Optimization of Fermentation Conditions for Cell Envelope Proteinase Produced by Lactobacillus Plantarum LP69

Cell-envelope proteinases (CEPs) can hydrolyze casein into functional peptides, which is beneficial to the health of the host. The single factor experiment screened out that the optimal conditions for CEP production by Lactobacillus plantarum LP69 were 37°C, 20h, initial pH of 7, and optimal inoculation amount of 5%. The best conditions for this experiment were obtained by orthogonal experiment: time 22h, temperature 39°C, initial pH value of 6, and inoculation amount of 5%. Under this culture condition, the target protease activity of Lactobacillus plantarum LP69 reached 22.31±0.82U/mL, the protein content was 19.07±0.36mg/mL, and the specific activity was 1.17±0.06U/mg. The specific activity significantly increased by 15.8% compared with the control (p<0.05).

The Characterization of Stenotrophomonas maltophilia Isolated from Marine Sponge Producing Salt Tolerant Proteases

Protease is an important industrial enzyme and salt tolerant protease in which has desirable properties that could enhance its uses not only in industries, but also in agriculture and environmental. Marine organisms usually harbour halophilic microorganisms which produce salt tolerant protease. In this study, salt tolerant protease producing bacteria from marine sponge were isolated and screened on skim milk marine agar supplemented with different NaCl concentrations (1.5 % w/v). Out of 11 isolates, 8 isolates (S1-1, S1-2, S1-3, S2-1, S2-3, S2-4, S2-5 and S2-6) showed clearing zones with ability to digest casein on the skim milk agar. Morphologically, these strains are gram negative bacilli which grow in yellow colonies and were found to be catalase-positive but oxidase-negative. They are also non-lactose fermenter that produce gelatinase but not α-amylase. The ribosomal 16S rRNA sequencing was used to identify each isolate (Acc. number of S1-1 for MT645770, S1-2 for MT645 771, S1-3 for MT645 772, S2-1 MT645 773, S2-3 MT645 774, S2-4 MT645 775, S2-5 MT645 776 and S2-6 MT645 7767). The 16S rRNA sequences showed that these isolates were highly similar to Stenotrophomonas maltophilia (S2-6, 99.87%) and to a related strain Pseudomonas hibiscicola (S1-1, S1-2, S1-3, S2-1, S2-3, S2-4, S2-5 (97.16-99.9 %). Further proteolytic studies were carried out using skim milk agar with 1.0 %, 2.0 %, 3.0 % and 4.o % (w/v) of NaCl concentrations. All isolates were able to hydrolyze casein which produced clear zones surrounding each colony at 1.0 % and 2.0 % (w/v) salt. However, only isolate S1-5 and S2-6 showed proteolytic activities at 3.0 % (w/v) salt but none of them at 4.0 % (w/v). The ability of these isolates to produce protease which active at higher salt may indicate their potential to be the sources for enzyme with useful properties.

Prevalence and toxicity characterization of Bacillus cereus in food products from Poland

The prevalence of Bacillus cereus in a total of 585 samples of food products (herbs and spices, breakfast cereals, pasta, rice, infant formulas, pasteurized milk, fresh acid and acid/rennet cheeses, mold cheeses and ripening rennet cheeses) marketed in Poland was investigated. The potential of 1022 selected isolates of B. cereus to hydrolyze casein, starch and tributyrin, to ferment lactose, to grow at 7 C/10 days, to produce Nhe and Hbl toxin and to possess the ces gene was verified. B. cereus was found in 38.8% of the analyzed samples, reaching levels from 0.3 to 3.8 log CFU g-1 or mL-1. From the 1022 isolates, 48.8%, 36.0%, 98.9%, 80.0% and 25.0% were capable of fermenting lactose, producing amylase, protease, lipase and growing at 7 C/10 days, respectively, indicating spoilage potentiality. The occurrence of toxigenic B. cereus strains in all tested market products, both of plant (55.8% Hbl(+), 70.7% Nhe(+) and 1.7% ces(+) isolates) and animal origin (84.9% Hbl(+), 82.7% Nhe(+) and 0.9% ces(+) isolates) indicates the possible risk of foodborne infections/intoxications that occur as a result of the possibility of the development of B. cereus in favorable conditions and consumption of these products.

