Study of physical and biological factors involved in the disruption of E. coli by hydrodynamic cavitation

Hydrodynamic cavitation results in flow restriction in a flow system causing rapid pressure fluctuations and significant fluid forces. These can be harnessed to mediate microbial cell damage. Hydrodynamic cavitation was studied for the partial disruption of E. coli and selective release of specific proteins relative to the total soluble protein. The effects of the cavitation number, the number of passes, and the specific growth rate of E. coli on the release of periplasmic and cytoplasmic proteins were studied. At the optimum cavitation number of 0.17 for this experimental configuration, 48% of the total soluble protein, 88% of acid phosphatase, and 67% of beta-galactosidase were released by hydrodynamic cavitation in comparison with the maximum release attained using multiple passes through the French Press. The higher release of the acid phosphatase over the total soluble protein suggested preferred release of periplasmic compounds. This was supported by SDS-PAGE analysis. The absence of micronization of cell material resulting in the potential for ease of solid-liquid separation downstream of the cell disruption operation was confirmed by TEM microscopy. E. coli cells cultivated at a higher specific growth rate (0.36 h(-1)) were more easily disrupted than slower grown cells (0.11 h(-1)). The specific activity of the enzyme of interest released by hydrodynamic cavitation, defined as the units of enzyme in solution per milligram of total soluble protein, was greater than that obtained on release by the French Press, high-pressure homogenization, osmotic shock, and EDTA treatment. The selectivity offered indicates the potential of enzyme release by hydrodynamic cavitation to ease the purification in the subsequent downstream processing.     

Some peculiarities of functioning of H+-ATPase from the membranes of the anaerobic bacterium Lactobacillus casei

Radiation inactivation analysis gave the target sizes of 176 +/- 5 kDa and 275 +/- 33 kDa for ATPase from anaerobic Lactobacillus casei and aerobic Micrococcus luteus bacteria respectively. The values are close to the known molecular masses of the enzymes. Thus, to function the L. casei ATPase, like the F1-ATPases, requires a complete structure composed of all the enzyme subunits. L. casei ATPase is inhibited by 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole owing to modification of an amino acid residue(s) with pK greater than 8.5. L. casei ATPase consists of six identical subunits and differs from alpha 3 beta 3 gamma delta epsilon-type F1-ATPases in a number of catalytic properties. Namely, ATP hydrolysis under the 'unisite' conditions proceeds at a relatively high rate suggesting the absence of cooperative interactions between the catalytic sites. Contrary to mitochondrial F1-ATPase. L. casei ATPase does not form an inactive complex with ADP. These findings imply essential differences in the operating mechanism for L. casei ATPase and F1 ATPase.     

The disruption of Alcaligenes eutrophus by high pressure homogenisation: key factors involved in the process.

The disruption of the Gram-negative bacterium Alcaligenes eutrophus by high pressure homogenisation, using the APV Gaulin 15M 8BA and 30CD homogenisers is reported. The operating parameters such as operating pressure, number of passes, temperature and biomass concentration, mimicked trends previously reported for yeasts. Extension of the study to consider the effect of cell characteristics, including the growth rate, size and shape, illustrated the dominant effect of the growth phase. The improved disruption of bacterial cultures in the logarithmic phase with respect to stationary phase cultures was confirmed by an increased dependence of actively growing cultures on the operating pressure. An increase in size in excess of 30% on the accumulation of the storage product, PHB in the stationary phase caused little change in the ease of disruption. The use of transmission electron microscopy to directly monitor the disruption on multiple passes shed light on the two-stage nature of this disruption process.     

Disruption of native and recombinant Escherichia coli in a high-pressure homogenizer

The disruption of native and recombinant strains of Escherichia coli was studied using a high-pressure homogenizer (Microfluidizer). The cells were grown in both batch and continuous fermentations. Cell suspensions ranging from 4 to 175 g dry wt/L were investigated at disruption pressures ranging from 30-95 MPa and at up to five passes. For both types of cells, the fraction of cells disrupted was dependent on the growth rate and concentration of the cells, the disruption pressure, and the number of passes through the disrupter. A model is presented that correlates the fractional disruption with these operating variables. The recombinant strain disrupted more readily than the native strain; 95 to 98% disruption of the former was achieved in two to three passes at a pressure of 95 MPa.     

