Novel fibrinolytic enzyme in fermented shrimp paste, a traditional asian fermented seasoning

A novel fibrinolytic enzyme was purified from fermented shrimp paste, a popular seasoning used in Asian countries. The enzyme is a monomer with an apparent molecular weight of 18 kDa, and it is composed primarily of beta-sheet and random coils. The N-terminal amino acid sequence was determined to be DPYEEPGPCENLQVA. It is a neutral protease with an optimal activity from pH 3 to 7. No inhibition was observed with PMSF, Pepstatin A, E64, and 1,10-phenanthroline, but the enzyme was slightly inhibited by EDTA and Cu(2+). It was relatively specific to fibrin or fibrinogen as a protein substrate, yet it hydrolyzed none of the plasma proteins in the studies. In vitro, the enzyme was resistant to pepsin and trypsin digestion. It also had an anticoagulant activity measured with activated partial thrombin time and prothrombin time tests. The novel fibrinolytic enzyme derived from traditional Asian foods is useful for thrombolytic therapy. In addition, this enzyme has a significant potential for food fortification and nutraceutical applications, such that its use could effectively prevent cardiovascular diseases.     

Purification and characterization of proteases from Bacillus amyloliquefaciens isolated from traditional soybean fermentation starter

Bacillus amyloliquefaciens FSE-68 isolated from meju, a Korean soybean fermentation starter, was identified on the basis of biophysical tests and 16S rRNA gene sequence. A neutral metalloprotease (NPR68) and an alkaline serine-protease (APR68) were purified by ammonium sulfate precipitation and cation exchange chromatography and identified on the basis of their activities at different pH values and the selective protease inhibitors. The molecular weights of NPR68 and APR68 measured with ESI-MS were 32743 (+/- 0.8) and 27443 (+/- 0.5) Da, respectively. Against oxidized insulin chains, the NPR68 has a cleavage preference at the site where leucine is located as a P1' residue followed by phenylalanine, and the APR68 has broad specificity and favors leucine at the P1 site. These results indicate that the proteases are natural variants of subtilisin and bacillolysin.     

Isolation and characterization of novel thermophilic nitrifying Bacillus sp. from compost

A thermophilic nitrifying bacterium, strain T3, was isolated from compost made of animal waste by using a novel selective medium. Strain T3 was classified into the genus Bacillus, close to Bacillus halodurans, but identified as a novel species. To evaluate the effect of adding strain T3 on ammonia emission during the process of composting animal waste, laboratory scale composting was done. Ammonia emission was lower when strain T3 was added than in the control material to which strain T3 was not added. Thermophilic nitrifying bacteria in the strain T3-containing material increased from 6.24 (log value) to 7.55 (log value) on average during the tests. These results suggested the possibility of reducing ammonia emission from composting of animal waste by adding strain T3.     

Potent fibrinolytic enzyme from a mutant of Bacillus subtilis IMR-NK1

A mutant of Bacillus subtilis IMR-NK1, which is used for the production of domestic "natto" in Taiwan, produced high fibrinolytic enzyme activity by solid-state fermentation using wheat bran as medium. In addition, a strong fibrinolytic enzyme was purified from the cultivation media. The purified enzyme was almost homogeneous, as examined by SDS-PAGE and capillary electrophoresis. The enzyme had an optimal pH of 7.8, an optimal temperature of 55 degrees C, and a K(m) of 0.15% for fibrin hydrolysis. The molecular mass estimated by gel filtration was 31.5 kDa, and the isoelectric point estimated by isoelectric focusing electrophoresis was 8.3. The enzyme also showed activity for hydrolysis of fibrinogen, casein, and several synthetic substrates. Among the synthetic substrates, the most sensitive substrate was N-succinyl-Ala-Ala-Pro-Phe-pNA. PMSF and NBS almost completely inhibited the activity of the enzyme. These results indicate that the enzyme is a subtilisin-like serine protease, similar to nattokinase from Bacillus natto.     

