Characterization of a novel marine microbial uricase from Priestia flexa and evaluation of the effects of CMCS conjugation on its enzymatic properties.

A novel uricase producing marine bacterium Priestia flexa alkaAU was isolated and identified. The 16S rDNA and the uricase coding gene were sequenced, analyzed and submitted to GenBank. The uricase from Priestia flexa alkaAU (PFU) was purified, determined to be 58.87 kDa, and conjugated with carboxymethyl chitosan (CMCS) by ionic gelation. CMCS conjugation had no effect on the optimum pH of PFU but decreased the optimum temperature by 10 °C. CMCS conjugation increased the specific activity of PFU by 53% at the human body temperature (37 °C) and small intestine's pH (pH 6.8). Uricase thermostabilizing ability of CMCS was significant in the range of 37-80 °C but not at lower temperatures. For improvement of the pH stability of PFU, CMCS was more effective at pHs 3-5 than pHs 6-11. CMCS increased the half-life of PFU against artificial intestinal fluid by 1.5 folds, which demonstrated the potential capability of CMCS-PFU for oral administration.

The impact of N-terminal nonessential domains on the enzymological properties of the pullulanase from a marine Bacillus megaterium.

OBJECTIVE: To determine the impact of the N-terminal nonessential domains on the enzymological properties of a pullulanase (BmP) from Bacillus megaterium strain P6. METHODS: The domains of BmP were identified by the conserved domain (CD) search online software. BmP was prepared by fermentation with the strain P6 and its N-terminal truncated form (BmNTP) was obtained by heterologous expression. Structure-property relations were analyzed by homology modeling. RESULTS: BmP showed a domain architecture consisting of CBM41a-CBM41b-X-CBM48-pulA. CBM41a-CBM41b-X was removed in BmNTP. In comparison with BmP, BmNTP was lower in pH stability, specific activity and optimum NaCl concentration, but higher in Km value, thermostability, optimum pH and temperature, and activity enhancement by NaCl. Particularly, BmNTP was active over 0-35% (w/v) NaCl concentrations and enhanced 8.7 folds in specific activity (from 17.6 to 170 U/mg) in 10% NaCl. The solvent accessible surface area (SASA) of the catalytic triad was found to be correlated positively to the substrate affinity and negatively to the optimum temperature and activity enhancement by NaCl. CONCLUSION: The impact of CBM41a-CBM41b-X on the pullulanase properties was extensive and distinguished from the previous reports. The decrease in SASA may be responsible for the enzymological changes.

Theoretical and experimental studies on the conformational changes of organic solvent-stable protease from Bacillus sphaericus DS11 in methanol/water mixtures.

If natural proteases are used in organic synthesis, they are often inactivated or give a low rate of reaction in non-aqueous or aqueous-organic media. Therefore, to reveal the molecular mechanism governing the stability of proteases in organic solvents and increase protease stability in those systems is of intriguing interest. In the present study, the activity and conformational changes of an organic solvent-stable protease (OSP) from Bacillus sphaericus DS11 in different concentrations of methanol were investigated by measuring fluorescence, UV-Vis spectra, circular dichroism (CD), and conducting molecular dynamics (MD) simulations. The OSP expanded with increasing methanol concentration. The methanol molecules were able to enter into the OSP, leading to microenvironmental changes around the aromatic amino acids. More hydrophobic groups were exposed to the solvents at high methanol concentrations, and the original hydrophobic interaction in the protein decreased, thus resulting in the secondary and tertiary structure change in the OSP. Our results provide helpful insight into the molecular mechanism of the OSP tolerance to organic solvent and indicate directions for future work to design and engineer proteases that are stable at high organic solvent concentrations.

Dextranase from Arthrobacter oxydans KQ11-1 inhibits biofilm formation by polysaccharide hydrolysis.

Dental plaque is a biofilm of water-soluble and water-insoluble polysaccharides, produced primarily by Streptococcus mutans. Dextranase can inhibit biofilm formation. Here, a dextranase gene from the marine microorganism Arthrobacter oxydans KQ11-1 is described, and cloned and expressed using E. coli DH5α competent cells. The recombinant enzyme was then purified and its properties were characterized. The optimal temperature and pH were determined to be 60°C and 6.5, respectively. High-performance liquid chromatography data show that the final hydrolysis products were glucose, maltose, maltotriose, and maltotetraose. Thus, dextranase can inhibit the adhesive ability of S. mutans. The minimum biofilm inhibition and reduction concentrations (MBIC50 and MBRC50) of dextranase were 2 U ml-1 and 5 U ml-1, respectively. Scanning electron microscopy and confocal laser scanning microscope (CLSM) observations confirmed that dextranase inhibited biofilm formation and removed previously formed biofilms.

