7K showed the highest identity of 57% to EstA3, which is an esterase from a drinking water metagenome, when compared with the enzymes with reported properties. Est7K had three motifs, SMTK, YSV, and WGG, which correspond to the typical motifs of family VIII esterases, SxxK, Yxx, and WGG, respectively. Est7K did not have the GxSxG motif in most lipolytic enzymes. Three additional motifs, LxxxPGxxW, PLGMxDTxF, and GGxG, were found to be conserved in family VIII enzymes. The results of the phylogenetic analysis and the alignment study suggest that family VIII enzymes could be classified into two subfamilies, VIII.1 and VIII.2. The purified Est7K was optimally active at 40°C and pH 10.0. It was activated to exhibit a 2.1-fold higher activity by the presence of 30% methanol. It preferred short-length p-nitrophenyl esters, particularly p-nitrophenyl butyrate, and efficiently hydrolyzed glyceryl tributyrate. It did not hydrolyze ß-lactamase substrates, tertiary alcohol esters, glyceryl trioleate, fish oil, and olive oil. Est7K preferred an Senantiomer, such as (S)-methyl-3-hydroxy-2-methylpropionate, as the substrate. The tolerance to methanol and the substrate specificity may provide potential advantage in the use of the enzyme in pharmaceutical and other biotechnological processes.

Characterization of an Alkaline Family I.4 Lipase from Bacillus sp. W130-35 Isolated from a Tidal Mud Flat with Broad Substrate Specificity.

A gene encoding lipolytic enzyme, lip7-3, was isolated from Bacillus sp. W130-35 isolated from a tidal mud flat. The gene encoded a protein of 215 amino acids with a signal peptide composed of 34 amino acid residues. Lip7-3 belonged to the family I.4 lipase and showed its maximal activity at pH 9.0 and 60°C. Its activity increased in the presence of 30% methanol and, remarkably, increased as well to 154.6% in the presence of Ca(2+). Lip7-3 preferred pnitrophenyl octanoate (C8) as a substrate and exhibited broad specificity for short- to longchain fatty acid esters. Additionally, Lip7-3 showed a low degree of enantioselectivity for an S-enantiomer (e.g., (S)-methyl-3-hydroxy-2-methylpropionate). It efficiently hydrolyzed glyceryl tributyrate, but did not hydrolyze glyceryl trioleate, fish oil, or olive oil. Its substrate specificity and activation by the solvent might offer a merit to the biotechnological enzyme applications like transesterification in the production of biodiesel.

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.