Simulation of particle size distribution changes occurring during high-pressure disruption of bakers' yeast

Measurements of size distributions are provided for the breakage of commercial packed bakers' yeast cells as a function of operating pressure and number of passes through a Manton Gaulin high-pressure homogenizer. A two parameter model was developed, based upon the use of a Boltzmann function, to simulate the changes in size distribution that accompany the cell breakage process. The effects of operating pressure and number of passes are incorporated in the model and the result is used to simulate the particle size distribution of the cell homogenate. The results show that there is little breakage below a threshold pressure of 115 bar and above which breakage is critically dependent upon the pressure and number of passes through the homogenizer. The analysis provides a means of studying the efficiency of centrifugation that may follow cell disruption and provides the basis for further studies of size distribution changes accompanying cell disruption. (c) 1996 John Wiley & Sons, Inc.     

Disruption of Saccharomyces cerevisiae using enzymatic lysis combined with high-pressure homogenization

The disruption of commercially-available pressed Bakers' yeast (Saccharomyces cerevisiae) was studied using a relatively new high-pressure homogenizer (the Microfluidizer). Initial experiments using only mechanical disruption generally gave low disruption yields (i.e., less than 40% disruption in 5 passes). Consequently combinations of two disruption methods, namely enzymatic lysis and subsequent homogenization, were tested to identify achievable levels of disruption. The enzyme preparation employed was Zymolyase, which has been shown to effectively lyse the walls of viable yeast. Yeast cell suspensions ranging in concentration from 0.6 to 15 gDW/L were disrupted with and without enzymatic pre-treatment. Final total disruption obtained using the combined protocol approached 100% with 4 passes at a pressure of 95 MPa, as compared to only 32% disruption with 4 passes at 95 MPa using only homogenization. A model is presented to predict the fraction disrupted while employing this novel enzymatic pretreatment.Nomenclature a exponent of pressure (-) - b exponent of number of passes (-) - K disruption constant (MPa^-a) - N number of passes (-) - P pressure (MPa) - R total fraction of cells disrupted (-) - Ro fraction of cells disrupted after enzymatic pre-treatment (-) - X cell concentration (dry weight) (gDW/L) abbreviation DW dry weight     

Combined chemical and mechanical processes for the disruption of bacteria.

Mechanical cell disruption by high pressure homogenisation or high speed bead mills is currently the general method of choice for the large scale disruption of micro-organisms; however, deleterious effects include the high energy requirement, the need for efficient cooling to prevent the excessive heating of the product and the micronisation of cell debris. Certain chemical treatments for microbial cell disruption alter the permeability of bacteria and yeasts, allowing partial release of soluble products. Such treatments are insufficient for the recovery of granular intracellular products. As cell wall strength has been cited as a major factor influencing the requirements for efficient mechanical disruption, the use of chemical pretreatment to decrease cell wall strength prior to mechanical breakage by homogenisation has been considered. The following treatments were shown to increase the sensitivity of the Gram-negative bacterium, Alcaligenes eutrophus, to disruption: alkaline pH shock, the addition of an anionic detergent, increase of the monovalent cation concentration, the addition of EDTA and enzymic lysis by lysozyme. These pretreatments allow equivalent disruption to be achieved at lower operating pressures or fewer passes through the homogeniser. Alkaline pH pretreatment at pH 10.5 allowed a 37.5% increase in soluble protein release on subsequent homogenisation. An increase of some 30% in soluble protein release was found following prior addition of 0.137 M monovalent cations (Na+ or K+) at 60 degrees C. Treatment with an anionic detergent showed a considerable decrease in the number of passes required through the homogeniser. Maximum cell rupture can thus be accomplished at reduced energy inputs.     

Purification and characterization of a prolidase from Lactobacillus casei subsp. casei IFPL 731.

A peptidase showing a high level of specificity towards dipeptides of the X-Pro type was purified to homogeneity from the cell extract of Lactobacillus casei subsp. casei IFPL 731. The enzyme was a monomer having a molecular mass of 41 kDa. The pH and temperature optima were 6.5 to 7.5 and 55 degrees C, respectively. Metal chelating agents completely inhibited enzyme activity, indicating that the prolidase was a metalloenzyme. The Michaelis constant (K(m)) and Vmax for several proline-containing dipeptides were determined.     

