Autolysis of Bacillus cereus cell walls and isolation of structural components

Autolysis of Bacillus cereus N.R.R.L. 569 cell walls was accompanied by hydrolysis of the majority of the 4-O-beta-N-acetylglucosaminyl-N-acetylmuramic acid linkages in mucopeptide, presumably by an endo-beta-N-acetylglucosaminidase. Hydrolysis of the N-acetylmuramyl-l-alanine linkages by an amidase also occurred. Free d-alanine residues were detected in isolated cell walls and the proportion of these residues increased during autolysis, presumably due to d-alanine carboxypeptidase action. Fractionation and analysis of the products of autolysis confirmed these results. Among the products originating from mucopeptide were a disaccharide, N-acetylmuramyl-N-acetylglucosamine, and a tetrapeptide of sequence l-Ala-d-Glu-meso-Dap-d-Ala (Dap=diaminopimelate). A dimer fraction containing a d-Ala-meso-Dap cross-link was also isolated. Two polysaccharides were obtained from the products of autolysed cell walls and from walls made soluble by Chalaropsis B glycosidase. A neutral polysaccharide accounted for about 40% of the wall and contained N-acetylglucosamine, N-acetylgalactosamine and glucose. The neutral polysaccharide isolated from wall autolysates was attached to a part of the glycan moiety of mucopeptide. The molecular weight of the complex was approx. 28000. Stoicheiometric amounts of phosphorus were present, possibly in linkages between the polysaccharide and mucopeptide moieties. The second polysaccharide accounted for 12% of the wall and was very acidic. After acidic hydrolysis of the polysaccharide, glucosamine, galactosamine and unidentified acidic substances were detected. The acid polysaccharide isolated from wall autolysates contained only traces of mucopeptide constituents and no phosphorus.     

Purification and characterization of an exo-1,4-beta-galactanase from a strain of Bacillus subtilis

An arabinogalactan 4-beta-D-galactanohydrolase was purified to a homogeneous state from the culture filtrate of a strain of Bacillus subtilis. The enzyme have a molecular mass of 36 kDa and an isoelectric point of pH 7.9. The enzyme is most active at around pH 6.5-7 and at 60 degrees C, and is stable between pH 6-10 and below 55 degrees C. Hg2+ and Cu2+ inhibit the activity. The enzyme hydrolyze soybean arabinogalactan which contains beta-1,4-galactosidic linkages in its main chain structure, but not other polysaccharides with beta-1,3-galactosidic linkages. The hydrolysis products from soybean arabinogalactan are predominantly galactobiose with a small amount of galactotetraose. The enzyme is an exo-enzyme and the ability to transfer galactobiose to other galactobiose molecules is indicated by the formation of galactotetraose.     

pH-induced structural changes of apo-lactoferrin and implications for its function: a molecular dynamics simulation study

The minor protein in milk, lactoferrin (Lf), is known for a variety of biological functions, and has been investigated as a protective encapsulant for probiotic bacteria in health-promoting food products. Lf is likely to be exposed to extreme pH conditions which are known to have disruptive influences on its functionality. The molecular mechanisms underlying these pH-dependent changes are not well-understood. To explore the potential of Lf as an encapsulant, molecular dynamics (MD) simulations were applied to study its conformational changes under extreme acidic (pH 1.0) or basic (pH 14.0) conditions, relative to neutral pH. Simulations indicate that the structure of apo-Lf is relatively stable at neutral pH, while acidic and basic pH result in substantially greater flexibility, partly induced by the loss of contacts between the N- and C-terminal lobes, causing them to undergo extensive relative bending and twisting motions. Basic pH causes greater structural disruption compared to acidic exposure. The latter has greater influence on the N-terminus, with increased fluctuations and disruptions of inter-residue contacts compared to those at neutral pH; while basic pH was found to more prominently disrupt contacts at the C-terminus. These results help elucidate possible functional consequences on Lf of exposure to extreme pH conditions.     