Fibrinogen/fibrin-specific enzymes from copperhead (Agkistrodon halys halys) and cobra (Naja oxiana eichwald) snake venoms

Ability of fractions of cobra’s (Naja oxiana Eichwald) and copperhead snake’s (Agkistrodon halys halys) venoms to hydrolyze fibrinogen/fibrin was studied. In cobra’s snake a component with molecular mass of nearly 60 kDa was found to hydrolyze a-chain of fibrinogen but failed to hydrolyze casein/azocasein and fibrin. A fibrinogen-specific metalloproteinase, the enzyme was inhibited by EDTA. Cobra’s venom reduced the mass of donor’s fresh blood clots. The copperhead snake’s venom and the fractions obtained by gel-filtration (HW-50) and ion exchange chromatography (DEAE-650) were found to hydrolyze casein/azocasein, a- and b-chains of fibrinogen/fibrin and donor’s blood clots. The results from the study of the venom and proteolytically active fractions are the evidence for a thrombolytic potential in a copperhead snake’s venom.

LYTIC ENZYMES OF SORANGIUM SP.: ISOLATION AND ENZYMATIC PROPERTIES OF THE α- AND β-LYTIC PROTEASES

Procedures are described for the isolation of two lytic enzymes from culture filtrates of a species of Sorangium. The enzymes, designated α-lytic protease and β-lytic protease, are responsible for most of the filtrate's lytic activity towards Arthrobacter globiformis cells. The enzymes were adsorbed from the filtrate by Amberlite CG50, separated by displacement from the resin with citrate buffer containing a gradient of sodium citrate concentration, and refractionated on columns of the same resin. Trace impurities in the β-enzyme were removed by precipitation of the enzyme with ammonium sulfate. The β-enzyme has been crystallized.On electrophoresis in Tris buffer of pH 8.0, the α-enzyme migrates slightly faster than egg-white lysozyme, the β-enzyme slightly slower. The absorptivity of the α-enzyme at 280 mμ was estimated to be 0.89; that of the β-enzyme 2.05.Low concentrations of the β-enzyme lyse suspensions of Arthrobacter globiformis cells completely, and moderately higher concentrations lyse suspensions of Micrococcus lysodeikticus cells completely; corresponding concentrations of the α-enzyme lyse the suspensions incompletely. The concomitant changes in A660 of the suspensions are consistent with zero-order kinetics for the β-enzyme and first-order kinetics for the α-enzyme. Untreated and partially lysed suspensions of Arthrobacter cells show little difference in the dependence of their absorbances on wavelength; corresponding suspensions of Micrococcus cells show marked differences in this respect, and the nature of the change suggests that the breakup of clumps of cells is responsible for a substantial part of the change in absorbance measured during lysis of suspensions of Micrococcus cells. Phase-contrast photomicrographs of cells undergoing lysis show that individual cells vary greatly in their rates of lysis; swelling and decreases in the refractive index precede fragmentation of the cell and may contribute appreciably to the change in absorbance of the suspension.Both enzymes hydrolyze casein. The α-enzyme has the greater activity towards this substrate.

STUDIES WITH STAPHYLOCOCCAL TOXINS: V. POSSIBLE IDENTIFICATION OF ALPHA HEMOLYSIN WITH A PROTEOLYTIC ENZYME

Alpha hemolysin has been separated from many of the other components present in culture filtrates of four strains of Staphylococcus aureus using a column chromatographic procedure with carboxymethyl-cellulose as the selective adsorbent, and graded levels of phosphate buffer at pH 6.0 to provide selective elution. The preparation of alpha hemolysin obtained in this manner hemolyzed rabbit and sheep erythrocytes, induced a dermonecrotic reaction in rabbits, was lethal to mice, and was proteolytic. The hemolytic activity of the preparation was stimulated by ethylenediamine tetraacetic acid, presumably by removing toxic ions from solution. Divalent cations inhibited activity of the alpha hemolysin, but stimulated the activity of a distinctive sheep hemolysin, which is shown to be a separate entity.Alpha hemolysin, obtained by the chromatographic procedure, and subjected further to resolution by zone electrophoresis retained its capacity to induce dermonecrosis, to hemolyze rabbit erythrocytes, and to hydrolyze casein. Apparent loss of the capacity of the preparation to hemolyze sheep erythrocytes was discussed.The possibility that alpha hemolysin is a specific proteolytic enzyme is suggested by the observation that under no conditions could a separation be effected between the hemolytic and proteolytic properties of the alpha hemolysin recovered from a number of different cultures.