Discovery a novel organic solvent tolerant esterase from Salinispora arenicola CNP193 through genome mining.

An esterase gene, encoding a 325-amino-acid protein (SAestA), was mined form obligate marine actinomycete strain Salinispora arenicola CNP193 genome sequence. Phylogenetic analysis of the deduced amino acid sequence showed that the enzyme belonged to the family IV of lipolytic enzymes. The gene was cloned, expressed in Escherichia coli as a His-tagged protein, purified and characterized. The molecular weight of His-tagged SAestA is ∼38 kDa. SAestA-His6 was active in a temperature (5-40 °C) and pH range (7.0-11.0), and maximal activity was determined at pH 9.0 and 30 °C. The activity was severely inhibited by Hg(2+), Cu(2+), and Zn(2+). In particular, this enzyme showed remarkable stability in presence of organic solvents (25%, v/v) with log P>2.0 even after incubation for 7 days. All these characteristics suggested that SAestA may be a potential candidate for application in industrial processes in aqueous/organic media.

Solid-state fermentation of Acanthogobius hasta processing by-products for the production of antioxidant protein hydrolysates with Aspergillus oryzae

Functional properties and antioxidative activity of a protein hydrolysate prepared from Acanthogobius hasta processing by-product protein during solid-state fermentation with Aspergillus oryzae were investigated. Overall, protease activity increased with the degree of hydrolysis (DH) decreased during solid-state fermentation. All the protein hydrolysate had excellent solubility, possessed interfacial properties, and varying degrees of antioxidant activity which were governed by their concentrations and DH, molecular weight distribution and amino acid composition. After 5 days fermentation, the DH of the protein hydrolysate was 31.23%. The protein hydrolysate had the highest total hydrophobic amino acid content, the highest DPPH scavenging activity, reducing power, and the chelating activity. The radical-scavenging activity of the hydrolysates at 6 mg/mL was 78.6%. The reducing power of protein hydrolysate at the range of 0-6 mg/mL was lower than that of BHA at the range of 0-60 µg/mL, while the chelating activity of APs was similar to that of BHA at the range of 0-60 µg/mL. Moreover, the protein hydrolysate showed good emulsifying and foaming properties over a wide pH range from 2 to 12. Therefore, solid state fermentation provided a suitable and low-cost method for converting Acanthogobius hasta processing by-product protein into antioxidant protein hydrolysates.

Purification and characterization of a novel marine Arthrobacter oxydans KQ11 dextranase.

Dextranases can hydrolyze dextran deposits and have been used in the sugar industry. Microbial strains which produce dextranases for industrial use are chiefly molds, which present safety issues, and dextranase production from them is impractically long. Thus, marine bacteria to produce dextranases may overcome these problems. Crude dextranase was purified by a combination of ammonium sulfate fractionation and ion-exchange chromatography, and then the enzyme was characterized. The enzyme was 66.2 kDa with an optimal temperature of 50°C and a pH of 7. The enzyme had greater than 60% activity at 60°C for 1h. Moreover, 10mM Co(2+) enhanced dextranase activity (196%), whereas Ni(2+) and Fe(3+) negatively affected activity. 0.02% xylitol and 1% alcohol enhanced activity (132.25% and 110.37%, respectively) whereas 0.05% SDS inhibited activity (14.07%). The thickness of S. mutans and mixed-species oral biofilm decreased from 54,340 nm to 36,670 nm and from 64,260 nm to 43,320 nm, respectively.

Improving stability of a novel dextran-degrading enzyme from marine Arthrobacter oxydans KQ11.

Dextranases can hydrolyze dextran, so they are used in the sugar industry to mitigate the milling problems associated with dextran contamination. Few studies have been carried out on the storage stability of dextranase, let alone the dextranase of Arthrobacter oxydans KQ11 isolated from sea mud samples. This study improved the storage stability of dextranase from marine A. oxydans KQ11 by adding enzymatic protective reagents (stabilizer and antiseptic). Initially, the conditions (55 °C and 30 min) for maintaining 50% dextranase activity were obtained. Then, the best stabilizers of dextranase were obtained, namely, glycerol (16%), sodium acetate (18%) and sodium citrate (20%). Results showed that p-hydroxybenzoic acid compound sodium acetate (0.05%), D-sodium isoascorbiate (0.03%), and potassium sorbate (0.05%) were the best antiseptics. Subsequent validation experiment showed that dextranase with enzymatic protective reagents maintained 70.8% and 28.96% activities at the 13th week at 25 and 37 °C, respectively.

