Anti-inflammatory effects of fish bone fermented using Monascus purpureus in LPS-induced RAW264.7 cells by regulating NF-κB pathway.

Fish bones are the by-products of aquatic and fishery processing, which are often discarded. However, it has been considered having health-promoting by containing many essential nutrients. This study investigates the anti-inflammatory effect of fish bone fermented by Monascus purpureus (FBF) and the NF-κB pathway regulation mechanism in lipopolysaccharides (LPS)-induced RAW 264.7 cells. FBF has inhibited the production of PGE2 (prostaglandin E2), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in LPS-induced RAW264.7 cells. The FBF has significantly inhibited mRNA expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Moreover, FBF has suppressed activation of NF-κB (nuclear factor kappa-B) by increasing IκB mRNA expression and reduced of p65, p50 mRNA expression, as well as nuclear NF-κB DNA binding activity in LPS-induced RAW 246.7 cells. These findings demonstrate that FBF has inhibited LPS-induced inflammation by subsiding the activation of NF-κB in RAW 246.7 cells, implying that FBF could be employed as a promising natural product.

Evaluation of Chemical Compositions, Antioxidant Capacity and Intracellular Antioxidant Action in Fish Bone Fermented with Monascus purpureus.

Fish bones (FBs) are aquatic by-products that are sources of antioxidant-active peptides, calcium dietary supplements, and biomedical materials. Usually, fermentation of these by-products via microorganisms brings desirable changes, enhancing their value. This study investigates the value addition of FB when fermented with Monascus purpureus (MP) for different time intervals, such as 3 days (F3) and 6 days (F6). The results indicate that the soluble protein, peptide, amino acid and total phenol content, as well as the antioxidant capacity (DPPH, ABTS+ radical scavenging activity, and relative reducing power), of F3 and F6 were significantly increased after fermentation. Furthermore, the ROS contents of F3 and F6 were reduced to a greater extent than that of hydrogen peroxide (H2O2) in Clone-9 cells. The MMP integrity, as well as the SOD, CAT, and GPx activity, of F3 and F6 were also increased significantly compared to the H2O2 in Clone-9 cells. Notably, F3 and F6 displayed significant reductions in ROS content, as well as elevate, SOD activity and MMP integrity in Clone-9 cells, when compared with the native FB. These results indicate that the FBs fermented with MP for 3 days (F3), and 6 days (F6) have antioxidant capacity, with possible applications as natural food supplements.

Production of functional peptides with inhibition ability against angiotensin I-Converting enzyme using P. pastoris expression system.

To obtain the angiotension-I converting enzyme inhibitor (ACEI), a fusion ACEI polypeptide encoded with 8 DNA sequences of GPL, GPM, IKW, IVY, IRPVQ, IWHHT, IYPRY and IAPG, which were selected and designed and cloned into pGAPZαC and then transformed into Pichia pastoris SMD1168H. After 3 days induction, the fraction with highest ACEI activity was expressed and purified using a Ni Sepharose™ 6 Fast Flow. The IC50 of recombinant ACEI polypeptide was 88.2 µM. A 128-fold increase of ACEI activity (0.69 µM) was obtained after pepsin digestion, which was equivalent to 0.022 µM of captopril. Reverse phase HPLC indicated all the 8 peptides contained in ACEI-hydrolysate after pepsin digestion.

Overexpression of Escherichia coli phytase in Pichia pastoris and its biochemical properties.

To obtain a Pichia pastoris mutant with an Escherichia coli phytase gene, which was synthesized according to P. pastoris codon preference, a mature phytase cDNA of E. coli being altered according to the codons usage preference of P. pastoris was artificially synthesized and cloned into an expression vector of pGAPZαC. The final extracellular phytase activity was 112.5 U/mL after 72 h of cultivation. The phytase, with a molecular mass of 46 kDa, was purified to electrophoretical homogeneity after Ni Sepharose 6 Fast Flow chromatography. The yield, purification fold, and specific activity were 63.97%, 26.17, and 1.57 kU/mg, respectively. It had an optimal pH and temperature of 4.0-6.0 and 50 °C, respectively, and was stable at pH 3.0-8.0 and 25-40 °C. The purified recombinant phytase was resistant to trypsin, highly inhibited by Cu(2+), Zn(2+), Hg(2+), Fe(2+), Fe(3+), phenylmethylsulfonyl fluoride, and N-tosyl-l-lysine chloromethyl ketone, but activated by Mg(2+), Ca(2+), Sr(2+), Ba(2+), glutathione, ethylenediaminetetraacetic acid, and N-ethylmaleimide. It revealed higher affinity to calcium phytate than to other phosphate conjugates.