Disruption of Candida utilis cells in high pressure flow devices*

The disruption of Candida utilis cells in suspensions subjected to different types of stress was investigated. Stresses caused by impingement of a high velocity jet of suspended cells against a stationary surface were found to be significantly more effective for disruption than either shear or normal stresses. The fraction of cells disrupted by impingement is a first order function of the number of passes through the disruptor and, over a prescribed range of operating pressures, is a power function of pressure. These results indicate that impingement is the predominant mechanism causing cells disruption in high pressure flow devices such as Manton–Gaulin homogenizers. The impingement results suggest that cells grown in cyclic batch culture are easier to disrupt than cells grown at a lower specific growth rate in continuous culture. In addition to determining the fraction of cells disrupted, the release of invertase activity was determined for the impingement experiments. The fraction of total invertase activity released was found to be somewhat greater than the fraction of cells disrupted.     

Disruption of Brewers' yeast by hydrodynamic cavitation: Process variables and their influence on selective release

Intracellular products, not secreted from the microbial cell, are released by breaking the cell envelope consisting of cytoplasmic membrane and an outer cell wall. Hydrodynamic cavitation has been reported to cause microbial cell disruption. By manipulating the operating variables involved, a wide range of intensity of cavitation can be achieved resulting in a varying extent of disruption. The effect of the process variables including cavitation number, initial cell concentration of the suspension and the number of passes across the cavitation zone on the release of enzymes from various locations of the Brewers' yeast was studied. The release profile of the enzymes studied include alpha-glucosidase (periplasmic), invertase (cell wall bound), alcohol dehydrogenase (ADH; cytoplasmic) and glucose-6-phosphate dehydrogenase (G6PDH; cytoplasmic). An optimum cavitation number Cv of 0.13 for maximum disruption was observed across the range Cv 0.09-0.99. The optimum cell concentration was found to be 0.5% (w/v, wet wt) when varying over the range 0.1%-5%. The sustained effect of cavitation on the yeast cell wall when re-circulating the suspension across the cavitation zone was found to release the cell wall bound enzyme invertase (86%) to a greater extent than the enzymes from other locations of the cell (e.g. periplasmic alpha-glucosidase at 17%). Localised damage to the cell wall could be observed using transmission electron microscopy (TEM) of cells subjected to less intense cavitation conditions. Absence of the release of cytoplasmic enzymes to a significant extent, absence of micronisation as observed by TEM and presence of a lower number of proteins bands in the culture supernatant on SDS-PAGE analysis following hydrodynamic cavitation compared to disruption by high-pressure homogenisation confirmed the selective release offered by hydrodynamic cavitation.     

Characterization of the DNA binding region recognized by dihydrofolate reductase from lactobacillus casei

Two specific DNA binding sites for the enzyme dihydrofolate reductase from Lactobacillus casei have been located by means of an immunoprecipitation assay within a 2900-base pair L. casei DNA fragment containing the L. casei dihydrofolate reductase structural gene, which was previously cloned into pBR322. The inserted L. casei DNA was mapped using restriction endonucleases, and the location and orientation of the structural gene coding for L. casei dihydrofolate reductase were determined. The two specific binding sites map at the 5' end of the structural gene, approximately 100 base pairs upstream from the start of the coding region.     

Membrane ATPases and acid tolerance of Actinomyces viscosus and Lactobacillus casei

Lactobacillus casei ATCC 4646 and Actinomyces viscosus OMZ105E were found to differ markedly in acid tolerance. For example, pH profiles for glycolysis of intact cells in dense suspensions indicated that glycolysis by L. casei had an optimal pH of about 6.0 and that glucose degradation was reduced by 50% at a pH of 4.2. Comparable values for A. viscosus cells were at pHs of about 7.0 and 5.6. The difference in acid tolerance appeared to depend mainly on membrane physiology, and the addition of 40 microM gramicidin to cell suspensions increased the sensitivity of the glycolytic system by as much as 1.5 pH units for L. casei and up to 0.5 pH unit for A. viscosus. L. casei cells were inherently somewhat more resistant to severe acid damage than were A. viscosus cells, in that Mg release from L. casei cells in medium with a pH of 3.0 occurred only after a lag of some 4 h, compared with rapid release from A. viscosus cells. However, the major differences pertinent to the physiology of the organisms appeared to be related to proton-translocating ATPases. Isolated membranes of L. casei had about 3.29 U of ATPase per mg of protein, compared with only about 0.06 U per mg of protein for those of A. viscosus. Moreover, the ATPase of L. casei had a pH optimum for hydrolytic activity of about 5, compared with an optimal pH of about 7 for that of A. viscosus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane ATPases and acid tolerance of Actinomyces viscosus and Lactobacillus casei.