中国传统细菌型豆豉溶栓酶的提取纯化技术研究(简报)

为深入开发利用中国传统发酵细菌型豆豉溶栓酶,对豆豉溶栓酶分离纯化工艺进行了研究探讨,确认了最佳的纯化条件:首先用饱和度75%的硫酸铵沉淀豆豉溶栓酶;然后利用DEAE-Sepharose FF (Diethylaminoethyl Sepharose Fast Flow,二乙基氨基琼脂糖快速凝胶)阴离子交换柱进行层析,优化的层析条件为用10 mmol/L Tris(Trishydroxymethylaminomen,三羟甲基氨基甲烷)-HCI (pH 8.7)作为上样缓冲液,采用线性梯度洗脱,洗脱液为10mmol/L Tris-HCI(pH 8.7)和0.6mol/LNaCl,洗脱流速为1 mL/min;最后经过Sephadex G-75(葡聚糖凝胶G-75)凝胶过滤,得到纯化倍数为11.29倍的豆豉溶栓酶.利用SDS-PAGE (Sodium dodecyl sulphate-polylamide gel electroresis,十二烷基硫酸钠-聚丙烯酰胺凝胶电泳) 显示经纯化后的酶无杂蛋白,初步鉴定其分子量接近31 ku.     

Purification and characterization of a lipoxygenase enzyme from durum wheat semolina.

Purification of a lipoxygenase enzyme from the cultivar Tresor of durum wheat semolina (Triticum turgidum var. durum Desf) was reinvestigated furnishing a new procedure. The 895-fold purified homogeneous enzyme showed a monomeric structure with a molecular mass of 95 +/- 5 kDa. Among the substrates tested, linoleic acid showed the highest k(cat)/K(m) value; a beta-carotene bleaching activity was also detected. The enzyme optimal activity was at pH 6. 8 on linoleic acid as substrate and at pH 5.2 for the bleaching activity on beta-carotene, both assayed at 25 degrees C. The dependence of lipoxygenase activity on temperature showed a maximum at 40 degrees C for linoleic acid and at 60 degrees C for bleaching activity on beta-carotene. The amino acid composition showed the presence of only one tryptophan residue per monomer. Far-UV circular dichroism studies carried out at 25 degrees C in acidic, neutral, and basic regions revealed that the protein possesses a secondary structure content with a high percentage of alpha- and beta-structures. Near-UV circular dichroism, at 25 degrees C and at the same pH values, pointed out a strong perturbation of the tertiary structure in the acidic and basic regions compared to the neutral pH condition. Moreover, far-UV CD spectra studying the effects of the temperature on alpha-helix content revealed that the melting point of the alpha-helix is at 60 degrees C at pH 5.0, whereas it was at 50 degrees C at pH 6.8 and 9.0. The NH(2)-terminal sequence allowed a homology comparison with other lipoxygenase sequences from mammalian and vegetable sources.     

Purification and characterization of a hexanol-degrading enzyme extracted from apple

An enzyme having activity toward n-hexanol was purified from apple, and its biochemical characteristics were analyzed. The purification steps consisted of sedimentation with ammonium sulfate, DEAE Sepharose Fast Flow ion exchange chromatography, and Sephadex G-100 column. The obtained enzyme had a yield of 16.00% with a specific activity of 18879.20 U/mg protein and overall purification of 142.77-fold. The enzyme showed activity to isoamylol, 1-propanol, n-hexanol, and isobutanol but not toward methanol and ethanol. With n-hexanol as a substrate, the optimum conditions were pH 4.0 and 30 °C for enzyme activity and pH 3.0-4.0 and temperatures below 40 °C for enzyme stability. The enzyme activity was increased significantly by adding l-cysteine and Fe(2+) at all tested concentrations and slightly by Zn(2+) at a high concentration but decreased by additions of EDTA, Ga(2+), K(+), Mg(2+), sodium dodecyl sulfate (SDS), sodium aluminum sulfate (SAS), dithiothreitol (DTT), and glutathione (GSH). The enzyme activities toward n-hexanol and n-hexanal were increased by NADH but decreased by NAD(+), in contrast to a decrease toward n-hexane by addition of both NAD(+) and NADH.     

Purification and characterization of a novel extracellular tripeptidyl peptidase from Rhizopus oligosporus

A novel extracellular tripeptidyl peptidase (TPP) was homogenously purified from the culture supernatant of Rhizopus oligosporus by sequential fast protein liquid chromatography. The purified enzyme was a 136.5 kDa dimer composed of identical subunits. The effects of inhibitors and metal ions indicated that TPP is a metallo- and serine protease. TPP was activated by divalent cations, such as Co(2+) and Mn(2+), and completely inhibited by Cu(2+). Enzyme activity was optimal at pH 7.0 and 45 °C with a specific activity of 281.9 units/mg for the substrate Ala-Ala-Phe-pNA. The purified enzyme catalyzed cleavage of various synthetic tripeptides but not when proline occupied the P1 position. Purified TPP cleaved the pentapeptide Ala-Ala-Phe-Tyr-Tyr and tripeptide Ala-Ala-Phe, confirming the TPP activity of the enzyme.     

