Rational design of PspAlgL to improve its thermostability and anti-biofilm activity against Pseudomonas aeruginosa.

Pseudomonas aeruginosa biofilm enhances tolerance to antimicrobials and immune system defenses. Alginate is an important component of biofilm and a virulence factor of P. aeruginosa. The degradation of alginate by alginate lyases has come to serve as an adjunctive therapeutic strategy against P. aeruginosa biofilm, but poor stability of the enzyme limited this application. Thus, PspAlgL, an alginate lyase, can degrade acetylated alginate but has poor thermostability. The 3D structure of PspAlgL was predicted, and the thermostability of PspAlgL was rationally designed by GRAPE strategy, resulting in two variants with better stability. These variants, PspAlgLS270F/E311P and PspAlgLG291S/E311P, effectively degraded the alginate in biofilm. In addition, compared with PspAlgL, these variants were more efficient in inhibiting biofilm formation and degrading the established biofilm of P. aeruginosa PAO1, and they were also able to destroy the biofilm attached to catheters and to increase the sensitivity of P. aeruginosa to the antibiotic amikacin. This study provides one potential anti-biofilm agent for P. aeruginosa infection.

[Spatial and temporal expression pattern of somatostatin receptor 2 in mouse].

Somatostatin (SST) is an inhibitory polypeptide hormone that plays an important role in a variety of biological processes. Somatostatin receptor 2 (SSTR2) is the most widely expressed somatostatin receptor. However, the specific cell types expressing Sstr2 in the tissues have not been investigated. In this study, we detected the expression pattern of SSTR2 protein in mouse at different development stages, including the embryonic 15.5 days and the postnatal 1, 7, 15 days as well as 3 and 6 months, by multicolour immunofluorescence analyses. We found that Sstr2 was expressed in some specific cells types of several tissues, including the neuronal cells and astrocytes in the brain, the mesenchymal cells, the hematopoietic cells, the early hematopoietic stem cells, and the B cells in the bone marrow, the macrophages, the type Ⅱ alveolar epithelial cells, and the airway ciliated cells in the lung, the epithelial cells and the neuronal cells in the intestine, the hair follicle cells, the gastric epithelial cells, the hematopoietic stem cells and the nerve fibre in the spleen, and the tubular epithelial cells in the kidney. This study identified the specific cell types expressing Sstr2 in mouse at different developmental stages, providing new insights into the physiological function of SST and SSTR2 in several cell types.

Anti-Biofilm Enzymes-Assisted Antibiotic Therapy against Burn Wound Infection by Pseudomonas aeruginosa.

Pseudomonas aeruginosa can form biofilms at the site of burn wound, leading to infection and the failure of treatment regimens. The previous in vitro study demonstrated that a combination of the quorum-quenching enzyme AidHA147G and the extracellular matrix hydrolase PslG was effective in inhibiting biofilm and promoting antibiotic synergy. The aim of the present study was to evaluate the efficacy of this combination of enzymes in conjunction with tobramycin in treating burn wound infected with P. aeruginosa. The results showed that this treatment was effective in quorum-quenching and biofilm inhibition on infected wounds. Compared with the tobramycin treatment only, simultaneous treatment with the enzymes and antibiotics significantly reduced the severity of tissue damage, decreased the bacterial load, and reduced the expression of the inflammatory indicators myeloperoxidase (MPO) and malondialdehyde (MDA). Topical application of the enzymes also reduced the bacterial load and inflammation to some extent. These results indicate that the combined-enzyme approach is a potentially effective treatment for P. aeruginosa biofilm infections of burn wounds.

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.

Enhancement degradation efficiency of pyrethroid-degrading esterase (Est816) through rational design and its application in bioremediation.

The pervasive use of pyrethroids is seriously hazardous to the environment and even human health. Enzymatic bioremediation is potentially a rapid and environmentally friendly technology to combat the pollution of pyrethroid pesticides. The hydrolysis of ester linkages is the initial and critical enzymatic step in microbial degradation pathways. Here, the versatile and thermostable esterase Est816 was cloned and its new function, pyrethroid-hydrolysis activity, was expanded. To further improve its pyrethroid-hydrolysis ability, Est816 was modified by rational design. After two rounds of mutation, the best-performing mutant, Est816A216V/K238N/M97V, was obtained, which could completely degrade 1 mg/L λ-cyhalothrin, cypermethrin, and deltamethrin within 20 min, and efficiently degrade fenvalerate, reaching over 80% conversion. Degradation activity analyses showed that three substitutions (A216V, K238 N and M97V) were beneficial for enhancing the activity of Est816. Enzymatic characterization showed that Est816A216V/K238N/M97V inherited broad substrate specificity and possessed excellent stability and adaptability over wide ranges of temperature and pH, which is essential for bioremediation in frequently changing conditions. Furthermore, Est816A216V/K238N/M97V had the best degradation effect on all four pyrethroid residues in Panax notoginseng root, with more than 87% conversion after 24 h. Pyrethroid residues in tea, cucumber, and soil were reduced by more than 76%, 80%, and 76%, respectively. Taken together, these findings highlight the great potential of Est816A216V/K238N/M97V in the bioremediation of pyrethroid-contaminated soil and agricultural products.