Characterization of acidic protease from Aspergillus niger BCRC 32720.

An acid protease from the broth of a 24 h cultivated Aspergillus niger BCRC 32720 was purified to electrophoretical homogeneity by CM Sepharose FF and Sephacryl S-100 HR chromatographs. The specific activity, purification fold, and yield were 23.29 kU/mg, 2.5, and 24.2%, respectively. Molecular mass (M) and N-terminal amino acid sequence were 47.5 kDa and SKGSAVTT, whereas the pH and temperature optima were at 2.5 and 50 °C, respectively. It was stable at pH 2.0-4.0 or ≤40 °C and activated by Fe(2+) and cysteine, but partially inhibited by phenylmethanesulfonyl fluoride and tosyllysine chloromethyl ketone and highly inhibited by Ag(+), Sn(2+), Fe(3+), Sb(3+), and pepstatin A. It was considered to be an aspartic protease.

Characterization of mannanase from a novel mannanase-producing bacterium.

Locust bean gum (LBG) was employed to screen mannanase-producing bacteria. The bacterium with highest mannanase ability was identified as Paenibacillus cookii. It revealed highest activity (6.67 U/mL) when cultivated in 0.1% LBG with 1.5% soytone and 0.5% tryptone after 4 days incubation at 27 °C. Its mannanase was purified to electrophoretical homogeneity after DEAE-Sepharose and Sephacryl S-100 separation. The purified mannanase, with an N-terminus of GLFGINAY, had pH and temperature optimum at 5.0 and 50 °C, respectively, and was stable at pH 5.0-7.0, ≤ 50 °C. It was strongly activated by ß-mercaptoethanol, dithiothreitol, cysteine, and glutathione, but inhibited by Hg(2+), Cu(2+), Zn(2+), Fe(3+), PMSF, iodoacetic acid, and EDTA. According to substrate specificity study, the purified mannanase had high specificity to LBG and konjac.

Isolation of cellulase-producing bacteria and characterization of the cellulase from the isolated bacterium Cellulomonas sp. YJ5.

A cellulase-producing bacterium was isolated from soil and identified as Cellulomonas sp. YJ5. Maximal cellulase activity was obtained after 48 h of incubation at 30 degrees C in a medium containing 1.0% carboxymethyl cellulose (CMC), 1.0% algae powder, 1.0% peptone, 0.24% (NH4)2SO4, 0.20% K2HPO4, and 0.03% MgSO(4).7H2O. The cellulase was purified after Sephacryl S-100 chromatography twice with a recovery of 27.9% and purification fold of 17.5. It was, with N-terminal amino acids of AGTKTPVAK, stable at pH 7.5-10.5 and 20-50 degrees C with optimal pH and temperature of 7.0 and 60 degrees C, respectively. Cu2+, Fe2+, Hg2+, Cr3+, and SDS highly inhibited, but cysteine and beta-mercaptoethanol activated, its activity. Substrate specificity indicated it to be an endo-beta-1,4-glucanase.

Bioproperties of potent nattokinase from Bacillus subtilis YJ1.

Fibrinolytic enzyme activity was observed during cultivation of Bacillus subtilis YJ1 in a medium containing 1% skim milk, 1% rice husk, 0.5% NaCl, and 0.25% glucose. It was purified to electrophoretical homogeneity after CM-sepharose FF chromatography. The specific activity and yield were 1791.9 FU/mg and 9.5%, respectively. This purified fibrinolytic enzyme had M of 27.5 kDa, optimal temperature and pH at 50 degrees C and 8.5, respectively. It was stable at pH 6.0-10.0 and 10-40 degrees C and inhibited by Fe(3+), Hg(2+), Cu(2+), Zn(2+), and PMSF. Compared the N terminal of amino acids and full DNA sequence with those in NCBI, it was considered to be a nattokinase.

Hydrolysis of Chlorella by Cellulomonas sp. YJ5 cellulases and its biofunctional properties.

