PURIFICATION AND CHARACTERIZATION OF BACILLUS SUBTILIS JM-3 PROTEASE FROM ANCHOVY SAUCE

Bacillus subtilis JM‐3 was isolated from anchovy sauce naturally fermented in an underground cellar at 15 ± 3C for 3 years. The activity of the B. subtilis protease was highest in the 40–60% ammonium sulfate fraction. The yield of the purified protease was 5.3%, and its purification ratio was 35.6 folds. The molecular weight of the B. subtilis protease was 17.1 kDa, and its K_m and V_maxvalues were 1.75 μg/mL and 318 μM 1/min, respectively. The optimal temperature for protease activity was 60C, but optimal stability temperature was 30C. The optimal pH for protease activity and stability was 5.5. Therefore, the B. subtilis JM‐3 protease was classified as an acid protease. The relative activities of the B. subtilis JM‐3 protease were 69, 21 and 1.3% at 10, 20 and 30% NaCl concentrations, respectively. The best substrate for the B. subtilis JM‐3 protease was benzyloxycarbonyl‐glycine‐p‐nitrophenyl ester followed by bovine serum albumin. p‐Toluene‐sulfonyl‐L‐lysine chloromethylketone was the strongest inhibitor followed by soybean trypsin inhibitor, but N‐ethylmaleimide did not inhibit this enzyme. The B. subtilis JM‐3 protease was therefore presumed to be a trypsin‐like serine protease.     

PURIFICATION AND PHYSICOCHEMICAL PROPERTIES OF AN EXTRA CELLULAR AMYLASE FROM A STRAIN OF BACILLUS AMYLOLIQUEFACIENS ISOLATED FROM DRY ONION POWDER

An extracellular α-amylase from Bacillus amyloliquefaciens, isolated from dry onion powder, has been purified to homogeneity by ammonium sulfate fractionation, adsorption on starch, column chromatography on DEAE-cellulose, and gel filtration on Sephadex G-100 column. The enzyme consisted of one polypeptide chain with a molecular weight of 60,000. The isoelectric point was pH 5.2, the pH optimum 5.5 and the temperature optimum ranging from 50°-70°C. Prolonged digestion by trypsin did not affect the catalytic properties of the enzyme. The K_m for starch was 6.9 mg/ml. The enzyme was quite stable at 50°C, but lost about 85% of its activity at 60° after 30 min (pH 6.0).     

PURIFICATION AND CHARACTERIZATION OF AN ALKALINE PROTEASE FROM THE VISCERA OF BOLTI FISH (TILAPIA NILOTICA)

An alkaline protease was extracted from acetone powder of the viscera of bolti fish (Tilapia nilotica) with distilled water, precipitated from the extract by 40–60% (NH₄)₂SO₄and then dialyzed. Enzyme homogeneity studies by polyacrylamide gel electrophoresis (PAGE) illustrated that the enzyme was homogenous. Sodium dodecyl sulfate–PAGE showed a molecular weight of 23.0 kDa. The optimal pH and optimal temperature were 8.0 and 45C, respectively, with casein as a substrate. A remarkable stability was observed under alkaline conditions (pH 6.0–10.0), where more than 90% of the enzyme activity was maintained. In the acidic region (pH 2.0–6.0), the enzyme retained more than 50% of its activity. Furthermore, the enzyme retained 85.4 and 41.8% of its activity after heating at 50 and 60C for 120 min, respectively, while the relative activity after heating at 70C for 120 min was 5.25%. The alkaline enzyme lost most of its activity when incubated at 80C for 30 min. A high percentage of total enzyme activity was inhibited when the enzyme was incubated with 50 mM of either soybean trypsin inhibitor or ethylenediaminetetraacetic acid. The presence of NaCl and CaCl₂at 10 mM concentration increased the enzyme activity by 6.9 and 10.6%, respectively.     

新疆番茄酱中枯草芽孢杆菌耐热性的研究

对从新疆番茄酱中分离出的优势耐热菌枯草芽孢杆菌,在不同温度下（93、98、103、108℃）加热不同时间（5、10、15、20min）,对其残留的活菌进行计数,进而计算出D值和Z值,以了解其耐热特性。结果表明：D108℃=5．75min;D103℃=6．1min;D98℃=6．8min;D93℃=10．2min。Z=6．41℃。     