Targeting Multidrug-Recalcitrant Pseudomonas aeruginosa Biofilms: Combined-Enzyme Treatment Enhances Antibiotic Efficacy.

Pseudomonas aeruginosa is an opportunistic pathogen that forms biofilms during infection, resulting in recalcitrance to antibiotic treatment. Biofilm inhibition is a promising research direction for the treatment of biofilm-associated infections. Here, a combined-enzyme biofilm-targeted strategy was put forward for the first time to simultaneously prevent biofilm formation and break down preformed biofilms. The N-acylhomoserine lactonase AidH was used as a quorum-sensing inhibitor and was modified to enhance the inhibitory effect on biofilms by rational design. Mutant AidHA147G exerted maximum activity at the human body temperature and pH and could reduce the expression of virulence factors as well as biofilm-related genes of P. aeruginosa. Subsequently, the P. aeruginosa self-produced glycosyl hydrolase PslG joined with AidHA147G to disrupt biofilms. Interestingly, under the combined-enzyme intervention for P. aeruginosa wild-type strain PAO1 and clinical strains, no biofilm was observed on the bottom of NEST glass-bottom cell culture dishes. The combination strategy also helped multidrug-resistant clinical strains change from resistant to intermediate or sensitive to many antibiotics commonly used in clinical practice. These results demonstrated that the combined-enzyme approach for inhibiting biofilms is a potential clinical treatment for P. aeruginosa infection.

Enhanced Production of (S)-2-arylpropionic Acids by Protein Engineering and Whole-Cell Catalysis.

Esterases are important biocatalysts for chemical synthesis. Several bHSL family esterases have been used to prepare (S)-2-arylpropionic acids with stronger anti-inflammatory effects via kinetic resolution. Here, we presented the discovery of key residues that controlled the enantioselectivity of bHSL family esterases to ethyl 2-arylpropionates, through careful analysis of the structural information and molecular docking. A new bHSL family esterase, Est924, was identified as a promising catalyst for kinetic resolution of racemic ethyl 2-arylpropionates with slight (R)-stereopreference. Using Est924 as the starting enzyme, protein engineering was conducted at hotspots, and the substitution of A203 was proved to enhance the enantioselectivity. The stereopreference of the mutant M1 (A203W) was inverted to ethyl (S)-2-arylpropionates, and this stereopreference was further improved in variant M3 (I202F/A203W/G208F). In addition, the optimal variant, M3, was also suitable for the resolution of ibuprofen ethyl ester and ketoprofen ethyl ester, and their efficient (S)-isomers were synthesized. Next, the whole-cell catalyst harboring M3 was used to prepare (S)-ketoprofen. (S)-ketoprofen with 86%ee was produced by whole-cell catalyst with a single freeze-thaw cycle, and the cells could be reused for at least five cycles. Our results suggested that Est924 variants could kinetically resolve economically important racemates for industrial production and further offer the opportunity for the rational design of enzyme enantioselectivity. Moreover, it is an economical process to prepare optically pure (S)-ketoprofen and (S)-naproxen by using an engineered strain harboring M3 as the catalyst.

Efficient Enzyme-Assisted Extraction and Conversion of Polydatin to Resveratrol From Polygonum cuspidatum Using Thermostable Cellulase and Immobilized ß-Glucosidase.

Resveratrol, a bioactive compound in high quantities in Polygonum cuspidatum, has well-known health benefits. However, it mainly exists in its glycosidic form, polydatin, in plants. To increase the production of resveratrol for various uses in medicine, foods, and cosmetics, an efficient deglycosylation technique is needed for converting polydatin into resveratrol. We screened a new cellulolytic strain of Bacillus from herb compost, and we optimized parameters within the fermentation process using response surface methodology with a Box-Behnken design. The yield of cellulase reached 2701.08 U/L, corresponding to values that were 5.4 times as high as those under unoptimized conditions. The Bacillus cellulase possessed good thermostablity and was stable under both acidic and neutral conditions. The cellulase was then used in the pretreatment of P. cuspidatum root. After incubation at 50°C for 4 h with shaking at 150 rpm, the contents of piceid and resveratrol were determined to be 7.60 ± 0.15 and 9.72 ± 0.29 mg/g, respectively. To obtain complete deglycosylation, immobilized ß-glucosidase (bgl2238) was added to the cellulase-treated extracts of P. cuspidatum root to convert residual polydatin into resveratrol. After the first cycle, the contents of piceid and resveratrol were determined to be 0 and 13.69 ± 0.30 mg/g, respectively. Moreover, enzyme activity showed little loss during up to 4 consecutive cycles. These results demonstrated that the immobilized ß-glucosidase possessed high deglycosylation activity and outstanding operational stability. The mixture of Bacillus cellulase and immobilized bgl2238 appears promising as a means to increase the supply of resveratrol in the medicine market worldwide.