Both 10% and 20% (w/w) Chlorella suspensions were hydrolyzed by 150 to 350 U/mL of cellulases from a 3-d cultivation of Cellulomonas sp. YJ5. Higher chlorophyll, reducing sugars and soluble proteins, and lower residual insoluble solid were observed on both samples after 30-min hydrolysis by various concentrations of cellulases at 50 °C. Decrease in insoluble solid, increases in soluble proteins, peptides and chlorophyll contents, and microscopic observation indicated obvious lysis of cell walls occurred during 60- to 180-min hydrolysis. Significant increases in soluble proteins, peptides, Fe(2+) chelating ability, trolox equivalent antioxidation capacity (TEAC), and reducing power was obtained after 3-h hydrolysis by 150 U/mL of cellulase. These data suggested that cellulolysis technology has high application potential in Chlorella industry.

Bioproperties and purification of xylanase from Bacillus sp. YJ6.

To characterize the xylanase from Bacillus sp. YJ6, broth after 4 days incubation at 25 degrees C was collected and purified to electrophoretical homogeneity after Sephacryl S-100 HR chromatograph. About 3.5% recovery and 678.1 purification fold were achieved. The purified xylanase, with a Mw of 19 kDa, had an optimal pH and temperature at 5.0 and 50 degrees C, respectively, and was stable at pH 5.0-9.0 or <50 degrees C. It was inhibited by Cu2+, Fe3+, Hg2+, phenylmethyl sulfonyl fluoride (PMSF), N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), N-ethylmaleimide (NEM), and leupeptin but activated by K+, Na+, Co2+, Mg2+, beta-mercaptoethanol (beta-ME), and glutathione (GSH). The purified xylanase had high specificity to beechwood, birchwood, and oat spelt xylans. The DNA fragment encoding this xylanase, corresponding to 213 amino acids, exhibited about 95% homology with seven strains of Bacillus in the NCBI database.

Expression and purification of pseudomonas aeruginosa keratinase in Bacillus subtilis DB104 expression system.

The DNA encoding Pseudomonas aeruginosa keratinase was ligated into pRPA expression vector and transformed into Bacillus subtilis DB104. Recombinant keratinase (rK), secreted by B. subtilis after 72 h of incubation, was purified to electrophoretical homogeneity by nickel affinity chromatography and found to have a molecular mass of 33 kDa. The rK had an optimal pH and temperature at 8.0 and 60 degrees C, respectively, and was stable at pH 6.0-9.0 and 10-50 degrees C. It was strongly inhibited by Cu(2+), Fe(2+), Hg(2+), Fe(3+), ethylene glycol tetraacetic acid, and ethylene diamine tetraacetic acid but activated by Ca(2+), Mg(2+), Zn(2+), dithiothreitol, glutathione, and beta-mercaptoethanol. According to substrate specificity, the rK was considered to be a metalloprotease.

Cloning, expression, and purification of Pseudomonas aeruginosa keratinase in Escherichia coli AD494(DE3)pLysS expression system.

The DNA encoding keratinase from Pseudomonas aeruginosa was ligated into pET-43b(+) expression vector and transformed into Escherichia coli AD494(DE3)pLysS. After isopropyl beta-d-thiogalactopyranoside induction, the soluble recombinant keratinase was expressed in E. coli. The keratinase with a molecular mass of 33 kDa was purified to electrophoretical homogeneity after nickel affinity chromatography. It had an optimal pH and temperature of 8.0 and 50 degrees C, respectively, and was stable at pH 6.0-9.0 and 10-60 degrees C. It was highly inhibited by Cd(2+), Cu(2+), Hg(2+), Ni(2+), Fe(3+), ethylene glycol tetraacetic acid, ethylenediaminetetraacetic acid, and p-chloromercuribenzoate, but activated by Ba(2+), Ca(2+), Mg(2+), Mn(2+), Zn(2+), dithiothreitol, glutathione, and beta-mercaptoethanol. According to substrate specificity results, the purified keratinase was considered to be a metalloprotease.

Functional expression and characterization of keratinase from Pseudomonas aeruginosa in Pichia pastoris.

Recombinant keratinase (rK) from Pseudomonas aeruginosa was secreted by Pichia pastoris SMD1168H with a final yield of 580 mg/L (1.03 kU/mL) after 72 h of induction. The rK was purified after nickel affinity chromatography and was stable at pH 6.0-9.0 and 10-60 degrees C. It was nonglycosylated protein with a molecular mass of 33 kDa and had an optimal pH and temperature at 8.0 and 60 degrees C, respectively. Ba(2+), Ca(2+), Mg(2+), Mn(2+), Zn(2+), dithiothreitol, glutathione, and beta-mercaptoethanol activated, while Cu(2+), Fe(2+), Hg(2+), Fe(3+), ethylene glycol tetraacetic acid, ethylene diamine tetraacetic acid, and p-chloromercuribenzoate inhibited its activity. rK could hydrolyze broad substrates and cleave hydrophobic and aromatic amino acids at P(1) position, behaving as those from the wild type strain and E. coli transformant.