Purification and characterization of two antifungal chitinases extracellularly produced by Bacillus amyloliquefaciens V656 in a shrimp and crab shell powder medium

A Gram-positive bacterium with antagonistic activity was isolated from the soil. It has been identified as Bacillus amyloliquefaciens strain V656 on the basis of 16S ribosomal DNA analysis and standard bacteriological tests. B. amyloliquefaciens V656 produced antifungal enzymes when it was grown in a medium containing shrimp and crab shell powder (SCSP) of marine waste. The antifungal enzymes displayed chitinase activities. Two extracellular antifungal chitinases (FI and FII) were purified and characterized, and their molecular weights, isoelectric points, pH and thermal stabilities, and antifungal activities were determined. The characteristics of V656 chitinases are similar to those of the known bacterial chitinases in terms of their isoelectric points, thermal instabilities, and lack of lysozyme activity. In contrast to other known bacterial chitinases, the unique characteristics of V656 chitinases include extremely low molecular weights and nearly neutral optimum pH. Furthermore, this is the first report of the isolation of chitinases from B. amyloliquefaciens that are active against fungi.     

Purification and properties of a novel phenolic antioxidant from Radix astragali fermented by Aspergillus oryzae M29

The Chinese herb Radix astragalus (RA) has been widely used as a dietary supplement in Asia, and there are numerous reports on its bioactivities. However, there are no reports to date regarding the use of Aspergillus spp. in the culture medium of the RA plant for the production of phenolic antioxidants. In this study, utilizing the fungus Aspergillus to ferment the native RA has successfully resulted in a significant increase in the phenolic contents of RA, and the fermented RA also revealed much better antioxidant activity toward 2,2'-azinobis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radicals, hydroxyl radical, superoxide radical and peroxyl radical than those of unfermented RA. Among these phenolics, a potent novel antioxidant was isolated and identified as 3,4-di(4'-hydroxyphenyl) isobutyric acid with a molecular weight of 272, by ESI-MS (electrospray ionization mass), 1H NMR (nuclear magnetic resonance), 13C NMR, DEPT (distortionless enhancement by polarization transfer)-NMR, HMQC (heteronuclear multiple quantum coherence), and HMBC (heteronuclear multiple bond correlation) spectra. These data demonstrated that the solid-state bioprocessing strategy could be an innovative approach to enhance the antioxidant activity of RA.     

Purification and characterization of a novel pyrethroid hydrolase from Aspergillus niger ZD11

The pyrethroid pesticides residues on foods and environmental contamination are a public safety concern. Pretreatment with pyrethroid hydrolase has the potential to alleviate the conditions. For this purpose, a fungus capable of using pyrethroid pesticides as a sole carbon source was isolated from the soil and characterized as Aspergillus niger ZD11. A novel pyrethroid hydrolase from cell extract was purified 41.5-fold to apparent homogeneity with 12.6% overall recovery. It is a monomeric structure with a molecular mass of 56 kDa, a pI of 5.4, and the enzyme activity was optimal at 45 degrees C and pH 6.5. The activities were strongly inhibited by Hg(2+), Ag(+), and rho-chloromercuribenzoate, whereas less pronounced effects (5-10% inhibition) were observed in the presence of the remaining divalent cations, the chelating agent EDTA and phenanthroline. The purified enzyme hydrolyzed various insecticides with similar carboxylester. trans-Permethrin is the preferred substrate.     

Purification and characterization of two novel beta-galactosidases from Lactobacillus reuteri

The intracellular beta-galactosidase (beta-gal) enzymes from two strains of Lactobacillus reuteri, L103 and L461, were purified by ammonium sulfate fractionation, hydrophobic interaction, and affinity chromatography. Both enzymes are heterodimers with a molecular mass of 105 kDa, consisting of a 35 kDa subunit and a 72 kDa subunit. Active staining of L. reuteri L103 and L461 beta-gal with 4-methylumbelliferyl beta-d-galactoside showed that the intact enzymes as well as the larger subunits possess beta-galactosidase activity. The isoelectric points of L. reuteri L461 and L103 beta-gal were found to be in the range of 3.8-4.0 and 4.6-4.8, respectively. Both enzymes are most active in the pH range of 6-8; however, they are not stable at pH 8. The L. reuteri beta-galactosidases are activated by various mono- and divalent cations, including Na(+), K(+), and Mn(2+), and are moderately inhibited by their reaction products d-glucose and d-galactose. Because of their origin from beneficial and potentially probiotic lactobacilli, these enzymes could be of interest for the synthesis of prebiotic galacto-oligosaccharides.     

