Cloning and rational modification of a cold-adapted esterase for phthalate esters and parabens degradation.

Phthalate esters (PAEs) and parabens are environmental pollutants that can be toxic to human health. Herein, a cold-adapted esterase from the Mao-tofu metagenome named Est1260 was screened for its PAE-hydrolyzing potential in cold temperatures. The results showed that purified Est1260 could degrade a variety of PAEs and parabens at temperatures as low as 0 °C. After careful analysis of the structural information and molecular docking, site-saturation mutation was conducted at the identified hotspots. Protein expression of variant A1B6 doubled, and its thermal stability significantly improved (24 times) without sacrificing activity at low temperatures. In addition, Est1260 and its variants were activated by NaCl and demonstrated resistance to high concentrations of saline (up to 5 M), making it a potential biocatalyst for bioremediation of PAE and paraben-polluted environments.

Engineering of a thermostable esterase Est816 to improve its quorum-quenching activity and the underlying structural basis.

N-acyl-homoserine lactones (AHLs) are small diffusible molecules called autoinducers that mediate cell-to-cell communications. Enzymatic degradation of AHLs is a promising bio-control strategy known as quorum-quenching. To improve the quorum-quenching activity of a thermostable esterase Est816, which had been previously cloned, we have engineered the enzyme by random mutagenesis. One of the mutants M2 with double amino acid substitutions (A216V/K238N) showed 3-fold improvement on catalytic efficiency. Based on the crystal structure determined at 2.64 Å, rational design of M2 was conducted, giving rise to the mutant M3 (A216V/K238N/L122A). The kcat/KM value of the mutant M3 is 21.6-fold higher than that of Est816. Furthermore, activity assays demonstrated that M3 reached 99% conversion of 10-µM N-octanoyl-DL-homoserine lactone (C8-HSL) to N-octanoyl- DL-homoserine (C8-Hse) in 20 min, in contrast to the 8 h required by wild type Est816. The dramatic activity enhancement may be attributed to the increased hydrophobic interactions with the lactone ring by the mutation A216V, and the reduced steric clashes between the long side chain of L122 and the aliphatic tail of HSL by the mutation L122A, according to the crystal structure. This study sheds lights on the activity-structure relationship of AHL-lactonases, and may provide useful information in engineering AHL-degrading enzymes.

Molecular cloning and characterization of a novel ß-glucosidase with high hydrolyzing ability for soybean isoflavone glycosides and glucose-tolerance from soil metagenomic library.

A novel ß-glucosidase (Bgl1269) was identified from a metagenomic library of mangrove soil by activity-based functional screening. Sequence analysis revealed that Bgl1269 encodes a protein of 422 amino acids. After being overexpressed in Escherichia coli and purified, the enzymatic properties of Bgl1269 were investigated. The recombinant enzyme displayed a pH optimum of 6.0 and a temperature optimum of 40°C, and the addition of most common metal ions (1 or 10mM) increased the enzymatic activity evidently. In addition, the enzyme showed high hydrolyzing ability for soybean isoflavone glycosides, and 0.8unit of enzyme could completely converted daidzin and genistin (0.5mg/mL) to daidzein and genistein at 40°C for 0.5h. Interestingly, Bgl1269 also exhibited a very high glucose-tolerance, with the highest inhibition constant K(i) (4.28M) among ß-glucosidases reported so far. These properties make it a good candidate in the production of soybean isoflavone aglycones after further study.

Isolation and characterization of a novel tannase from a metagenomic library.

A novel gene (designated as tan410) encoding tannase was isolated from a cotton field metagenomic library by functional screening. Sequence analysis revealed that tan410 encoded a protein of 521 amino acids. SDS-PAGE and gel filtration chromatography analysis of purified tannase suggested that Tan410 was a monomeric enzyme with a molecular mass of 55 kDa. The optimum temperature and pH of Tan410 were 30 °C and 6.4. The activity was enhanced by addition of Ca(2+), Mg(2+) and Cd(2+). In addition, Tan410 was stable in the presence of 4 M NaCl. Chlorogenic acid, rosmarinic acid, ethyl ferulate, tannic acid, epicatechin gallate and epigallocathchin gallate were efficiently hydrolyzed by recombinant tannase. All of these excellent properties make Tan410 an interesting enzyme for biotechnological application.