Cellular Location and Some Properties of Proteolytic Enzymes of Rumen Bacteria

Several physical and chemical techniques were used to extract, and to identify the location of, proteolytic enzymes associated with mixed rumen bacteria. Most activity was removable by gentle physical methods such as shaking and brief blending, without cell disruption, indicating that it was associated with coat and capsular material. Proteases were present also in the cell envelope, corresponding to the inner membrane fraction of gram-negative bacteria, and intracellularly and were removable by detergent and French press treatment. Temperature and pH profiles were obtained for the coat enzymes, likely to be the most important in the digestion of food protein. Inhibition studies indicated that these proteases, and those of the whole bacterial fraction from rumen fluid, were predominantly of the cysteine protease type.     

Fractionation,Purification and Some Properties of Proteolytic Enzymes from Stem Bromelain

The heterogeneity of the proteolytic enzymes in the stem bromelain was investigated by the isoelectric focusing with carrier ampholytes. The isoelectric focusing of the stem bromelain demonstrated the presence of two types of proteolytic enzymes which were distinguishable from each other by their isoelectric points. One of these was a basic protein having an isoelectric point of 9.45. This basic enzyme comprised almost all of basic protein which are found in stem bromelain. The other was an acidic protein having an isoelectric point near pH 4.7. This was a minor compooent. The purification of the two enzymes was carried out by use of chromatographies on CM-Sephadex, DEAE-Sephadex and Sephadex at pH 7.0.     

Some Properties of Proteolytic Enzymes and Storage Proteins in Recalcitrant and Orthodox Seeds of Araucaria

Araucaria bidwillii Hook. and Araucaria cunninghamii Don D. are two species of conifers whose seeds belong to different physiological categories: A. bidwillii seeds are recalcitrant, while A. cunninghamii seeds are orthodox. The extraction of enzymes and storage proteins was carried out from A. bidwillii and A. cunninghamii megagametophytes. The endopeptidase activities of both species were assayed with azocasein and with haemoglobin; the exopeptidase activities were detected by various N-carbobenzyloxy-dipeptides and L-leucine p-nitroanilide. The use of appropriate proteinase inhibitors, i.e. pepstatin A, ethylenediaminetetraacetic acid and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, showed the presence of aspartic and metallo proteinases and the absence of the cysteine ones both in A. bidwillii and in A. cunninghamii ungerminated seeds. Since the results do not show differences between the types of enzymes in the ungerminated araucarian seeds and those present in some ungerminated angiosperm seeds (barley, wheat, maize, rice, buckwheat), we conclude that their physiological role is similar. The electrophoretical analyses of soluble and insoluble storage proteins of A. cunninghamii showed patterns similar to those found in other gymnosperms, while the storage protein patterns of A. bidwillii seeds were rather atypical.     

Purification and Properties of Proteolytic Enzymes from Thermophilic Actinomycetes

The enzymes isolated from two selected cultures of thermophilic actinomycetes-Thermomonospora fusca (A 29) and Thermoactinomyces vulgaris (A 60)-possess proteolytic activity. The enzymes were purified more than 35- to 40-fold and showed three bands each upon cellulose acetate electrophoresis at several pH values. Based upon Sephadex gel filtration, molecular weights of 21,500 and 23,800 were calculated for the active peaks of the enzymes. The purified enzymes lysed heat-killed cells of gram-positive and gram-negative bacteria, mycobacteria, and fungi and also hydrolyzed casein. The enzymes were most active between a temperature range of 60 and 70 C and pH 8.0 and 9.0, and were significantly inhibited by potassium permanganate, potassium ferricyanide, and iodine.     

Properties of Chitosanase from Bacillus cereus S1

Chitosanase from Bacillus cereus S1 was purified, and the enzymatic properties were investigated. The molecular weight was estimated to 45,000 on SDS-PAGE. Optimum pH was about 6, and stable pH in the incubation at 40°C for 60 min was 6–11. This chitosanase was stable in alkaline side. Optimum temperature was around 60°C, and enzyme activity was relatively stable below 60°C. The degradations of colloidal chitosan and carboxymethyl cellulose (CMC) were about 30 and 20% relative to the value of soluble chitosan, respectively, but colloidal chitin and crystalline cellulose were not almost hydrolyzed. On the other hand, S1 chitosanase adsorbed on colloidal chitin completely and by about 50% also on crystalline cellulose, in contrast to colloidal chitosan, which it did not adsorb. S1 chitosanase finally hydrolyzed 100% N-deacetylated chitosan (soluble state) to chitobiose (27.2%), chitotriose (40.6%), and chitotetraose (32.2%). In the hydrolysis of various chitooligosaccharides, chitobiose and chitotriose were not hydrolyzed, and chitotetraose was hydrolyzed to chitobiose. Chitobiose and chitotriose were released from chitopentaose and chitohexaose. From this specificity, it was hypothesized that the active site of S1 chitosanase recognized more than two glucosamine residues posited in both sides against splitting point for glucosamine polymer. Received: 8 June 1999 / Accepted: 20 July 1999     

