Shann-Tzong Jiang

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.

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.

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.

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.

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.

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.

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.

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 β-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.

Biopreservative effect of pediocin ACCEL on refrigerated seafood

To investigate the biopreservative effectiveness of pediocin ACCEL on refrigerated seafoods, fresh fish fillets were immersed in various concentrations of pediocin ACCEL and then stored at either 4° or 0°C. Samples treated with nisin were used as a positive control. The aerobic plate counts (APC) of samples with bacteriocins were <2.0 log10cfu/g (log cfu/g) after 2 days storage at 0°C, except that with 1500 IU/mL of pediocin ACCEL. The APC of samples with nisin were >2.0 log cfu/g after 2 days storage, while those with pediocin ACCEL occurred after 1 day storage at 4°C. In refrigerated seafoods, pediocin ACCEL and nisin suppressed the growth of inoculated Listeria monocytogenes during 2- and 1-week storage at 4°C, respectively. Compared with nisin, the pediocin ACCEL was considered to be more effective on the suppression of L. monocytogenes growth in refrigerated seafoods during 2-week storage at 4°C.

Purification and Characterization of Acidic Protease from Aspergillus oryzae BCRC 30118

The acidic protease was purified from 4-day cultivation of Aspergillus oryzae BCRC 30118 by DEAE Sephacel and Sephacryl S-200 HR chromatographs. The specific activity, yield and purification fold were 117.62 kU/mg, 15.1% and 6.6, respectively. The molecular weight (M) was 41.0 kDa, while the optimal pH and temperature were 3.0 and 60°C, respectively. It was stable at pH 3.0-6.0 and 4-35°C. However, it was inhibited by Fe^2+, Hg^2+, Fe^3+ and pepstatin A, and slightly by leupeptin and TPCK. According to substrate specificity and inhibitor study, it was a cysteine protease with activation energy of 37.5 kcal/mol. Its Km, Vmax, Kcat and Kcat/Km for the hydrolysis of hemoglobin were 0.12 mM, 14.29 μmol/min, 14.55 sec^(-1) and 125.80 (sec^(-1) mM^(-1)), respectively.