Possible role of hydrogen cyanide in chemical evolution investigation of the proposed direct synthesis of peptides from hydrogen cyanide

The compounds formed upon oligomerization of cyanide in aqueous solution have been separated into acidic, basic, amphoteric and neutral fractions. Urea is the major constituent of the neutral fraction and oxalic acid is present in the acidic fraction. The oligomerization mixtures isolated in the acid, basic and amphoteric fractions consist of low molecular weight substances which yield amino acids on acid hydrolysis. Citrulline has been identified as a major amino acid released on acid hydrolysis of the product mixture. No amino acids are released from the oligomerization mixture by pronase or carboxypeptidase A. This catalyzed hydrolysis demonstrates that this system does not contain compounds with peptide links. The oligomerization products are susceptible to oxidizing agents but are affected little by reducing agents.     

Solid phase synthesis of oligodeoxyribonucleoside phosphorodithioates from thiophosphoramidites.

Oligonucleoside phosphorodithioates 1 are modified DNA sequences with potential use as antisense oligonucleotides. The preparation of up to 20-mers containing all four bases by solid phase synthesis is described, with details on the preparation of the four monomer units (protected nucleoside thiophosphoramidites 2), the conditions used for the assembly of the strands with up to 19 phosphorodithioate linkages, and the purification and characterisation of the products. Full-length homogeneity of HPLC-purified all-phosphorodithioate products is demonstrated by PAGE, but 31P NMR discloses the presence of phosphorothioate impurities (typically 8-9%), the origin of which is discussed. Oligonucleoside phosphorodithioates are freely soluble in water at neutral or basic pH, and are very stable towards oxidation, hydrolysis, and nuclease cleavage. Their ability to hybridize to complementary DNA has been studied by UV melting point (Tm) measurements. The observed depression of Tm, 0.5-2 degrees C per phosphorodithioate linkage, is higher that the 0.4-0.6 degrees C found for phosphorothioates.     

Studies on the chemical stability of propylene glycol alginate esters

The chemical stability of propylene glycol alginates (PGAs) has been examined. Under acidic conditions the ester groups in PGA are stable to hydrolysis but hydrolytic degradation of the glycosidic linkages in the polysaccharide backbone occurs. Under alkaline conditions the ester groups are hydrolysed with the primary 2-hydroxyprop-1-yl ester groups being more susceptible than secondary 1-hydroxyprop-2-yl ester groups, with little degradation of the polysaccharide backbone. Sodium carbonate-bicarbonate buffer was a much more effective hydrolysing reagent than sodium hydroxide at the same concentration and pH, and the rate of hydrolysis was greatly accelerated by increasing the hydrolysis temperature. Acetate, citrate and phosphate ions accelerated the rate of hydrolysis of the ester groups in PGA when added to the sodium hydroxide hydrolysing reagent. Hydrolysis of the ester groups in PGA with sodium hydroxide was unaffected by the addition of imidazole. However hydrolysis of the ester groups in PGA with sodium hydroxide in the presence of 1-aminobutane led to the formation of an alginate amide in which only the primary 2-hydroxylprop-1-yl ester groups were present, suggesting that a nucleophilic substitution of primary ester groups by amine groups is involved in the reaction.     

Enhanced intracellular delivery of quantum dot and adenovirus nanoparticles triggered by acidic pH via surface charge reversal

