Optimization of pH for high molecular weight pullulan

Summary Pullulan is a polysaccharide produced by Aureobasidium pullulans. In this study, the effect of pH on the molecular weight of pullulan was investigated. High concentration of pullulan was obtained when initial pH was 6. Pullulan having molecular weight of 500,000–600,000 was produced at initial pH of 3.0, while pullulan with molecular weight of 200,000–300,000 was produced at pH above 4.5. To obtain high molecular weight pullulan with high concentration, pH was initially controlled at pH 6, followed by pH shift from pH 6 to pH 3. Transition of pH at 2 days of fermentation was observed to be optimum. Higher molecular weight pullulan was also obtained when sucrose concentration was 50 g/l compared to the result obtained at initial sucrose concentration of 20 g/l. Sucrose concentration and pH of the fermentation broth seem to be important parameters in obtaining high molecular weight of pullulan.     

Effect of the pH of the lysing solution on the sedimentation properties of the nucleoid of intact and irradiated Ehrlich ascites tumor cells

A study was made of the sedimentation properties of Ehrlich ascites tumor cell nucleotide obtained by cell lysis in 1.95 M NaCl, 0.1 M EDTA, 0.02 M tris, and 0.5% triton X-100, at pH 5.0 and 8.0. It was shown that the nucleotide obtained at pH 5.0 has a more compact structure than that obtained at pH 8.0. Irradiation of cells leads to relaxation of the nucleotide decreasing the rate of its sedimentation in a neutral sucrose gradient. The influence of irradiation on the sedimentation properties is more pronounced with pH 5.0 than 8.0.     

Effect on fertility of storing fowl semen for 24 h at 5 degrees C in fluids of different pH.

Improved storage of fowl semen above 0 degree C was achieved by adjusting the pH of the diluent. The fertility obtained with semen stored for 24 h at 5 degrees C in diluents buffered at different pH values was compared with that of semen stored in a basic, unbuffered solution. The most satisfactory result was achieved with diluent buffered at pH 6.8 OR 7.1. Worst fertility was obtained at pH 5.8 and pH 7.4 did not prove very satisfactory. There were indications that the effect of pH under the conditions of the experiment was to regulate metabolism and thereby influence the maintenance of the fertilizing ability of the spermatozoa.     

Improvement for the production of clavulanic acid by mutant Streptomyces clavuligerus

AIMS: To improve the production of clavulanic acid through the development of strains, the selection of a production medium and a pH shift strategy in a bioreactor. METHODS AND RESULTS: Streptomyces clavuligerus mutant 15 was selected by antibacterial activities. As a result of pH control in a 2.5 l bioreactor, the highest productivity (3.37 microg x ml(-1) x h(-1)) was obtained at a controlled pH of 7.0. CONCLUSIONS: The highest level of production obtained was an increase of about 36% compared with a non-controlled pH. When the production of clavulanic acid reached the maximum level, the pH was shifted from 7.0 to 6.0 for reduction in decomposition rate. The maximum concentration of clavulanic acid was maintained for 24 h as a result of the pH shift control, and a significant reduction in the decomposition of clavulanic acid was obtained. SIGNIFICANCE AND IMPACT OF THE STUDY: Clavulanic acid decomposition was considerably reduced as a result of the pH shift control. The results of this study can be applied for the efficient production of beta-lactamase inhibitory antibiotics.     

Effect of pH on exocellular riboflavin production byEremothecium ashbyii

Summary The production of riboflavin byEremothecium ashbyii grown in a chemically defined medium in batch culture was affected by pH of the medium. Highest yields were obtained at constant pH of 4.5 and 5.5, while little or no riboflavin was detected at either pH 3.5 or 8.5. The medium pH also affected cell morphology. When the organism was grown in a stirred tank reactor and an airlift vessel at pH 4.5 very similar levels of riboflavin were obtained.     

Kinetic analysis of catechin oxidation by polyphenol oxidase at neutral pH

Catechin oxidation by peach polyphenol oxidase was performed in a pH range of 3.5-8.0. At acidic pH, maximal spectral changes were observed at 390nm and at pH 7.5, at 430nm. Catechin oxidation was studied at pH 7.5 to avoid the formation of free radicals. The results obtained allowed us to propose a pathway for the enzymatic oxidation of catechin, according to which enzymatic oxidation produces the corresponding catechin-o-quinone, which suffers the nucleophilic attack of another catechin unit, leading to the formation of a dimer. This dimer is then oxidized by the enzymatically generated o-quinone. The progress curves obtained for catechin oxidation by PPO showed a lag period, whose length changed with enzyme and substrate concentrations, and which must have been caused by the chemical reactions taking place after the enzymatic reaction. The results obtained by simulation of the model produced the same qualitative dependences as obtained experimentally.     