Lactobacillus casei ATCC 4646 and Actinomyces viscosus OMZ105E were found to differ markedly in acid tolerance. For example, pH profiles for glycolysis of intact cells in dense suspensions indicated that glycolysis by L. casei had an optimal pH of about 6.0 and that glucose degradation was reduced by 50% at a pH of 4.2. Comparable values for A. viscosus cells were at pHs of about 7.0 and 5.6. The difference in acid tolerance appeared to depend mainly on membrane physiology, and the addition of 40 microM gramicidin to cell suspensions increased the sensitivity of the glycolytic system by as much as 1.5 pH units for L. casei and up to 0.5 pH unit for A. viscosus. L. casei cells were inherently somewhat more resistant to severe acid damage than were A. viscosus cells, in that Mg release from L. casei cells in medium with a pH of 3.0 occurred only after a lag of some 4 h, compared with rapid release from A. viscosus cells. However, the major differences pertinent to the physiology of the organisms appeared to be related to proton-translocating ATPases. Isolated membranes of L. casei had about 3.29 U of ATPase per mg of protein, compared with only about 0.06 U per mg of protein for those of A. viscosus. Moreover, the ATPase of L. casei had a pH optimum for hydrolytic activity of about 5, compared with an optimal pH of about 7 for that of A. viscosus.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new synbiotic, Lactobacillus casei subsp. casei together with dextran, reduces murine and human allergic reaction

We studied the development of atopic dermatitis-like skin lesions in NC/Nga mice and the allergic symptoms and blood patterns of healthy volunteers during the cedar (Cryptomeria japonica) pollen season in Japan following oral administration of a new synbiotic, Lactobacillus casei subsp. casei together with dextran. The combination of L. casei subsp. casei and dextran significantly decreased clinical skin severity scores and total immunoglobulin E levels in sera of NC/Nga mice that had developed picryl chloride-induced and Dermatophagoides pteronyssinus crude extract-swabbed atopic dermatitis-like skin lesions. During the most common Japanese cedar pollen season, synbiotic L. casei subsp. casei and dextran in humans led to no significant changes in total nasal and ocular symptom scores, in the levels of cedar pollen-specific immunoglobulin E, interferon-gamma and thymus and activation regulated chemokine or in the number of eosinophils in sera, whereas the placebo group showed a tendency for increased levels of cedar pollen-specific immunoglobulin E, thymus and activation regulated chemokine and number of eosinophils, and a decrease in interferon-gamma levels. Thus, the oral administration of synbiotic L. casei subsp. casei together with dextran appears to be an effective supplement for the prevention and treatment of allergic reactions.     

Isolation and characterization of an intracellular esterase from Lactobacillus casei subsp. casei IFPL731

An intracellular esterase from Lactobacillus casei subsp. casei IFPL731 was purified 1000-fold by ion exchange chromatography and gel filtration chromatography. The relative molecular mass of the native enzyme was 105 kDa, while the subunit molecular mass was estimated to be 38 kDa. The esterase hydrolysed tributyrin and had a preference for esters of short-chain fatty acids (butyrate, caproate and caprylate), while it did not hydrolyse palmitate and sterate esters. The apparent Michaelis-Menten constant of the enzyme on p -nitrophenyl butyrate was 0·3 mmol l⁻¹ while on p -nitrophenyl caprylate, it was 0·04 mmol l⁻¹. The esterase was active over a broad range of pH and temperature values, and retained about 50% of maximal activity at pH 5·0 and 12 °C. Activity was strongly inhibited by 5 mmol l⁻¹ phenylmethylsulphonyl fluoride, β-mercaptoethanol and N -ethylmaleimide, and was stimulated by Zn²⁺ at 1 mmol l⁻¹.     