Quantum dot (QD) and adenovirus (ADV) nanoparticles were surface-modified with graft copolymers that exhibited a charge reversal behavior under acidic condition. Poly(L-lysine) (PLL) was grafted with multiple biotin-PEG chains (biotin-PEG-PLL graft copolymer), and the remaining primary amine groups in the PLL backbone were postmodified using citraconic anhydride, a pH-sensitive primary amine blocker, to generate carboxylate groups. The surfaces of streptavidin-conjugated QDs were modified with citraconylated biotin-PEG-PLL copolymer, producing net negatively charged QD nanoparticles. Under acidic conditions, citraconylated amide linkages were cleaved, resulting in the recovery of positively charged amine groups with subsequent alteration of surface charge values. Intracellular delivery of QD nanoparticles was greatly enhanced in an acidic pH condition due to the surface charge reversal. The surface of avidin-conjugated adenovirus (ADV-Avi) encoding an exogenous green fluorescent protein (GFP) gene was also modified in the same fashion. The expression extent of GFP was significantly increased at more acidic pH than pH 7.4. This study demonstrates that various nanosized drug carriers, imaging agents, and viruses could be surface-engineered to enhance their cellular uptake specifically at a low pH microenvironment like solid tumor tissue.     

Characterization and comparison of leuprolide degradation profiles in water and dimethyl sulfoxide.

The effect of solvent on the rate of leuprolide degradation and on the structure of the degradation products was explored. Leuprolide solutions (370 mg/mL) were prepared in water and dimethyl sulfoxide (DMSO) for delivery in DUROS osmotic implants. Both solvent systems demonstrated better than 90% stability after 1 year at 37 degrees C, where the DMSO formulation afforded better stability than the aqueous formulation and was used in subsequent clinical trials. The rate of leuprolide degradation in DMSO was also observed to accelerate with increasing moisture content, indicating that the aprotic solvent minimized chemical degradation. Interestingly, leuprolide degradation products varied with formulation vehicle. The proportions of leuprolide degradation products observed to form in water and DMSO at 37 degrees C were hydrolysis > aggregation > isomerization > oxidation and aggregation > oxidation > hydrolysis > isomerization, respectively. Specifically, more N-terminal hydrolysis and acetylation were observed under aqueous conditions, and increased Trp oxidation and Ser beta-elimination were seen under non-aqueous conditions. Furthermore, the major chemical degradation pathway changed with temperature in the DMSO formulation (decreasing oxidation with increasing temperature), but not in the aqueous formulation.     

Isolation and analysis of a novel acidic polysaccharide from the case of squid pen.

A new non-sulphated acidic polysaccharide with an average molecular mass of 55 kDa was isolated from squid pen case after papain digestion and beta-elimination. This polysaccharide contains mainly L-iduronic acid, D-glucuronic acid, D-galactosamine, D-glucosamine and significant amounts of neutral sugars as glucose, galactose and fucose. The polysaccharide was not degraded to the relative disaccharides by chondroitinases ABC, AC and B, hyaluronidase and keratanase or by treatment with heparinases, suggesting a structure different from those of known glycosaminoglycans. The polysaccharide cannot form self aggregates.     

Hydrolysis of poly(alkylene amidophosphate)s containing amino acid or peptide residues in the side groups. Kinetics and selectivity of hydrolysis

Kinetics of hydrolysis of poly(alkylene amidophosphate)s with amino acids or dipeptides as the side groups was studied by 31P NMR at pH 1.5, 6.5, and 8.5. The direction of hydrolysis and the relative rate coefficients of breaking P-O bonds in the main chain and P-N bonds in the side groups depend strongly on the pH of the medium of hydrolysis. The P-N (amide) bond hydrolyzes much faster than the P-O (ester) bond in acidic and close to neutral conditions (negligible P-O hydrolysis), whereas above pH > or = 8.5 these differences are much smaller. For instance, for 4-Ala the rate coefficients of hydrolysis are equal (in H2O at 37 degrees C and pH 8.5) to 1.9 x 10(-8) s(-1) and 1.0 x 10(-9) s(-1) for the P-N and P-O bonds, respectively, quite different from the values found for the low molecular model 2 (0 and 1.4 x 10(-7) s(-1), respectively).     