PURIFICATION AND PROPERTIES OF CYCLODEXTRIN GLUCANOTRANSFERASE FROM AN ALKALOPHILIC BACILLUS sp. 7-12

Cyclodextrin glucanotransferases (EC 2.4.1.19) (CGTase) are industrially important enzymes for production of cyclodextrin (CD) from starch. γ‐CD yield of CGTase from alkalophilic Bacillus species is usually much lower than β‐CD, while from alkalophilic Bacillus sp. 7‐12. γ‐CD yield is close to β‐CD. A CGTase from alkalophilic Bacillus sp. 7‐12 was purified and characterized. When purified by ammonium sulfate fractionation, DEAE‐cellulose column chromatography and Sepharose CL‐6B column chromatography, the enzyme obtained consisted of a single band that did not dissociate into subunits by SDS polyacrylamide gel electrophoresis. Molecular weight of the purified enzyme was determined to be 69,000 Da by SDS‐PAGE. The enzyme showed a K_mof 1.24 mg/mL and V_max0.101 µM/min when potato starch was used as substrate. The enzyme was stable below 70C with an optimum activity at 60C, and stable at pH range 6–10 with an optimum pH at 8.5. The enzyme activity was strongly inhibited by Ag⁺, Cu²⁺, Mg²⁺, Al³⁺, Co²⁺, Zn²⁺, Fe²⁺and slightly inhibited by Sn²⁺, Mn²⁺. The ions Ca²⁺and K⁺, EDTA and DTT had no influence on the enzyme activity.     

PROPERTIES OF AN ALKALINE PROTEASE FROM THE SKELETAL MUSCLE OF ATLANTIC CROAKER1

An alkaline protease was partially purified from the skeletal muscle of Atlantic croaker. The protease is a cytoplasmic enzyme and heat stable. The enzyme preparation was shown to degrade fish actomyosin in vitro between 50–60°C. The enzyme is a sulfhydryl protease and does not require Ca⁺⁺ ions for its activity. Preparations of the enzyme do not hydrolyze TAME, BTEE or denatured hemoglobin. Column chromatographic analyses suggest an apparent molecular weight of 80,000 ± 4,000 and the isoelectric point is 6.0 ± 0.2.     

PARTIAL PURIFICATION AND PROPERTIES OF A SERINE PROTEASE FROM TRANSFORMED C3H/10T1/2 CELLS

A 23-kDa serine protease was partially purified (1800-fold) from the cytosolic fraction of transformed C3H/10T1/2 cells. The purification procedure included chromatography with diethylaminoethyl (DEAE)-Sephacel, BBI-Sepharose, and C18 reverse phase high performance liquid chromatography (HPLC). The partially purified protease had a pH optimum of 7.8 for the hydrolysis of the synthetic tetrapeptide succinyl-Ala-Ala-Pro-Phe-AFC (K_m= 0.27 mM). The protease showed no other hydrolyzing activity against a number of various protease-specific synthetic peptides. The protease was inhibited by a number of serine protease inhibitors including Bowman-Birk inhibitor (BBI) (K_i= 0.7 nM) and chymostatin (K_i= 20 nM).     

PURIFICATION OF PEACH POLYPHENOL OXIDASE IN THE PRESENCE OF ADDED PROTEASE INHIBITORS1

Crude preparations of peach fruit (Prunus persica Batsch cv. Redskin) polyphenol oxidase (PPO) showed many apparent isoenzyme forms. Some of these forms were probably the result of proteolytic action of peach proteases while other forms were the result of association of PPO with carbohydrate materials. In the presence of protease inhibitors, Trasylol and phenylmethylsul-fonyl fluoride, three apparent isoenzyme forms of PPO were purified to homogeneity. The purification scheme included hydrophobic chromatography on phenyl sepharose CL-4B, hydroxylapatite chromatography, DEAE cellulose chromatography, and gel filtration on Ultrogel AcA 34. Minor contaminants remaining after these steps were separated from PPO by gel electrophoresis. The major PPO isoenzyme form (A) was purified 44 fold with an overall yield of 5.6% and contained no detectable carbohydrates. Isoenzyme forms A' and A' were purified 104 and 67 fold respectively, but still were associated with carbohydrate material. Cesium chloride centrifugation partially removed the carbohydrates associated with PPO A' and A'. Purified peach PPO A showed greater activity toward D-catechin (539%) and pyrogallol(l82%) than to catechol (100%). An apparent K₃ of 4, 0.3, and 2 mM was obtained with D-catechin, pyrogallol and catechol, respectively. The enzyme was severely inhibited by 10 μM 2,3-naphthalenediol (91%) and by 10 pM diethyl dithiocarbamate (100%).     

MACRODONTIN, A NEW PROTEASE ISOLATED FROM FRUITS OF PSEUDANANAS MACRODONTES (MORR.) HARMS (BROMELIACEAE)

A cysteine proteinase was isolated from immature fruits of Pseudananas macrodontes (Morr.) Harms, a species taxonomically close to pineapple grown in the northeast of Argentina. The protease is active at pH 7.0 to 10.5, and rather unstable at temperatures over 45C. Crude extracts were purified by acetone fractionation followed by chromatofocusing at the pH range 4.5 to 6.5. Two main proteolytic fractions were obtained: fraction I (M_r= 20.6 kDa, pI = 5.9) and fraction II (M_r= 19.3 kDa, pI = 5.3), both homogeneous by gel-filtration and SDS-PAGE criteria. This new plant protease (“macrodontin”) could be valuable in processes carried out in neutral-weak alkaline media. The thermal behavior of the crude enzyme is also a useful property, since it could be easily inactivated in foodstuffs by heating 10 min at 75C. Comparison of macrodontin and alcalase proteolytic behavior on soy concentrates is presented.     