Modelling and optimization of environmental conditions for kefiran production by Lactobacillus kefiranofaciens

A mathematical model for kefiran production by Lactobacillus kefiranofaciens was established, in which the effects of pH, substrate and product on cell growth, exopolysaccharide formation and substrate assimilation were considered. The model gave a good representation both of the formation of exopolysaccharides (which are not only attached to cells but also released into the medium) and of the time courses of the production of galactose and glucose in the medium (which are produced and consumed by the cells). Since pH and both lactose and lactic acid concentrations differently affected production and growth activity, the model included the effects of pH and the concentrations of lactose and lactic acid. Based on the mathematical model, an optimal pH profile for the maximum production of kefiran in batch culture was obtained. In this study, a simplified optimization method was developed, in which the optimal pH profile was determined at a particular final fermentation time. This was based on the principle that, at a certain time, switching from the maximum specific growth rate to the critical one (which yields the maximum specific production rate) results in maximum production. Maximum kefiran production was obtained, which was 20% higher than that obtained in the constant-pH control fermentation. A genetic algorithm (GA) was also applied to obtain the optimal pH profile; and it was found that practically the same solution was obtained using the GA.     

Rapid Ornithine Decarboxylase Test for the Identification of Enterobacteriaceae

Conventional methods for detecting ornithine decarboxylase activity require an extended period of incubation. However, with a few simple modifications, accurate results were obtained within a few hours rather than several days. The broth medium was modified, primarily by omitting glucose and by decreasing the pH to 5.5. A 1-ml amount of this broth was inoculated with one colony and then overlaid with sterile mineral oil. Within 2 to 4 hr, the pH increased if ornithine was decarboxylated, thus changing the color of the internal pH indicator to a dark purple. If the amino acid was not decarboxylated, the pH decreased to pH 5.0 to 5.2, enough to give a definite yellow color. With 347 selected clinical isolates, the rapid test gave results identical to those obtained in 1 to 4 days with Moeller's decarboxylase medium. Less reliable results were obtained with Difco's decarboxylase medium with 0.3% agar which was stabinoculated and read after 18 to 24 hr without a mineral oil seal. The rapid ornithine decarboxylase test represents a simple, accurate technique which is well suited for the clinical microbiology laboratory.     

Production and Purification of the Metalloprotease of Bacillus polymyxa

The nutritional and environmental factors relating to the production of an extracellular protease by Bacillus polymyxa were investigated. The enzyme was produced in all media that supported growth of the microorganism, irrespective of the carbon source used. Arabinose and hydrolyzed starch, however, gave highest yields. The nature of the peptone had a significant effect on the level of protease produced. Calcium and manganous ions exerted a beneficial effect on protease production. Highest enzyme levels were obtained when the initial pH of the medium was within the range 5.9 to 7.0. When the pH of the medium was not controlled during the fermentation, the accumulation of the enzyme paralleled the growth of the microorganism and reached a maximum towards the end of the exponential phase. With a fixed pH of 6.8, the level of protease was only one-fifteenth of that obtained when the culture was allowed to maintain its own pH. In addition, accumulation of the protease reached a maximum somewhat earlier, i.e., in the mid-log phase of growth. A 70-fold increase in the specific activity of the protease was obtained by ammonium sulfate and acetone fractionation followed by gel filtration on Sephadex G-100. The purified protease behaved as a homogenous entity when eluted by a sodium chloride gradient from CM-cellulose at pH 6.9. An overall enzyme recovery of 60% was obtained.     

Activation of proenzyme of acidic protease from human seminal plasma.