The xylanase system of Coniophora cerebella

1. The culture filtrate of the fungus Coniophora cerebella grown on poplar 4-O-methylglucuronoxylan as carbon source and enzyme inducer contained an enzyme system that degraded the polysaccharide to xylose, acidic and neutral oligosaccharides and an enzyme-resistant polymer. Free uronic acid was not produced. 2. Cold ethanol fractionation of the culture filtrate yielded two active fractions, one of which had only xylanase (EC 3.2.1.8) and the other both xylanase and xylosidase (EC 3.2.1.37) activities. Further fractionation on DEAE-cellulose resolved the xylanase and xylosidase activities. 3. The xylanase degraded poplar 4-O-methylglucuronoxylan in an essentially random manner, producing oligosaccharides, but some xylose residues in the vicinity of uronic acid side groups were protected from hydrolysis, preventing a truly random attack. The xylosidase attacked the polysaccharide very slowly, releasing xylose, but the oligosaccharides produced by the action of the xylanase were much more susceptible to hydrolysis by the xylosidase. 4. The products of xylanase action were separated into neutral and acidic fractions. The neutral oligosaccharides were separated by chromatography on charcoal-Celite, and the major products were characterized as xylobiose, xylotriose, xylotetraose and xylopentaose. Some of the acidic sugars were branched, having the uronic acid residue attached to a xylose residue other than the terminal non-reducing one. 5. Gel filtration of various xylanase fractions gave values for the molecular weight of the enzyme from 34000 to 38000.     

Biosynthesis and characterization of lipid-linked sugars and glycoproteins in aorta.

A particulate enzyme from bovine aorta catalyzes the incorporation of mannose from GDP-D-[14C]mannose into three products as follows: 1. Most of the radioactivity which is incorporated in short term incubations is into a product that is soluble in CHCl3/CH3OH (2/1, v/v). This product was purified by chromatography on DEAE-cellulose and Sephadex LH-20. The purified glycolipid was stable to alkaline saponification but released [14C]mannose when subjected to mild acid hydrolysis (1/2 = 7 min at 100 degrees in 0.01 N HCl). The purified glycolipid had the same mobility on silica gel plates in an acidic, basic, or neutral solvent system as did glycolipid had the same mobility on silica gel plates in an acidic, basic, or neutral solvent system as did authentic dolichyl mannopyranosyl phosphate. The synthesis of the 14C-mannolipid was reversed by the addition of GDP and Mg2+. 2. [14C]mannose is also incorporated, although at a slower rate into products which are soluble in CHCl3/CH3OH/H2O (1/1/0.3, v/v). When the 1/10.3 soluble material was chromatographed on Avicel plates, it gave rise to three distinct radioactive bands which appear to be lipid-linked oligosaccharides. Mild acid hydrolysis of the 1/10.3 soluble material released water-soluble, neutral 14C-oligosaccharides which eluted from Sephadex G-50 in two or three peaks between the standards cytochrome c and GDP-mannose...     

Mass spectrometry of uronic acid derivatives. XI--Structure elucidation by mass spectrometry of aldobiouronic and pseudoaldobiouronic acids and their beta-elimination products as per-o-methyl derivatives

Electron impact mass spectra of a series of aldobiouronic and pseudoaldobiouronic acid per-O-methyl derivatives and of the corresponding 4,5-unsaturated analogues, found normally among the products of methylation of uronic acid containing disaccharides as a result of methylation accompanying beta-elimination, have been studied. Using labelling experiments, metastable transition measurements and high resolution mass spectrometry, the fragmentation mechanisms of substances of this class have been deduced. Application of the information to the structure elucidation of this type of compound is discussed. It is concluded that from the mass spectra alone it is possible to determine the molecular weight, the cycle masses as well as the mode of linkage between the monomeric units. The appearance potentials of ions formed by cleavage of the glycosidic linkages have also been determined and the energetic differences encountered in the fission of the glycosidic linkages of various types of uronic acid containing oligosaccharides are discussed.     