Cloning and expression of antibacterial goat lactoferricin from Escherichia coli AD494(DE3)pLysS expression system.

Goat lactoferricin (GLfcin), an antibacterial peptide, is released from the N terminus of goat lactoferrin by pepsin digestion. Two GLfcin-related cDNAs, GLfcin L and GLfcin S, encoding Ala20-Ser60 and Ser36-Ser60 of goat lactoferrin, respectively, were cloned into the pET-23a(+) expression vector upstream from (His)6-Tag gene and transformed into Escherichia coli AD494(DE3)pLysS expression host. After being induced by isopropyl-beta-D-thiogalactopyranoside (IPTG), two (His)6-Tag fused recombinant lactoferricins, GLfcin L-His*Tag and GLfcin S-His*Tag, were expressed in soluble form within the E. coli cytoplasm. The GLfcin L-His*Tag and GLfcin S-His*Tag were purified using HisTrap affinity chromatography. According to an antibacterial activity assay using the agar diffusion method, GLfcin L-His*Tag had antibacterial activity against E. coli BCRC 11549, Staphylococcus aureus BCRC 25923, and Propionibacterium acnes BCRC 10723, while GLfcin S-His*Tag was able to inhibit the growth of E. coli BCRC 11549 and P. acnes BCRC 10723. These two recombinant lactoferricins behaved as thermostable peptides, which could retain their activity for up to 30 min of exposure at 100 degrees C.

Expression and purification of goat lactoferrin from Pichia pastoris expression system.

The recombinant goat lactoferrin (rGLF) was expressed in the methylotropic yeast Pichia pastoris using pGAPZalphaC vector, GAP as promoter, and Zeocin as the selective marker. After transformation of the GLF-pGAPZalphaC into Pichia pastoris X-33 expression host, the GLF-pGAPZalphaC vector was integrated into the GAP promoter locus of Pichia pastoris X-33 chromosome. The rGLF was expressed and secreted into the broth using alpha-factor preprosequence. SDS-PAGE and PAS staining analysis indicated that the rGLF could be purified to electrophoretic homogeneity by heparin-Sepharose 6 Fast Flow affinity chromatography and glycosylated by the expression host. The yield of purified rGLF was approximately 2.0 mg/L of culture broth. The N-terminal sequence was identical to the native goat lactoferrin (nGLF). The iron-binding behavior, papain-inhibiting property, and thermal stability of the purified rGLF were comparable to nGLF. This is the 1st report of intact goat lactoferrin expression using the P. pastoris system.

Purification and characterization of bacteriocin from Pediococcus pentosaceus ACCEL.

The Pediococcus pentosaceus ACCEL bacteriocin was purified to electrophoretical homogeneity by cell adsorption-desorption and Superose 12 fast performance liquid chromatography (FPLC). The purified bacteriocin, with a molecular mass of 17.5 kDa and an N-terminal sequence of -KYYGNGVTXGKHSXXVDXG-, belongs to class IIa and is designated pediocin ACCEL. It was inactivated by various proteases and stable at pH 2.0-6.0 and <100 degrees C. More than 80% activity was left even after 15 min of heating at 121 degrees C and pH 2.0-4.0. Gram-positive food-borne pathogens were inhibited by this bacteriocin, but Gram-negative ones were not. According to the storage stability study, the purified pediocin was stable at pH <6.0 and low temperature. No significant change in bactericidal activity was observed after freeze-drying and subsequent 1-month storage at room temperature.

Bacteriocins from Pediococcus pentosaceus L and S from pork meat.

Two strains of Pediococcus pentosaceus were isolated from refrigerated pork and found to produce antimicrobial substances that may inhibit foodborne pathogens and have potential as natural food preservatives. They were named P. pentosaceus L and S. The antimicrobial substances were purified to electrophoretical homogeneity by chloroform extraction and designated pentocins L and S with molecular masses (M) of 27 and 25 kDa, respectively. Both pentocins also had broad inhibition spectra and were thermostable. They inhibit the growth of tested spore-forming G+ and G- strains and the germination of Bacillus subtilis ATCC 10225, B. subtilis ATCC 10254, and Bacillus cereus ATCC 11778 spores. The inhibition activities decreased as the glucose in the medium decreased from 8.0 to 2.0%.