STUDIES ON CELL DEFORMABILITY : II. Effects of Some Proteolytic Enzymes

Murine sarcoma 37 ascites cells were treated with the proteolytic enzymes, trypsin and chymotrypsin, after which cellular deformability and electrophoretic mobility were measured. It was shown that incubation with trypsin increased the ease with which the cells could be deformed without changing electrophoretic mobility, and that diisopropylfluorophosphate (DFP)-trypsin was inactive, a fact which suggests that trypsin-sensitive peptide linkages help to maintain the "tension" at the cell periphery. On the other hand, chymotrypsin reduced cellular electrophoretic mobility without appreciably altering deformability. This suggests that, although chymotrypsin-sensitive bonds do not contribute to "tension," they are in some way associated with charged groups at the cell periphery.     

Characterization of Proteinases Excreted by Bacillus subtilis Marburg Strain during Sporulation

S ummary . This study has characterized 3 proteolytic enzymes during sporulation by Bacillus subtilis Marburg strain when grown in nutrient broth. A method of purification is described which permits the separation of 2 different proteinases: one belonging to the metal enzyme group and the other to the serine enzyme group. The third enzyme, probably an esterase, showed a high esterolytic activity, but only low proteolytic activity. Determination of the 3 enzymes in a mixture was accomplished by using specific substrates and inhibitors. They were excreted simultaneously between the end of the growth phase until the appearance of the prespores. During this entire period, 20% of the total proteolytic activity was due to the metal proteinase; 80% of the proteolytic activity and 15% of the esterolytic activity was due to the serine proteinase; 85% of the esterolytic activity was the result of the esterase. These findings will contribute to a more complete phenotypic characterization of those mutants of sporulation that appear to be involved in the production of extracellular hydrolytic enzymes.     

Enzymes of the Tryptophan Pathway in Three Bacillus Species

The tryptophan synthetic pathway was characterized in three species of Bacillus, B. subtilis, B. pumilus, and B. alvei. They share the common features of a pathway which is subject to tryptophan repression, contains no unexpected complexes among the five enzymes, exhibits dissociable anthranilate synthase enzymes which do not require phosphoribosyl transferase for amidetransfer activity, contains separate indoleglycerol phosphate synthase and phosphoribosylanthranilate isomerase enzymes, and contains similar tryptophan synthetase multimers. In looking at these characteristics in detail however, differences among the three species became apparent, as, for example, in the complementation observed between the alpha and beta(2) components of tryptophan synthetase, and the dissociation patterns of the large and small components of anthranilate synthase. The results demonstrate some pitfalls in attempting to compare multimeric enzymes in crude extracts from different organisms.     

Microbial Transformation of Quercetin by Bacillus cereus

Biotransformation of quercetin was examined with a number of bacterial cultures. In the presence of a bacterial culture (Bacillus cereus), quercetin was transformed into two crystalline products, identified as protocatechuic acid and quercetin-3-glucoside (isoquercitrin).     

Induction of Calcium Carbonate by Bacillus cereus

The purpose of this research was to study how the bacteria Bacillus cereus (DCB1) utilizes calcium ions in a culture medium with carbon dioxide (CO₂) to yield calcium carbonate (CaCO₃). The bacteria strain DCB1 was a dominant strain isolated from dolomitic surfaces in areas of Karst topographies. The experimental method was as follows: a modified beef extract-peptone medium (beef extract 3.0 g, peptone 10 g, NaCl 5.0 g, CaCl₂ 2.0 g, glass powder 2.0 g, distilled water 1 L, and a pH between 6.5 and 7.5) was inoculated with B. cereus to attempt to induce the synthesis of CaCO₃. The sample was then processed by centrifugation every 24 h during the 7-day cultivation period. The pH, carbonic anhydrase (CA) activity, and the concentrations of both HCO^- ₃ and Ca²⁺ in the supernatant fluid were measured. Subsequently, precipitation in the culture medium was analyzed to confirm, or otherwise, the presence and if present, the formation, of CaCO₃. Methods used included X-ray diffraction (XRD), Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Energy Dispersive Spectroscopy (EDS). Meanwhile, the carbon source in the carbonate was classified by its isotope composition. Results showed that B. cereus can improve its pH value in this culture medium; concentrations of HCO^- ₃ and Ca²⁺ showed a significant decline over the duration of the cultivation period. CA activity reached its maximum during the second day; XRD, SEM, TEM, and isotope analysis all revealed the presence of CaCO₃ as a precipitate. Additionally, these results did not occur in an aseptic control group: no detectable level of CaCO₃ was produced therein. In conclusion: B. cereus can metabolize active materials, such as secretase, by its own growth and metabolism, and can either utilize atmospheric CO₂, or respire, to induce CaCO₃ production. Experimental evidence is offered for a concomitant CO₂ reduction and CaCO₃ induction by microorganisms.