Differences in the pattern of attack of acidic,neutral, and basic phospholipases A2 of A. halys blomhofii on human erythrocyte membranes: Problems in interpretation of phospholipid location

Purified acidic (pI 4.9), neutral (pI 6.9), and basic (pI 8.7) phospholipase A₂ from Agkistrodon halys blomhofii showed characteristically different patterns of hemolysis and phospholipid hydrolysis of intact human erthyrocytes. Acidic and neutral enzymes were nonlytic in the early periods of incubations with intact erythrocytes whereas the basic enzyme caused immediate hemolysis (5–8%). Under nonlytic conditions acidic and neutral enzymes hydrolyzed only phosphatidyl choline (PC) (20 and 50%, respectively), whereas basic enzyme hydrolyzed not only PC (60%) but nearly 15% of the phosphatidylethanolamine (PE). Both PC and PE were hydrolyzed significantly when the three phospholipases A₂ were incubated individually with erythrocyte lysate or hypotonic ghosts (sealed or unsealed). The order of substrate preference for acidic and neutral enzymes was always PC > PE. On the contrary basic enzyme exhibited the property of substrate specificity reversal. It hydrolyzed PC faster than PE when the membranes were sealed whereas PE hydrolysis was faster than PC hydrolysis in unsealed membranes. Interestingly only the basic enzyme showed activity in the absence of Ca²⁺ and in the presence of 0.5 mm EDTA. Phospholipase C (Bacillus cereus or Clostridium perfringens) did not show the property of substrate specificity reversal although their ability to hydrolyze PC and PE was different. In general this study demonstrates the unique activity patterns of three physically different pure phospholipases A₂ on human erythrocyte membranes which could be of value in selectively modifying membrane phospholipids. In addition it also throws an important light on the fact that results obtained with phospholipases should be interpreted with caution particularly as regards the localization of phospholipids in membranes.     

Comparative study of acidic glycosphingolipids by field desorption and secondary ion mass spectrometry

Acidic glycosphingolipids were analyzed by field desorption (FD-MS) and secondary ion mass spectrometry (SI-MS) using the primary ion Xe+ with a glycerol matrix. In the analysis of underivatized gangliosides by FD-MS, the fragment corresponding to the asialo residue resulting from the cationized cluster ion (M + Na)+ was the base peak, and ions due to cleavage at the glycosidic linkages were detected, as in the neutral glycosphingolipids. In the case of sulfatide, the ceramide fragment showed the highest intensity in the spectrum. In SI-MS spectra of acidic glycosphingolipids, (M + Na)+, (M + 2Na-H)+, and (M + K)+ were continuously detected as relatively high intensity ions during analysis of gangliosides and sulfatide. Other ions were mostly similar to those obtained by FD-MS. In FD-MS spectra of permethylated gangliosides, the cationized molecular ion (M + Na)+ was the base peak, and fragment ions due to asialo gangliosides were prominent. Other peaks were hard to detect. In SI-MS, molecular ions (M + H)+ and (M + H-32)+ and other ions due to cleavage of the glycosidic linkages were clearly detected. In this case, the sensitivity was greatly improved. Ions due to the non reducing end sugars were clearly detected, because of the relatively low intensity of ion peaks due to the glycerol matrix. It is concluded that the combination with FD-MS and SI-MS is particularly useful for the determination of molecular weight, sugar sequence and ceramide structure with sample amounting to only a few micrograms order.     

Selective production of organic acids in anaerobic acid reactor by pH control

The selective production of organic acids by anaerobic acidogenesis with pH control was examined using a chemostat culture. The results showed that the product spectrum in the acid reactor strongly depended on the culture pH. Under acidic and neutral conditions, the main products were butyric acid, while acetic and propionic acids were the main products under the basic condition. This phenomenon was reversible between the acidic and basic conditions, and was not affected by the dilution rate. The change in the main products was caused by the change in the dominant microbial populations, from butyric acid-producing bacteria to propionic acid-producing bacteria in the acid reactor due to the pH shift. The control of culture pH was considered to be a useful way for controlling the product spectrum in the anaerobic acid reactor.     