Characterization of a marine-derived dextranase and its application to the prevention of dental caries.

The dextranase added in current commercial dextranase-containing mouthwashes is largely from fungi. However, fungal dextranase has shown much higher optimum temperature than bacterial dextranase and relatively low activity when used in human oral cavities. Bacterial dextranase has been considered to be more effective and suitable for dental caries prevention. In this study, a dextranase (Dex410) from marine Arthrobacter sp. was purified and characterized. Dex410 is a 64-kDa endoglycosidase. The specific activity of Dex410 was 11.9 U/mg at optimum pH 5.5 and 45 °C. The main end-product of Dex410 was isomaltotriose, isomaltoteraose, and isomaltopentaose by hydrolyzing dextran T2000. In vitro studies showed that Dex410 effectively inhibited the Streptococcus mutans biofilm growth in coverage, biomass, and water-soluble glucan (WSG) by more than 80, 90, and 95 %, respectively. The animal experiment revealed that for short-term use (1.5 months), both Dex410 and the commercial mouthwash Biotene (Laclede Professional Products, Gardena, CA, USA) had a significant inhibitory effect on caries (p = 0.0008 and 0.0001, respectively), while for long-term use (3 months), only Dex410 showed significant inhibitory effect on dental caries (p = 0.005). The dextranase Dex410 from a marine-derived Arthrobacter sp. strain possessed the enzyme properties suitable to human oral environment and applicable to oral hygiene products.

Optimization of antioxidant exopolysaccharidess production by Bacillus licheniformis in solid state fermentation.

Response surface methodology was applied to optimize physical and nutritional variables for the production of antioxidant exopolysaccharidess (EPSs) by Bacillus licheniformis UD061 in solid state fermentation with squid processing byproduct and maize cob meal used as a carbon and nitrogen source and solid matrix. The factors noted with Plackett-Burman design for optimization of EPSs production were NaCl, MgSO4·7H2O, and moisture level. These factors were further optimized using Box-Behnken design and response surface methodology. Using this methodology, the quadratic regression model of EPSs production was built. Maximum EPSs production was obtained under the optimal conditions of 4.08 g L(-1) NaCl, 0.71 g L(-1) MgSO4·7H2O, and 60.49% moisture level. A production of 14.68 mg gds(-1), which was well in agreement with the predicted value, was achieved by this optimized procedure.

A novel method for promoting antioxidant exopolysaccharidess production of Bacillus licheniformis.

A novel method was described for improving the production of antioxidant extracellular polysaccharides from Bacillus licheniformis. Firstly, the tolerances of the strains to the organic solvents were investigated. Wild type strain of B. licheniformis OSTK95 and mutant strain UD061 can grow in a liquid medium in the presence of organic solvents with the logP value equal to or higher than 3.5 and 3.1, respectively. Secondly, the effects of different concentrations of n-hexane and xylene treatment on the extracellular polysaccharides excretion of both strains were studied. The maximum yield of the extracellular polysaccharides of B. licheniformis OSTK95 was 68.59 mg L(-1) after treated by 10% n-hexane or 1% xylene for 3h, while the maximum yield of the extracellular polysaccharides of strain UD061 was 185.01 mg L(-1) after treated by 12.5% n-hexane or 5% xylene for 3h. Finally, the continuous passage experiment showed that the strains have high genetic stability.

An evolutionary analysis of the GH57 amylopullulanases based on the DOMON_glucodextranase_like domains.

Thermostable amylopullulanase (TAPU) is valuable in starch saccharification industry for its capability to catalyze both α-1,4 and α-1,6 glucosidic bonds under the industrial starch liquefication condition. The majority of TAPUs belong to glycoside hydrolase family 57 (GH57). In this study, we performed a phylogenetic analysis of GH57 amylopullulanase (APU) based on the highly conserved DOMON_glucodextranase_like (DDL) domain and classified APUs according to their multidomain architectures, phylogenetic analysis and enzymatic characters. This study revealed that amylopullulanase, pullulanase, andα-amylase had passed through a long joint evolution process, in which DDL played an important role. The phylogenetic analysis of DDL domain showed that the GH57 APU is directly sharing a common ancestor with pullulanase, and the DDL domains in some species undergo evolution scenarios such as domain duplication and recombination.

[Structural and functional analysis of GH57 family thermostable amylopullulanase--a review].