AN ANTHOCYANIN COMPLEX ISOLATED FROM THE SYRUP OF CANNED BLUEBERRIES

SUMMARY -The addition of 5 vol ethanol to syrup from canned blueberries caused the precipitation of a blue-colored anthocyanin complex. The complex, which readily redissolved in water, was decomposed under strongly acid conditions. The complex contained glycosides of the antho-cyanidins, delphinidin and petunidin. It also contained a high proportion of a polysaccharide resembling pectin and a small proportion of proteinaceous material. Cations detected by X-ray fluorescence spectroscopy included: aluminum (0.13%), calcium (0.13%), iron (0.12%) and potassium (0.67%). Tin was not detected in the complex. There was some evidence that the complex also contained leucoanthocyanins. The results of ultracentrifugation studies suggested that the molecular weight was between 77,000,000 and 490,000,000.     

PURIFICATION AND PARTIAL CHARACTERIZATION OF AN ENDO-POLYGALACTURONASE FROM ASPERGILLUS NIGER

A pectinase was identified and isolated from a commercial Aspergillus niger pectinase preparation. The crude enzyme preparation, which was prepared by precipitation of the water extract of the culture of A. niger with ammonium sulfate, was further fractionated by three steps of chromatography, i. e., cation exchange, hydrophobic interaction and onion exchange, to obtain an electrophoretically homogeneous pectinase. The molecular weight of the purified enzyme was estimated by SDS‐PAGE to be about 40.4 kDa under both nonreducing and reducing conditions, with the optimum pH at 5.0 and the optimum temperature at 36C. The enzyme was stable at temperatures below 35C. The partial N‐terminal ammo acid sequence data analysis of the first 19 amina acids of the obtained pectinase revealed 94.7% and 89.5% homology with two reported pectinases from A. niger.     

CYTOTOXICITY ASSESSMENT OF BACILLUS STRAINS ISOLATED FROM STREET-VENDED FOODS IN JOHANNESBURG, SOUTH AFRICA

Twenty-one isolates each of Bacillus (B.) cereus, B. licheniformis and B. subtilis from street foods, collected in central Johannesburg, were randomly selected to test for cytotoxicity against McCoy 5A Mouse cells using a 3-(4,5-dimethylthizol-2-yl)-2,5-diphenyltetrazolium bromide assay, and observation by confocal scanning laser microscopy (CSLM) and scanning electron microscopy (SEM). Forty-eight percent of B. cereus, 33% of B. licheniformis and 19% of B. subtilis strains produced cytotoxic compounds. For B. cereus strains, all supernatants exhibiting cytotoxic effects were inactivated by heat treatment at 121C for 15 min. By contrast, 24% of B. licheniformis and 10% of B. subtilis supernatants exhibited cytotoxic effects following heat treatment. CSLM and SEM showed that McCoy cells treated with cytotoxic supernatants exhibited leakage and necrosis. Presence of B. cereus, B. licheniformis and B. subtilis in street foods in high numbers may pose potetnial safety risks due to production of cytotoxic compounds.     

THE PURIFICATION OF PROTEASE FROM COWSLIP (PRIMULA VERIS) AND ITS USE IN FOOD PROCESSING

Proteases perform very important functions and are used in different objectives as in vitro and in vivo. In recent years, proteases have been used for clinical, pharmaceutical and industrial applications. Most of these proteases are obtained from animal sources. News of diseases passing over from animals to human (severe acute respiratory syndrome [SARS], mad cow) has created suspicion of contamination in animal food and its product. Therefore, a new vegetal protease source for use in the food industry was studied. Ammonium sulfate fractionation and CM‐sephadex ion exchange chromatography were used in the purification of the enzyme. The purified enzyme has an optimum pH of 7.0 and its optimum temperature was 70C. The V_max and K_m values determined by Lineweaver–Burk graphics were 0.44 mg/mL.min and 40 mM, respectively. The purification degree was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE ). The native molecular weight of the enzyme was 46 kDa using a gel filtration chromatograph. SDS‐PAGE results show that the enzyme has two subunits, which have protease activity at 32 and 14 kD. It was investigated whether the purified and characterized protease enzyme would cause to congeal milk or could produce digestion of chicken and cow meat. The results showed that the protease enzyme can be used for industrial production.