Characterization of a Novel, Cold-Adapted, and Thermostable Laccase-Like Enzyme With High Tolerance for Organic Solvents and Salt and Potent Dye Decolorization Ability, Derived From a Marine Metagenomic Library.

Synthetic dyes are widely used in many industries, but they cause serious environmental problems due to their carcinogenic and mutagenic properties. In contrast to traditional physical and chemical treatments, biodegradation is generally considered an environmental-friendly, efficient, and inexpensive way to eliminate dye contaminants. Here, a novel laccase-like enzyme Lac1326 was cloned from a marine metagenomic library. It showed a maximum activity at 60°C, and it retained more than 40% of its maximal activity at 10°C and more than 50% at 20-70°C. Interestingly, the laccase behaved stably below 50°C, even in commonly used water-miscible organic solvents. The enzyme decolorized all tested dyes with high decolorization efficiency. This thermostable enzyme with high decolorization activity and excellent tolerance of organic solvents and salt has remarkable potential for bioremediation of dye wastewater. It is thus proposed as an industrial enzyme.

Aii810, a Novel Cold-Adapted N-Acylhomoserine Lactonase Discovered in a Metagenome, Can Strongly Attenuate Pseudomonas aeruginosa Virulence Factors and Biofilm Formation.

The pathogen Pseudomonas aeruginosa uses quorum sensing (QS) to control virulence and biofilm formation. Enzymatic disruption of quorum sensing is a promising anti-infection therapeutic strategy that does not rely on antibiotics. Here, a novel gene (aii810) encoding an N-acylhomoserine lactonase was isolated from the Mao-tofu metagenome for the first time. Aii810 encoded a protein of 269 amino acids and was expressed in Escherichia coli BL21 (DE3) in soluble form. It showed the highest activity at 20°C, and it maintained 76.5% of activity at 0°C and more than 50% activity at 0-40°C. The optimal pH was 8.0. It was stable in both neutral and slightly alkaline conditions and at temperatures below 40°C. The enzyme hydrolyzed several ρ-nitrophenyl esters, but its best substrate was ρ-nitrophenyl acetate. Its kcat and Km values were 347.7 S-1 and 205.1 µM, respectively. It efficiently degraded N-butyryl-L-homoserine lactone and N-(3-oxododecanoyl)-L-homoserine lactone, exceeding hydrolysis rates of 72.3 and 100%, respectively. Moreover, Aii810 strongly attenuated P. aeruginosa virulence and biofilm formation. This enzyme with high anti-QS activity was the most cold-adapted N-acylhomoserine lactonase reported, which makes it an attractive enzyme for use as a therapeutic agent against P. aeruginosa infection.

Identification and immobilization of a novel cold-adapted esterase, and its potential for bioremediation of pyrethroid-contaminated vegetables.

BACKGROUND: Pyrethroids are potentially harmful to living organisms and ecosystems. Thus, concerns have been raised about pyrethroid residues and their persistence in agricultural products. To date, although several pyrethroid-hydrolyzing enzymes have been cloned, very few reports are available on pyrethroid-hydrolyzing enzymes with cold adaptation, high hydrolytic activity and good reusability, indispensable properties in practical bioremediation of pyrethroid-contaminated vegetables. RESULTS: Here, a novel gene (est684) encoding pyrethroid-hydrolyzing esterase was isolated from the Mao-tofu metagenome for the first time. Est684 encoded a protein of 227 amino acids and was expressed in Escherichia coli BL21 (DE3) in soluble form. The optimum temperature was 18 °C. It maintained 46.1% of activity at 0 °C and over 50% of its maximal activity at 4-35 °C. With the goal of enhancing stability and recycling biocatalysts, we used mesoporous silica SBA-15 as a nanometer carrier for the efficient immobilization of Est684 by the absorption method. The best conditions were an esterase-to-silica ratio of 0.96 mg/g (w/w) and an adsorption time of 30 min at 10 °C. Under these conditions, the recovery of enzyme activity was 81.3%. A large improvement in the thermostability of Est684 was achieved. The half-life (T1/2) of the immobilized enzyme at 35 °C was 6 h, 4 times longer than the soluble enzyme. Interestingly, the immobilized Est684 had less loss in enzyme activity up to 12 consecutive cycles, and it retained nearly 54% of its activity after 28 cycles, indicating excellent operational stability. Another noteworthy characteristic was its high catalytic activity. It efficiently hydrolyzed cyhalothrin, cypermethrin, and fenvalreate in pyrethroid-contaminated cucumber within 5 min, reaching over 85% degradation efficiency after four cycles. CONCLUSIONS: A novel cold-adapted pyrethroid-hydrolyzing esterase was screened from the Mao-tofu metagenome. This report is the first on immobilizing pyrethroid-hydrolyzing enzyme on mesoporous silica. The immobilized enzyme with high hydrolytic activity and outstanding reusability has a remarkable potential for bioremediation of pyrethroid-contaminated vegetables, and it is proposed as an industrial enzyme.