Cloning and characterization of two Lactobacillus casei genes encoding a cystathionine lyase

Volatile sulfur compounds are key flavor compounds in several cheese types. To better understand the metabolism of sulfur-containing amino acids, which certainly plays a key role in the release of volatile sulfur compounds, we searched the genome database of Lactobacillus casei ATCC 334 for genes encoding putative homologs of enzymes known to degrade cysteine, cystathionine, and methionine. The search revealed that L. casei possesses two genes that putatively encode a cystathionine beta-lyase (CBL; EC 4.4.1.8). The enzyme has been implicated in the degradation of not only cystathionine but also cysteine and methionine. Recombinant CBL proteins catalyzed the degradation of L-cystathionine, O-succinyl-L-homoserine, L-cysteine, L-serine, and L-methionine to form alpha-keto acid, hydrogen sulfide, or methanethiol. The two enzymes showed notable differences in substrate specificity and pH optimum.     

Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei. Sequences of the cyanogen bromide peptides and complete sequences of the enzyme.

The complete amino acid sequence of dihydrofolate reductase from an amethopterin-resistant strain of Lactobacillus casei has been determined by sequence analysis of peptides produced by cleavage with cyanogen bromide, trypsin, staphylococcal protease, and myxobacter protease. Comparison of this sequence with those of reductases from other bacterial sources shows that the enzymes are homologous. The Lactobacillus casei reductase sequences shows a 29% sequence identity with that of the Escherichia coli enzyme and a 34% identity with the sequence of the enzyme from Streptococcus faecium. The NH2-terminal 68 residues of the L. casei reductase show a 54% sequence identity with that of the enzyme from S. faecium.     

Increased disruption resistance of Candida utilis grown from survivors of enzymatic lysis combined with high-pressure homogenization

The resistance of Candida utilis (ATCC 9226) to disruption as a result of enzymatic pretreatment combined with high-pressure homogenization was found to increase when the yeast was grown from an inoculum which had previously been subjected to enzymatic pretreatment combined with high-pressure homogenization. The inoculum thus consisted of a mixture of undisrupted, viable cells and non-viable cells. The enzyme preparation employed was Zymolyase, which depolymerizes various components of the cell walls of viable yeast. A Microfluidizer was used for the high-pressure homogenization step. In order to obtain the 'disruption-resistant' cell fraction for use as an inoculum, 'normal' C. utilis was enzymatically pretreated, and subsequently homogenized (herein referred to as Microfluidization) using either three or 10 passes through the Microfluidizer at an operating pressure of 95 MPa. Yeast grown from the survivors of the enzyme/3-pass treatment were found to be somewhat more resistant to disruption by either enzymatic pretreatment alone or to enzymatic pretreatment followed by Microfluidization. Cells grown from enzyme/ 10-pass treated inocula exhibited the highest resistance to disruption. The 'disruption-resistant' fraction exhibited this characteristic through three serial re-cultivations. Possible mechanisms for the increased 'disruption-resistance' of this isolated population of C. utilis are presented.     

Characteristics of subunit composition of H+-ATPase from the anaerobic bacterium Lactobacillus casei

In the presence of Mg2+ or Ca2+ the membranes of the anaerobic glycolytic bacterium Lactobacillus casei hydrolyze 0.1-0.2 mumole ATP/min/mg of protein with a pH optimum 6.4. This activity is inhibited by N,N'-dicyclohexylcarbodiimide and is insensitive to oligomycin, ouabain, vanadate and hydroxylamine. A soluble ATPase was isolated and purified from L. casei membranes. The specific activity of this ATPase is 3.0-4.0 mumole ATP/min/mg of protein. The enzyme homogeneity was established by analytical polyacrylamide gel disc electrophoresis and by analytical centrifugation (S20, omega = 12 +/- 0,5). The molecular weight of the enzyme is 270 000. Polyacrylamide gel electrophoresis of ATPase denaturated by 1% SDS and 8 M urea in the presence of SDS revealed one type of subunits with Mr = 43 000. These subunits could not be separated by isoelectrofocusing in polyacrylamide gel in the presence of 8 M urea and migrated as a single peptide with pI at 4.2. The experimental results suggest that the soluble ATPase from L. casei consists of six identical subunits with Mr of 43 000.