The kinetics and the extent of the conversion of the proenzyme into the active acidic protease (EC 3.4.23.--) of human seminal plasma were dependent on acidic pH. Between pH 2 and 4, the initial rate of the activation was first-order with respect to the proenzyme. Between pH 4.5 and 5, the rate deviated from the first-order with an initial lag period which can be abolished by adding an excess amount of the acidic protease or pepsin. The extent of the activation was complete between pH 2 and 3 and became incomplete between pH 4 and 5. Addition of the acidic protease or pepsin did not alter the extent of the activation at the high pH values. According to the chromatographic profile on a Sephadex G-75 column, the activation products (namely active acidic protease and an activation peptide) obtained at pH 3 and those obtained at pH 4.5 were identical. The molecular weight of the activation peptide obtained at pH 3 was 6900; its amino acid composition was analyzed and compared with those of the proenzyme and the acidic protease. Remarkable similarity between the amino acid composition of the acidic protease and that of human pepsin was observed. In the presence of an excess amount of hemoglobin, the conversion of the proenzyme was self-activated and showed an initial lag period. Addition of acidic protease did not change the rate of self activation or the lag period.     

The Effects of pH on the Labellar Sugar Receptor of the Fleshfly

Reproducible results describing the effects of pH on the response of the labellar sugar receptor of the fleshfly, Boettcherisca peregrina, were obtained. The response to sucrose was independent over a wide range of pH (3.0 to 10.0 for sucrose stimulation), but was inhibited fairly sharply on both sides of this range. Similar results were obtained for monosaccharide stimulation. The receptor was excited on stimulation by water above pH 12.0. The effects of high pH, both inhibitory and excitatory, were affected by the presence of salts. In the presence of 0.5 molar NaCl, for example, the pH-inhibition curve was shifted toward lower pH's by about one pH unit. The effects of low pH, on the other hand, were not affected by salts. Following Dixon's theory, it was concluded that at least five ionizable groups (loosing positive charges above pH 10.5) were located at the receptor site.     

Insights into pH‐induced metabolic switch by flux balance analysis

Lactate accumulation in mammalian cell culture is known to impede cellular growth and productivity. The control of lactate formation and consumption in a hybridoma cell line was achieved by pH alteration during the early exponential growth phase. In particular, lactate consumption was induced even at high glucose concentrations at pH 6.8, whereas highly increased production of lactate was obtained at pH 7.8. Consequently, constraint‐based metabolic flux analysis was used to examine pH‐induced metabolic states in the same growth state. We demonstrated that lactate influx at pH 6.8 led cells to maintain high fluxes in the TCA cycle and malate‐aspartate shuttle resulting in a high ATP production rate. In contrast, under increased pH conditions, less ATP was generated and different ATP sources were utilized. Gene expression analysis led to the conclusion that lactate formation at high pH was enabled by gluconeogenic pathways in addition to facilitated glucose uptake. The obtained results provide new insights into the influence of pH on cellular metabolism, and are of importance when considering pH heterogeneities typically present in large scale industrial bioreactors. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:347–357, 2015     

pH-sensitive genipin-cross-linked chitosan microspheres for heparin removal

Chitosan hydrogel microspheres were obtained by cross-linking chitosan in its inverse emulsion using genipin as cross-linker. The genipin-cross-linked chitosan microspheres (ChGp) swell significantly in water at pH values below 6.5 and shrink to a smaller extent at pH values above 6.5. ChGp microspheres bind heparin in water. The kinetics of heparin binding was found to be pH dependent and was faster and more efficient at a lower pH. That can be also controlled by the weight of ChGp microspheres used. Rate and efficiency of heparin adsorption at pH 7.4, which is typical of blood, could be increased by quaternization of ChGp microspheres using glycidyltrimethylammonium chloride (GTMAC). The polymeric material obtained thus can be potentially useful for heparin removal in biomedical applications.     

Activity of glutaraldehyde at low concentrations (less than 2%) against poliovirus and its relevance to gastrointestinal endoscope disinfection procedures.

The activity of glutaraldehyde (GTA) at low concentrations (less than 2%) against poliovirus was assessed by a suspension procedure. The inactivation kinetics showed that concentrations of less than or equal to 0.10% were effective against purified poliovirus at pH 7.2; a 1 log10 reduction was obtained in 70 min with 0.02% GTA, and a 3 log10 reduction was obtained in 30 min with 0.10% GTA. GTA activity at low concentrations was greatly enhanced at alkaline pH, but was completely abolished at acid pH. In contrast, the inactivation assays on poliovirus RNA showed that it was highly resistant to GTA at concentrations up to 1.0% at pH 7.2. At pH 8.3 a low inactivation was noticed with 1.0% GTA. Our results are of relevance to hospital practice in digestive endoscopy investigations because there has been an increasing tendency to use low concentrations of GTA and very short contact times in disinfection procedures.     