Directed evolution and secretory expression of a pyrethroid-hydrolyzing esterase with enhanced catalytic activity and thermostability.

BACKGROUND: Pyrethroids are potentially harmful to human health and ecosystems. It is necessary to develop some efficient strategies to degrade pyrethroid residues. Biodegradation is generally considered as a safe, efficient, and inexpensive way to eliminate environmental contaminants. To date, although several pyrethroid-hydrolyzing esterases have been cloned, there has been no report about a pyrethroid hydrolase with high hydrolytic activity, good stability, and high productivity, indispensable enzymatic properties in practical biodegradation. Almost all pyrethroid hydrolases are intracellular enzymes, which require complex extraction protocols and present issues in terms of easy inactivation and low production. RESULTS: In this study, random mutagenesis was performed on one pyrethroid-hydrolyzing esterase, Sys410, to enhance its activity and thermostability. Two beneficial mutations, A171V and D256N, were obtained by random mutagenesis and gave rise to the mutant M2. The mutant displayed ~1.5-fold improvement in the kcat/Km value and 2.46-fold higher catalytic activity. The optimal temperature was 10 °C higher than that of the wild-type enzyme (55 °C). The half-life at 40-65 °C was 3.3-310 times longer. It was surprising that M2 has a half-life of 12 h at 70 °C while Sys410 was completely inactivated at 70 °C. In addition, the desired gene was extracellularly expressed in a Pichia pastoris host system. The soluble expression level reached up to 689.7 mg/L. Remarkably, the enzyme could efficiently degrade various pyrethroids at moderate temperature for 15 min, exceeding a hydrolysis rate of 98%, which is the highest value ever reported. CONCLUSIONS: This is the first report about random mutagenesis and secretory expression of pyrethroid-hydrolyzing esterase with high-level productivity and purity in P. pastoris. Broad substrate specificity, enhanced activity and thermostability make M2 an ideal candidate for the biodegradation of pyrethroid residues.

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.

Identification and characterization of a novel thermostable pyrethroid-hydrolyzing enzyme isolated through metagenomic approach.

BACKGROUND: Pyrethroid pesticides are broad-spectrum pest control agents in agricultural production. Both agricultural and residential usage is continuing to grow, leading to the development of insecticide resistance in the pest and toxic effects on a number of nontarget organisms. Thus, it is necessary to hunt suitable enzymes including hydrolases for degrading pesticide residues, which is an efficient "green" solution to biodegrade polluting chemicals. Although many pyrethroid esterases have consistently been purified and characterized from various resources including metagenomes and organisms, the thermostable pyrethroid esterases have not been reported up to the present. RESULTS: In this study, we identified a novel pyrethroid-hydrolyzing enzyme Sys410 belonging to familyV esterases/lipases with activity-based functional screening from Turban Basin metagenomic library. Sys410 contained 280 amino acids with a predicted molecular mass (Mr) of 30.8 kDa and was overexpressed in Escherichia coli BL21 (DE3) in soluble form. The optimum pH and temperature of the recombinant Sys410 were 6.5 and 55°C, respectively. The enzyme was stable in the pH range of 4.5-8.5 and at temperatures below 50°C. The activity of Sys410 decreased a little when stored at 4°C for 10 weeks, and the residual activity reached 94.1%. Even after incubation at 25°C for 10 weeks, it kept 68.3% of its activity. The recombinant Sys410 could hydrolyze a wide range of ρ-nitrophenyl esters, but its best substrate is ρ-nitrophenyl acetate with the highest activity (772.9 U/mg). The enzyme efficiently degraded cyhalothrin, cypermethrin, sumicidin, and deltamethrin under assay conditions of 37°C for 15 min, with exceeding 95% hydrolysis rate. CONCLUSION: This is the first report to construct metagenomic libraries from Turban Basin to obtain the thermostable pyrethroid-hydrolyzing enzyme. The recombinant Sys410 with broad substrate specificities and high activity was the most thermostable one of the pyrethroid-hydrolyzing esterases studied before, which made it an ideal candidate for the detoxification of pyrethroids.