Purification and characterization of novel proteinase inhibitors from dried figs

Two types of the natural organic matter P and B were isolated from dried figs by gel permeation and high-performance liquid chromatography. The characterizations of their molecular structures were also performed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and infrared absorption spectrometry. As a result, these samples were revealed to inhibit the serine and cysteine proteinases chymotrypsin and papain (K(i) = 10(-)(6)-10(-)(4) M). The optimal inhibitory pH values of the P and B samples were observed to be approximately 5.5 and 5.0, respectively. The analyses of their UV-vis absorption spectra and infrared absorption spectra indicated that they would be a kind of humic substance. The mass spectrometry analyses showed that they possessed relatively small heterogeneous molecules with molecular masses around 692, 845, and 1389 Da for the P sample and around 551, 704, and 909 Da for the B sample.     

Purification and characterization of a phytate-degrading enzyme from germinated faba beans (Vicia faba Var. Alameda)

A phytate-degrading enzyme was purified approximately 2190-fold from germinated 4-day-old faba bean seedlings to apparent homogeneity with a recovery of 6% referred to the phytase activity in the crude extract. It behaves as a monomeric protein of a molecular mass of approximately 65 kDa. The phytate-degrading enzyme belongs to the acidic phytases. It exhibits a single pH optimum at 5.0. Optimal temperature for the degradation of sodium phytate is 50 degrees C. Kinetic parameters for the hydrolysis of sodium phytate are K(M) = 148 micromol L(-1) and k(cat) = 704 s(-1) at 35 degrees C and pH 5.0. The faba bean phytase exhibits a broad affinity for various phosphorylated compounds and hydrolyzes phytate in a stepwise manner. The first hydrolysis product was identified as D/L-myo-inositol(1,2,3,4,5)pentakisphosphate.     

Purification, characterization, and gene cloning of a chitosanase from Bacillus species strain s65

For the production of oligosaccharides from chitosan, a chitosanase-producing bacterium, S65, was isolated from soil. On the basis of phylogenetic analysis of the 16S rDNA gene sequence and phenotypic analysis, S65 was identified as a Bacillus sp. strain. This bacterium constitutively produced chitosanase in a culture medium without chitosan as an inducer. S65 chitosanase was homogeneously purified by DEAE Sepharose fast flow anion exchange followed by Superdex 75 size exclusion, and the molecular weight was 45 kDa according to SDS-PAGE. Enzyme analysis showed that the optimum pH and temperature of S65 were 6.0 and 65 degrees C, respectively. Catalytic activity was stable from pH 5.5-6.5 at temperatures below 40 degrees C, and the pI of chitosanase was about 6.0 as determined by a test tube method. S65 chitosanase degraded carboxymethyl cellulose (CMC) at the degree of about 5.3% relative to the value of soluble chitosan, but it cannot hydrolyze colloidal chitin and crystalline cellulose. Gene encoding was cloned and sequenced. The deduced amino acid sequence of the S65 exhibited the highest homology to those of family 8 glycanase, suggesting that the enzyme belonged to family 8.     

Purification and characterization of collagenolytic proteases from the hepatopancreas of northern shrimp (Pandalus eous)

Three gelatinolytic proteases (A1, A2, and B) were purified using a synthetic substrate, DNP-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, from the hepatopancreas of Northern shrimp (Pandalus eous) by several chromatographic steps involving hydroxyapatite column chromatography, gel filtration on Superdex75, and ion-exchange chromatography on a MonoQ column. Collagenolytic proteases A2 and B, but not protease A1, were demonstrated to digest native porcine type I collagen at 25 degrees C and pH 7.5. Further characterizations of these two collagenolytic proteases showed that the pH optimum of enzyme A2 against DNP-peptide was found to be 11, whereas that of enzyme B was 8.5. The optimum temperature ranged between 40 and 45 degrees C for both enzymes, although enzyme B appeared to be thermally more stable than enzyme A2 at pH 7.5. Both enzymes were strongly inhibited by PMSF and antipain, which suggests that they belong to collagenolytic serine proteases.     