A natural inhibitor of lignin peroxidase activity from Phanerochaete chrysosporium,active at low pH and inactivated by divalent metal ions

Summary The pH optimum of a crude preparation of lignin peroxidase was pH 3.1, whereas those of the three main isozymes of the enzyme purified from it were pH 2.2, pH 2.7 and pH 2.0. During the purification of the crude enzyme, an anionic polysaccharide containing fraction (PCF) was also separated. The latter was found to inhibit lignin peroxidase activity at pH values less than pH 3.2, thus resulting in a shift in the pH optimum of the purified isozymes back to a similar value as that obtained for the crude enzyme. Addition of divalent metal ions at 1.0 mM relieved the inhibition.     

Rapid acidification of endocytic vesicles containing alpha 2-macroglobulin

We have used fluorescein-labeled alpha 2-macroglobulin (F-alpha 2M) to measure pH changes in the microenvironment of internalized ligands following receptor-mediated endocytosis. Fluorescence intensities of single BALB/c 3T3 mouse fibroblasts were measured by using a microscope spectrofluorometer with narrow bandpass excitation filters. The pH was determined from the ratio of fluorescein fluorescence intensities with 450 nm and 490 nm excitation. A standard pH curve was obtained by incubating cells with F-alpha 2M for 30 min at 37 degrees C followed by fixation and incubation in buffers of varying pH. To measure the pH of endocytic vesicles, cells were incubated with F-alpha 2M for 15 min at 37 degrees C. Fluorescence intensities were measured on living cells within 5 min of rinsing. Under these conditions, the pH of the F-alpha 2M microenvironment was 5.0 +/- 0.2. Using colloidal gold-alpha 2M for electron microscopic localizations we have verified that, under these conditions, alpha 2M is predominantly in uncoated vesicles that are negative for acid phosphatase activity. With further incubation for 1/2 hr, we obtained a pH of 5.0 +/- 0.2 for the F-alpha 2M. Using fluorescein dextran, we obtained a lysosomal pH of 4.6 +/- 0.2. These results indicate that endocytic vesicles become acidic prior to fusion with lysosomes.     

Fermentation of cheese whey by a mixed culture ofClostridium beijerinckii andBacillus cereus

Summary Fermentation of cheese whey to produce butanol and butyric acid was carried out using a mixed culture ofClostridium beijerinkii andBacillus cereus. Fermentation selectivities were studied by controlling the pH of the system. Controlled pH values higher than 6.5 as well as those below 5.0 were not conducive to butanol production. Maximum product formation was obtained by controlling the pH at 5.5. When compared with the results obtained using the pure culture ofC. beijerinckii, a higher butanol concentration was obtained in the mixed culture without sacrificing the level of butyric acid formed.     

Activity of glutaraldehyde at low concentrations (less than 2%) against poliovirus and its relevance to gastrointestinal endoscope disinfection procedures.

The activity of glutaraldehyde (GTA) at low concentrations (less than 2%) against poliovirus was assessed by a suspension procedure. The inactivation kinetics showed that concentrations of less than or equal to 0.10% were effective against purified poliovirus at pH 7.2; a 1 log10 reduction was obtained in 70 min with 0.02% GTA, and a 3 log10 reduction was obtained in 30 min with 0.10% GTA. GTA activity at low concentrations was greatly enhanced at alkaline pH, but was completely abolished at acid pH. In contrast, the inactivation assays on poliovirus RNA showed that it was highly resistant to GTA at concentrations up to 1.0% at pH 7.2. At pH 8.3 a low inactivation was noticed with 1.0% GTA. Our results are of relevance to hospital practice in digestive endoscopy investigations because there has been an increasing tendency to use low concentrations of GTA and very short contact times in disinfection procedures.     

Effect of pH on the Protective Action of Interferon in L Cells

The pH of the solution in which interferon was applied to L cells determined the level of resistance developed against challenge with vesicular stomatitis virus (VSV). No inhibition of challenge virus was observed when interferon was applied to cells at pH 6.0. At pH 6.5, partial inhibition of VSV replication was observed and inhibition was maximum at pH 7.0. Evidence was obtained that interferon interacted with L cells at pH 6.0, but that resistance did not develop until the cells were placed in a medium at pH 7.0. These effects were explained by data showing that exposure of cells to a medium at pH 6.0 reversibly inhibited both ribonucleic acid and protein synthesis.