Amylopullulanse (E. C. 3.2. 1. 1/41) has the enzymatic activities of both alpha-amylase (E. C. 3.2. 1. 1) and pullulanase (E. C. 3. 2. 1. 41), and is classified into the glycoside hydrolase 13 and 57 families. Amylopullulanse can hydrolyze both the alpha-1,4 and alpha-1,6 glucosidic bonds and is valuable for decreasing cost, increasing both efficiency and dextrose equivalent in starch processing industry. Thermostable amylopullulanase is more valuable in starch saccharification industry due to its capability to catalyze both the liquefaction and saccharification processes under the industrial starch liquefaction condition. In addition, the special bifunctional catalytic mechanism of amylopullulanase is also of great value in enzymology research. The present review focuses on the structure and function of amylopullulanases and provides a brief overview on the latest studies.

A GH57 family amylopullulanase from deep-sea Thermococcus siculi: expression of the gene and characterization of the recombinant enzyme.

The gene encoding a new extracellular amylopullulanase (type II pullulanase) was cloned from an extremely thermophilic anaerobic archaeon Thermococcus siculi strain HJ21 isolated previously from a deep-sea hydrothermal vent. The functional hydrolytic domain of the amylopullulanase (TsiApuN) and its MalE fusion protein (MTsiApuN) were expressed heterologously. The complete amylopullulanase (TsiApu) was also purified from fermentation broth of the strain. The pullulanase and amylase activities of the three enzymes were characterized. TsiApu had optimum temperature of 95°C for the both activities, while MTsiApuN and TsiApuN had a higher optimum temperature of 100°C. The residual total activities of MTsiApuN and TsiApuN were both 89% after incubation at 100°C for 1 h, while that of TsiApu was 70%. For all the three enzymes the optimum pHs for amylase and pullulanase activities were 5.0 and 6.0, respectively. By analyzing enzymatic properties of the three enzymes, this study suggests that the carboxy terminal region of TsiApu might interfere with the thermoactivity. The acidic thermoactive amylopullulanases MTsiApuN and TsiApuN could be further employed for industrial saccharification of starch.

Isolation of new polyketide synthase gene fragments and a partial gene cluster from East China Sea and function analysis of a new acyltransferase.

Using the consensus-degenerate hybrid oligonucleotide primer polymerase chain reaction method, 26 new ketoacyl synthase (KS) fragments were isolated from a marine sediment sample in the East China Sea (ECS) and analyzed by construction of a phylogenetic tree. With a digoxigenin-labeled KS gene fragment used as a probe, a partial polyketide synthase (PKS) gene cluster was isolated and identified by hybridization screening of a marine sediment sample metagenome fosmid library constructed for this study. A new acyltransferase (AT) gene was cloned from the PKS gene cluster and heterogeneously expressed as a protein fused to maltose-binding protein (MBP). Ultraviolet spectrophotometry was used to study the binding of the MBP-AT fusion protein and single AT domain to substrates using MBP and bovine serum albumin as control proteins. Binding constants (Ka, per micromolar) were calculated and used to analyze the substrate specificity of the acyltransferase. We concluded that there are many unrevealed new PKS gene clusters in marine sediments in the ECS. The acyltransferase is presumably an acetyltransferase from a new PKS gene cluster.

Antinociceptive activity of petroleum ether fraction from the MeOH extracts of Paederia scandens in mice.

The petroleum ether fraction of MeOH extract from Paederia scandens was evaluated on anti-nociceptive activity in mice using chemical and thermal models of nociception. Given orally, the petroleum ether fraction (PEF) at doses of 20, 40 and 80mg/kg produced significant inhibitions on chemical nociception induced by intraperitoneal acetic acid and subplantar formalin or capsaicin injections and on thermal nociception in the tail-flick test and in the hot plate test. More significant inhibition of nociception was observed at dose of 80mg/kg of the petroleum ether fraction. In the pentobarbital sodium-induced sleeping time test and the open-field test, the petroleum ether fraction neither significantly enhanced the pentobarbital sodium-induced sleeping time nor impaired the motor performance, indicating that the observed anti-nociception was unlikely due to sedation or motor abnormality. Moreover, the petroleum ether fraction-induced anti-nociception in both capsaicin and formalin tests was insensitive to naloxone, but was significantly antagonized by glibenclamide. These results suggested that the petroleum ether fraction produced anti-nociception possibly related to glibenclamide-sensitive K(+)-ATP channels, which merited further studies regarding the precise site and mechanism of action. The major constituents of the petroleum ether fraction (PEF) determined by GC/MS analysis, are linoleic acid, the sterols and vitamin E. Therefore it can be suggested that they exert synergetic effects and are together responsible for the antinociceptive activity of the PEF-fraction.