Bioreduction of chromium (VI) by Bacillus sp. isolated from soils of iron mineral area

Extensive use of chromium in industry has caused environmental contamination. Chromium-resistant bacteria are capable of reducing toxic Cr (VI) to less toxic Cr (III). Eight isolates, which can grow on LB agar containing 500 mg/L of Cr (VI), were isolated from soil samples of iron mineral area. The bacterial isolates were identified as Bacillus sp. by the 16S rRNA gene sequences. Phylogenetic tree analysis indicates the isolates can be divided into two groups. The bacterial isolates can be resistant to other heavy metals and reduce Cr (VI) at different levels. One bacterial isolate (MDS05), which can tolerate 2500 mg/L Cr (VI) and was able to reduce almost 100% of Cr (VI) at the concentration of 10 mg/L in 24 h, was selected to study the effects of some environmental factors such as pH, temperature, and time on Cr (VI) reduction and growth. The cell growth of MDS05 was affected by the presence of Cr (VI), especially at the concentration of 100 mg/L. It reduced more amount of Cr (VI) under a wide range of concentrations from 5 to 50 mg/L, and reduction was optimum at 37 °C and pH 8. MDS05 showed great promise for use in Cr (VI) detoxification under a wide range of environmental conditions.     

Purification and some properties of an Aspergillus niger beta-apiosidase from an enzyme preparation hydrolyzing aroma precursors.

A beta-apiosidase was isolated and purified to electrophoretic homogeneity from an enzyme preparation, Klerzyme 200, through ammonium sulfate precipitation, gel filtration chromatography, ion-exchange chromatography, and HPLC on ion-exchange and size exclusion columns. The purification of the enzyme was aided by the synthesis of 4-methylumbelliferyl beta-D-apiofuranoside for the specific detection of activity on electrophoresis gels. The molecular mass estimated by SDS-PAGE was 120 kDa. The optimum activity of the beta-apiosidase was found at pH 5 and 40 degrees C. The K(m) and V(max) for p-nitrophenyl beta-D-apiofuranoside were 4.2 mM and 2460 nkat/mg of protein, respectively. The enzyme was not inhibited by glucose and ethanol. This enzyme hydrolyzed the intersugar linkages of apiofuranosylglucosides, aroma precursors from grape.     

Purification and characterization of an isoflavone-conjugates-hydrolyzing beta-glucosidase from endophytic bacterium

An isoflavone conjugates hydrolyzing beta-glucosidase (ICHG) from endophytic bacterium, Pseudomonas ZD-8 was purified to homogeneity by successive ammonium sulfate precipitation, gel filtration on SephadexG-100, DEAE-sephrose CL-6B and DEAE-Sephacel chromatography. The enzyme was a monomeric protein with an apparent molecular mass of 33 kDa as determined by SDS-PAGE and gel filtration. It was optimally active at pH 6.0 and 40 degrees C and had a specific activity of 1485 U mg of protein(-1) against genistin. The ICHG readily hydrolyzed rho-nitrophenyl-beta-glucoside, rho-nitrophenyl-beta-galactoside, genistin, daidzin, with Km values of 1.64, 1.87, 0.012, 0.014 mM, respectively. The ICHG showed a pronounced specificity for glucose in the 7-position of isoflavone and flavone conjugates and hydrolyzed effectively malonyl isoflavone glucosides as well as isoflavone glucosides with similar kinetics. Glucose and glucono-delta-lactone inhibited the enzyme competitively with Ki values of 84 mM and 23 mM, respectively. The enzyme did not require divalent cations for activity, and its activity was strongly inhibited by Hg2+, Ag+, rho-chloromercuribenzoate, iodoacetic acid, and N-ethylmaleimide while reducing agents such as beta-mercaptoethanol, dithiothreitol, dithioerythritol, glutathione slightly activated the enzyme.     

Purification and characterization of an antimicrobial chitinase extracellularly produced by Monascus purpureus CCRC31499 in a shrimp and crab shell powder medium

Monascus purpureus CCRC31499 produced an antimicrobial chitinase when it was grown in a medium containing shrimp and crab shell powder (SCSP) of marine wastes. An extracellular antimicrobial chitinase was purified from the culture supernatant to homology. The chitinase had a molecular weight of approximately 81,000 and a pI of 5.4. The optimal pH, optimum temperature, and pH stability of the chitinase were pH 7, 40 degrees C, and pH 6-8, respectively. The activity of the chitinase was activated by Fe(2+) and strongly inhibited by Hg(2+). The unique characteristics of the purified chitinase include high molecular weight, nearly neutral optimum pH, protease activity, and antimicrobial activity with bacteria and fungal phytopathogens. This is also the first report of isolation of a chitinase from a Monascus species.