Amino acid and soluble protein cocktail from waste keratin hydrolysed by a fungal keratinase of Paecilomyces marquandii

Waste bovine hooves and horns were enzymatically hydrolysed into soluble products intended for foliar fertilizer. With the powdered keratin at 50°C and pH 8 between 34 to nearly 60% of nitrogen was solubilized in 5 h, depending on the enzyme concentration. The reaction could further be improved by steam pretreatment of the keratin, resulting in 98% solubilisation of the nitrogen. The products of hydrolysis consisted of a mixture of soluble proteins, peptides, and free amino acids. Among the latter, 18 common amino acids were detected. Several of them were previously recognized to have a positive effect on plants. Nonpolar neutral, basic, and sulphur amino acids were present in relatively large amounts, while proline and tryptophan were not found. Comparison with other protein hydrolysates aimed for fertilizer suggests that keratin degradation products, obtained by enzymatic hydrolysis, have potential to be used for foliar fertilization, alone or in a combination with another complementary hydrolysate of a different source, such as skin or plant proteins.     

Kinetic study of partially purified cellulase enzyme produced by Trichoderma viride FCBP-142 and its hyperactive mutants

Cellulases are the enzymes that cleave beta-1,4 linkages of cellulose, and carbohydrate that is main part of plants' cell walls. Presently, cellulase isolation and partial purification was executed through ammonium sulfate precipitation. The isolated protein of parental and derived mutants conferred molecular weights of 30, 45 and 55 kDa. The optimum temperature for maximal cellulase activity was 50 degrees C with Ea for substrate hydrolysis of 77.73, 83.97 and 83.14 kJ mol(-1) and temperature quotient of 1.0020, 1.0022 and 1.0022 by Trichoderma viride FCBP-142, Tv-UV-5.6 and Tv-Ch-4.3, respectively. The enzyme was stable at 50 degrees C for about 60 min but rapid denaturation occurred above 55 degrees C. The enzyme showed optimum activity at pH 4.0 and involved two types of acidic and basic limbs with pKa1 and pKa2. The pKa1 of active site presented a significant shift from 2.55 to 2.9 and 3.1 by Tv-UV-5.6 and Tv-Ch-4.3, respectively in comparison to parental strain. Likewise, pKa2 moved from 6.05 to 6.5 and 6.4. Enzyme kinetics displayed Michaelis-Menten constant Km 0.6, 0.5 and 0.28 mg mL(-1) and Vmax value of 8.33, 10 and 9.09 Units mL(-1) for parental, Tv-UV-5.6 and Tv-Ch-4.3, respectively.     

Evidence for the synthesis of soluble peptidoglycan fragments by protoplasts of Streptococcus faecalis.

Growing protoplasts of Streptococcus faecalis 9790 were found to synthesize and excrete soluble peptidoglycan fragments. The presence of soluble peptidoglycan derivatives in culture supernatants was determined by (i) incorporation of three different radioactively labeled precursors (L-lysine, D-alanine, and acetate) into products which, after hen egg-white lysozyme hydrolysis, had the same KD values on gel filtration as muramidase hydrolysis products of isolated walls; (ii) inhibition of net synthesis of these products by cycloserine and vancomycin; and (iii) identification of disaccharide-peptide monomer using the beta-elimination reaction, gel filtration, and high-voltage paper electrophoresis. Under the conditions of these experiments the presence of newly synthesized, acid-precipitable (macromolecular) peptidoglycan was not detected. The predominance of monomer (70 to 80%) in lysozyme digests of peptidoglycan synthesized by protoplasts was in sharp contrast to digest of walls from intact streptococci which contain mostly peptide cross-linked products. Biosynthesis and release of relatively uncross-linked, soluble peptidoglycan fragments by protoplasts was related to the absence of suitable, preexisting acceptor wall.